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Ahmed U, Ochsenreither K, Eisele T. Production and application of peptidyl-lys metalloendopeptidase: advances, challenges, and future perspectives. Appl Microbiol Biotechnol 2025; 109:88. [PMID: 40208312 PMCID: PMC11985622 DOI: 10.1007/s00253-025-13473-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2024] [Revised: 03/30/2025] [Accepted: 03/31/2025] [Indexed: 04/11/2025]
Abstract
Peptidyl-lys metalloendopeptidases (PKMs) are enzymes that selectively cleave peptide bonds at the N-terminus of lysine residues present in the P1' position, making them valuable tools in proteomics. This mini-review presents an overview of PKMs, covering their traditional production from native sources, recent advances in recombinant production, and the current limitations in availability. The historical and current applications of PKMs in proteomics are discussed, highlighting their role in protein sequencing, peptide mapping, and mass spectrometry-based studies. Advances in recombinant technology now enable tailored modifications to PKM, allowing it to function not only as a sister enzyme to LysC but also to trypsin, thereby enhancing its suitability for specific analytical applications. The mini-review concludes with a forward-looking statement on PKM research, emphasizing the potential to broaden its use in novel proteomic methods and other applications.
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Affiliation(s)
- Uzair Ahmed
- Faculty of Mechanical and Process Engineering, Hochschule Offenburg, 77652, Offenburg, Germany
- Department of Chemical and Process Engineering, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany
| | - Katrin Ochsenreither
- Department of Chemical and Process Engineering, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany
| | - Thomas Eisele
- Faculty of Mechanical and Process Engineering, Hochschule Offenburg, 77652, Offenburg, Germany.
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Wu Y, Feng S, Sun Z, Hu Y, Jia X, Zeng B. An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system. Front Bioeng Biotechnol 2023; 11:1249841. [PMID: 37869712 PMCID: PMC10586203 DOI: 10.3389/fbioe.2023.1249841] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Accepted: 09/04/2023] [Indexed: 10/24/2023] Open
Abstract
Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.
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Affiliation(s)
| | | | | | | | | | - Bin Zeng
- College of Pharmacy, Shenzhen Technology University, Shenzhen, Guangdong, China
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den Haan R, Rose SH, Cripwell RA, Trollope KM, Myburgh MW, Viljoen-Bloom M, van Zyl WH. Heterologous production of cellulose- and starch-degrading hydrolases to expand Saccharomyces cerevisiae substrate utilization: Lessons learnt. Biotechnol Adv 2021; 53:107859. [PMID: 34678441 DOI: 10.1016/j.biotechadv.2021.107859] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Revised: 10/14/2021] [Accepted: 10/15/2021] [Indexed: 12/28/2022]
Abstract
Selected strains of Saccharomyces cerevisiae are used for commercial bioethanol production from cellulose and starch, but the high cost of exogenous enzymes for substrate hydrolysis remains a challenge. This can be addressed through consolidated bioprocessing (CBP) where S. cerevisiae strains are engineered to express recombinant glycoside hydrolases during fermentation. Looking back at numerous strategies undertaken over the past four decades to improve recombinant protein production in S. cerevisiae, it is evident that various steps in the protein production "pipeline" can be manipulated depending on the protein of interest and its anticipated application. In this review, we briefly introduce some of the strategies and highlight lessons learned with regards to improved transcription, translation, post-translational modification and protein secretion of heterologous hydrolases. We examine how host strain selection and modification, as well as enzyme compatibility, are crucial determinants for overall success. Finally, we discuss how lessons from heterologous hydrolase expression can inform modern synthetic biology and genome editing tools to provide process-ready yeast strains in future. However, it is clear that the successful expression of any particular enzyme is still unpredictable and requires a trial-and-error approach.
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Affiliation(s)
- Riaan den Haan
- Department of Biotechnology, University of the Western Cape, Bellville, South Africa
| | - Shaunita H Rose
- Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa
| | - Rosemary A Cripwell
- Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa
| | - Kim M Trollope
- Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa
| | - Marthinus W Myburgh
- Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa
| | | | - Willem H van Zyl
- Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa.
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Ngandu Mpoyi E, Cantini M, Sin YY, Fleming L, Zhou DW, Costell M, Lu Y, Kadler K, García AJ, Van Agtmael T, Salmeron-Sanchez M. Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts. Biomaterials 2020; 252:120090. [PMID: 32413593 DOI: 10.1016/j.biomaterials.2020.120090] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2020] [Accepted: 05/02/2020] [Indexed: 01/01/2023]
Abstract
Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells.
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Affiliation(s)
- Elie Ngandu Mpoyi
- Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, G12 8LT, UK
| | - Marco Cantini
- Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, G12 8LT, UK
| | - Yuan Yan Sin
- Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
| | - Lauren Fleming
- Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
| | - Dennis W Zhou
- Woodruff School of Mechanical Engineering & Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Mercedes Costell
- Departament de Bioquimica i Biologia Molecular, Universitat de València, Doctor Moliner s/n, 46100, Burjassot, Spain
| | - Yinhui Lu
- Wellcome Trust Centre for Cell Matrix Research, Faculty of Biology, Medicine & Health, University of Manchester, Manchester, M13 9PT, UK
| | - Karl Kadler
- Wellcome Trust Centre for Cell Matrix Research, Faculty of Biology, Medicine & Health, University of Manchester, Manchester, M13 9PT, UK
| | - Andrés J García
- Woodruff School of Mechanical Engineering & Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Tom Van Agtmael
- Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
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Exploiting strain diversity and rational engineering strategies to enhance recombinant cellulase secretion by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2020; 104:5163-5184. [PMID: 32337628 DOI: 10.1007/s00253-020-10602-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Revised: 03/26/2020] [Accepted: 03/31/2020] [Indexed: 12/14/2022]
Abstract
Consolidated bioprocessing (CBP) of lignocellulosic material into bioethanol has progressed in the past decades; however, several challenges still exist which impede the industrial application of this technology. Identifying the challenges that exist in all unit operations is crucial and needs to be optimised, but only the barriers related to the secretion of recombinant cellulolytic enzymes in Saccharomyces cerevisiae will be addressed in this review. Fundamental principles surrounding CBP as a biomass conversion platform have been established through the successful expression of core cellulolytic enzymes, namely β-glucosidases, endoglucanases, and exoglucanases (cellobiohydrolases) in S. cerevisiae. This review will briefly address the challenges involved in the construction of an efficient cellulolytic yeast, with particular focus on the secretion efficiency of cellulases from this host. Additionally, strategies for studying enhanced cellulolytic enzyme secretion, which include both rational and reverse engineering approaches, will be discussed. One such technique includes bio-engineering within genetically diverse strains, combining the strengths of both natural strain diversity and rational strain development. Furthermore, with the advancement in next-generation sequencing, studies that utilise this method of exploiting intra-strain diversity for industrially relevant traits will be reviewed. Finally, future prospects are discussed for the creation of ideal CBP strains with high enzyme production levels.Key Points• Several challenges are involved in the construction of efficient cellulolytic yeast, in particular, the secretion efficiency of cellulases from the hosts.• Strategies for enhancing cellulolytic enzyme secretion, a core requirement for CBP host microorganism development, include both rational and reverse engineering approaches.• One such technique includes bio-engineering within genetically diverse strains, combining the strengths of both natural strain diversity and rational strain development.
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Song X, Li Y, Wu Y, Cai M, Liu Q, Gao K, Zhang X, Bai Y, Xu H, Qiao M. Metabolic engineering strategies for improvement of ethanol production in cellulolytic Saccharomyces cerevisiae. FEMS Yeast Res 2019; 18:5071949. [PMID: 30107496 DOI: 10.1093/femsyr/foy090] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Accepted: 08/09/2018] [Indexed: 01/31/2023] Open
Abstract
As a traditional ethanol-producing microorganism, Saccharomyces cerevisiae is an ideal host for consolidated bioprocessing. However, expression of heterologous cellulase increases the metabolic burden in yeast, which results in low cellulase activity and poor cellulose degradation efficiency. In this study, cellulase-expressing yeast strains that could efficiently degrade different cellulosic substrates were created by optimizing cellulase ratios through a POT1-mediated δ-integration strategy. Metabolic engineering strategies, including optimization of codon usage, promoter and signal peptide, were also included in this system. We also confirmed that heterologous cellulase expression in cellulosic yeast induced the unfolded protein response. To enhance protein folding capacity, the endoplasmic reticulum chaperone protein BiP and the disulfide isomerase Pdi1p were overexpressed, and the Golgi membrane protein Ca2+/Mn2+ ATPase Pmr1p was disrupted to decrease the glycosylation of cellulase. The resultant strain, SK18-3, could produce 5.4 g L-1 ethanol with carboxymethyl-cellulose. Strain SK12-50 achieved 4.7 g L-1 ethanol production with phosphoric acid swollen cellulose hydrolysis. When Avicel was used as the substrate, 3.8 g L-1 ethanol (75% of the theoretical maximum yield) was produced in SK13-34. This work will significantly increase our knowledge of how to engineer optimal yeast strains for biofuel production from cellulosic biomass.
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Affiliation(s)
- Xiaofei Song
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Yuanzi Li
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Yuzhen Wu
- Department of Microbiology, College of Life Sciences, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Miao Cai
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Quanli Liu
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Kai Gao
- Tianjin Academy of Environmental Sciences, No. 17 Fukang Road, Nankai District, Tianjin 300071, China
| | - Xiuming Zhang
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Yanling Bai
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Haijin Xu
- Department of Microbiology, College of Life Sciences, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
| | - Mingqiang Qiao
- The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin 300071, China
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Vieira Gomes AM, Souza Carmo T, Silva Carvalho L, Mendonça Bahia F, Parachin NS. Comparison of Yeasts as Hosts for Recombinant Protein Production. Microorganisms 2018; 6:microorganisms6020038. [PMID: 29710826 PMCID: PMC6027275 DOI: 10.3390/microorganisms6020038] [Citation(s) in RCA: 150] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2018] [Revised: 04/23/2018] [Accepted: 04/24/2018] [Indexed: 12/21/2022] Open
Abstract
Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns.
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Affiliation(s)
- Antonio Milton Vieira Gomes
- Grupo Engenharia de Biocatalisadores, Departamento de Biologia Celular, Instituto de Ciências Biológicas Bloco K 1º andar, Universidade de Brasília, Campus Darcy Ribeiro, CEP 70.790-900 Brasília-DF, Brazil.
| | - Talita Souza Carmo
- Grupo Engenharia de Biocatalisadores, Departamento de Biologia Celular, Instituto de Ciências Biológicas Bloco K 1º andar, Universidade de Brasília, Campus Darcy Ribeiro, CEP 70.790-900 Brasília-DF, Brazil.
| | - Lucas Silva Carvalho
- Grupo Engenharia de Biocatalisadores, Departamento de Biologia Celular, Instituto de Ciências Biológicas Bloco K 1º andar, Universidade de Brasília, Campus Darcy Ribeiro, CEP 70.790-900 Brasília-DF, Brazil.
| | - Frederico Mendonça Bahia
- Grupo Engenharia de Biocatalisadores, Departamento de Biologia Celular, Instituto de Ciências Biológicas Bloco K 1º andar, Universidade de Brasília, Campus Darcy Ribeiro, CEP 70.790-900 Brasília-DF, Brazil.
| | - Nádia Skorupa Parachin
- Grupo Engenharia de Biocatalisadores, Departamento de Biologia Celular, Instituto de Ciências Biológicas Bloco K 1º andar, Universidade de Brasília, Campus Darcy Ribeiro, CEP 70.790-900 Brasília-DF, Brazil.
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Ottoz DSM, Rudolf F. Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae. Synth Biol (Oxf) 2018. [DOI: 10.1002/9783527688104.ch6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Affiliation(s)
- Diana S. M. Ottoz
- ETH Zurich; Department of Biosystems Science and Engineering; Mattenstrasse 26 4058 Basel Switzerland
- Yale University; Department of Molecular Biophysics and Biochemistry; 333 Cedar street SHM C-111 New Haven CT 06520 USA
| | - Fabian Rudolf
- ETH Zurich; Department of Biosystems Science and Engineering; Mattenstrasse 26 4058 Basel Switzerland
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Yagudin TA, Klyatchko EV, Zatsepin SS, Morozkina EV, Benevolensky SV, Shemchukova OB, Pozdnyakova LP, Solopova ON, Sveshnikov PG. Production of humanized F(ab’)2 fragment of rabies blocking antibodies in Pichia pastoris yeast. APPL BIOCHEM MICRO+ 2016. [DOI: 10.1134/s0003683816040165] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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Roongsawang N, Puseenam A, Kitikhun S, Sae-Tang K, Harnpicharnchai P, Ohashi T, Fujiyama K, Tirasophon W, Tanapongpipat S. A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica. Appl Biochem Biotechnol 2015; 178:710-24. [DOI: 10.1007/s12010-015-1904-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2015] [Accepted: 10/21/2015] [Indexed: 11/29/2022]
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Heinzelman P, Schoborg JA, Jewett MC. pH responsive granulocyte colony-stimulating factor variants with implications for treating Alzheimer's disease and other central nervous system disorders. Protein Eng Des Sel 2015; 28:481-9. [PMID: 25877663 PMCID: PMC4596278 DOI: 10.1093/protein/gzv022] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Revised: 02/27/2015] [Accepted: 03/06/2015] [Indexed: 12/14/2022] Open
Abstract
Systemic injection of granulocyte colony-stimulating factor (G-CSF) has yielded encouraging results in treating Alzheimer's Disease (AD) and other central nervous system (CNS) disorders. Making G-CSF a viable AD therapeutic will, however, require increasing G-CSF's ability to stimulate neurons within the brain. This objective could be realized by increasing transcytosis of G-CSF across the blood brain barrier (BBB). An established correlation between G-CSF receptor (G-CSFR) binding pH responsiveness and increased recycling of G-CSF to the cell exterior after endocytosis motivated development of G-CSF variants with highly pH responsive G-CSFR binding affinities. These variants will be used in future validation of our hypothesis that increased BBB transcytosis can enhance G-CSF therapeutic efficacy. Flow cytometric screening of a yeast-displayed library in which G-CSF/G-CSFR interface residues were mutated to histidine yielded a G-CSF triple His mutant (L109H/D110H/Q120H) with highly pH responsive binding affinity. This variant's KD, measured by surface plasmon resonance (SPR), increases ∼20-fold as pH decreases from 7.4 to below histidine's pKa of ∼6.0; an increase 2-fold greater than for previously reported G-CSF His mutants. Cell-free protein synthesis (CFPS) enabled expression and purification of soluble, bioactive G-CSF triple His variant protein, an outcome inaccessible via Escherichia coli inclusion body refolding. This purification and bioactivity validation will enable future identification of correlations between pH responsiveness and transcytosis in BBB cell culture model and animal experiments. Furthermore, the library screening and CFPS methods employed here could be applied to developing other pH responsive hematopoietic or neurotrophic factors for treating CNS disorders.
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Affiliation(s)
- Pete Heinzelman
- Department of Chemical, Biological and Materials Engineering, University of Oklahoma, Sarkeys Energy Center, 100 East Boyd Street, Room T-301, Norman, OK 73019, USA
| | - Jennifer A Schoborg
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208-3120, USA
| | - Michael C Jewett
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208-3120, USA
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Li C, Lin Y, Zheng X, Pang N, Liao X, Liu X, Huang Y, Liang S. Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris. BMC Biotechnol 2015; 15:88. [PMID: 26410558 PMCID: PMC4584009 DOI: 10.1186/s12896-015-0204-2] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 09/09/2015] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Phytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications. METHODS To improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production. RESULTS The phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12%, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141% higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 (i) (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412% higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. DISCUSSION The production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris. CONCLUSIONS Using modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412% relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.
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Affiliation(s)
- Cheng Li
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Ying Lin
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Xueyun Zheng
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Nuo Pang
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Xihao Liao
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Xiaoxiao Liu
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Yuanyuan Huang
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
| | - Shuli Liang
- Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China. .,Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
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Thomas DR, Walmsley AM. The effect of the unfolded protein response on the production of recombinant proteins in plants. PLANT CELL REPORTS 2015; 34:179-87. [PMID: 25187294 DOI: 10.1007/s00299-014-1680-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/10/2014] [Revised: 08/21/2014] [Accepted: 08/26/2014] [Indexed: 05/16/2023]
Abstract
Recombinant proteins are currently produced through a wide variety of host systems, including yeast, E. coli, insect and mammalian cells. One of the most recent systems developed uses plant cells. While considerable advances have been made in the yields and fidelity of plant-made recombinant proteins, many of these gains have arisen from the development of recombinant factors. This includes elements such as highly effective promoters and untranslated regions, deconstructed viral vectors, silencing inhibitors, and improved DNA delivery techniques. However, unlike other host systems, much of the work on recombinant protein production in plants uses wild-type hosts that have not been modified to facilitate recombinant protein expression. As such, there are still endogenous mechanisms functioning to maintain the health of the cell. The result is that these pathways, such as the unfolded protein response, can actively work to reduce recombinant protein production to maintain the integrity of the cell. This review examines how issues arising from the unfolded protein response have been addressed in other systems, and how these methods may be transferable to plant systems. We further identify several areas of host plant biology that present attractive targets for modification to facilitate recombinant protein production.
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Affiliation(s)
- David Rhys Thomas
- School of Biological Sciences, Monash University, Clayton, VIC, 3800, Australia,
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14
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Gu L, Zhang J, Du G, Chen J. Multivariate modular engineering of the protein secretory pathway for production of heterologous glucose oxidase in Pichia pastoris. Enzyme Microb Technol 2014; 68:33-42. [PMID: 25435503 DOI: 10.1016/j.enzmictec.2014.10.006] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Revised: 10/17/2014] [Accepted: 10/20/2014] [Indexed: 11/26/2022]
Abstract
Limitations in protein production and secretion have been attributed to the inefficient folding rate of overexpressed proteins and the cellular response to the presence of overexpressed proteins in the endoplasmic reticulum (ER). In this study, we improved the yield of glucose oxidase (GOD) by manipulating genes involved in protein folding machinery and abnormal folding stress responses. First, genes with folding and secretion functions were used to modulate the folding rate of GOD in the ER and its secretion level in the cytoplasm. Next, the potential benefits of the ERAD elements were determined. Cellular resistance to ER derived stress was then strengthened by overexpressing the stress response gene GCN4. Furthermore, a module combination strategy, which co-expressed the SEC53, CNE1 and GCN4 genes, was employed to construct the Pichia pastoris strain S17. This increased the yield of GOD to 21.81g/L, with an activity of 1972.9U/mL, which were 2.53- and 5.11-fold higher, respectively, than the control strain. The work described here improved GOD production significantly, and the strategies employed in this study provide novel information for the large-scale production of heterologous proteins.
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Affiliation(s)
- Lei Gu
- Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China
| | - Juan Zhang
- Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
| | - Guocheng Du
- The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China
| | - Jian Chen
- National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
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15
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Delic M, Göngrich R, Mattanovich D, Gasser B. Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins. Antioxid Redox Signal 2014; 21:414-37. [PMID: 24483278 DOI: 10.1089/ars.2014.5844] [Citation(s) in RCA: 75] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
SIGNIFICANCE Recombinant protein production has developed into a huge market with enormous positive implications for human health and for the future direction of a biobased economy. Limitations in the economic and technical feasibility of production processes are often related to bottlenecks of in vivo protein folding. RECENT ADVANCES Based on cell biological knowledge, some major bottlenecks have been overcome by the overexpression of molecular chaperones and other folding related proteins, or by the deletion of deleterious pathways that may lead to misfolding, mistargeting, or degradation. CRITICAL ISSUES While important success could be achieved by this strategy, the list of reported unsuccessful cases is disappointingly long and obviously dependent on the recombinant protein to be produced. Singular engineering of protein folding steps may not lead to desired results if the pathway suffers from several limitations. In particular, the connection between folding quality control and proteolytic degradation needs further attention. FUTURE DIRECTIONS Based on recent understanding that multiple steps in the folding and secretion pathways limit productivity, synergistic combinations of the cell engineering approaches mentioned earlier need to be explored. In addition, systems biology-based whole cell analysis that also takes energy and redox metabolism into consideration will broaden the knowledge base for future rational engineering strategies.
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Affiliation(s)
- Marizela Delic
- 1 Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) , Vienna, Austria
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Feizi A, Österlund T, Petranovic D, Bordel S, Nielsen J. Genome-scale modeling of the protein secretory machinery in yeast. PLoS One 2013; 8:e63284. [PMID: 23667601 PMCID: PMC3646752 DOI: 10.1371/journal.pone.0063284] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2012] [Accepted: 03/31/2013] [Indexed: 11/19/2022] Open
Abstract
The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking. Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm was developed which mimics secretory machinery and assigns each secretory protein to a particular secretory class that determines the set of PTMs and transport steps specific to each protein. Protein abundances were integrated with the model in order to gain system level estimation of the metabolic demands associated with the processing of each specific protein as well as a quantitative estimation of the activity of each component of the secretory machinery.
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Affiliation(s)
- Amir Feizi
- Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
| | - Tobias Österlund
- Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
| | - Dina Petranovic
- Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
| | - Sergio Bordel
- Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
| | - Jens Nielsen
- Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark
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Zubkova ES, Semenkova LN, Dudich IV, Dudich EI, Khromykh LM, Makarevich PI, Parfenova EV, Men'shikov MI. [Recombinant human alpha-fetoprotein as a regulator of adipose tissue stromal cell activity]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY 2013; 38:524-34. [PMID: 23342486 DOI: 10.1134/s1068162012050147] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Recombinant human alpha-fetoprotein (rhAFP) expressed in yeast system as a glycoprotein, was isolated and purified to 98% by multistep method. The testing of the rhAFP in the culture of adipose tissue stromal cells (hASC) has revealed its ability to enhance hASC proliferation and migration as well as vascular endothelial growth factor production, with no significant influence on cell invasion and matrix metalloproteinase-2 and -9 secretion. It has been also estimated that rhAFP is internalized in hASC via clathrin-dependent mechanism. A study in the murine experimental model of hindlimb ischemia has shown the capability of rhAFP to enhance blood flow recovery. These data suggest that rhAFP is a promising agent for enhancement of the hASC regenerative ability.
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18
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Hou J, Tyo KE, Liu Z, Petranovic D, Nielsen J. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae. FEMS Yeast Res 2012; 12:491-510. [DOI: 10.1111/j.1567-1364.2012.00810.x] [Citation(s) in RCA: 118] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2012] [Revised: 04/19/2012] [Accepted: 04/22/2012] [Indexed: 01/02/2023] Open
Affiliation(s)
| | | | - Zihe Liu
- Department of Chemical and Biological Engineering; Chalmers University of Technology; Göteborg; Sweden
| | - Dina Petranovic
- Department of Chemical and Biological Engineering; Chalmers University of Technology; Göteborg; Sweden
| | - Jens Nielsen
- Department of Chemical and Biological Engineering; Chalmers University of Technology; Göteborg; Sweden
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Dudich E, Dudich I, Semenkova L, Benevolensky S, Morozkina E, Marchenko A, Zatcepin S, Dudich D, Soboleva G, Khromikh L, Roslovtceva O, Tatulov E. Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization. Protein Expr Purif 2012; 84:94-107. [PMID: 22561245 DOI: 10.1016/j.pep.2012.04.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2011] [Revised: 04/11/2012] [Accepted: 04/14/2012] [Indexed: 11/20/2022]
Abstract
Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.
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Affiliation(s)
- Elena Dudich
- Institute of Immunological Engineering, Lyubuchany, Moscow Region, Chekhov District 142380, Russia.
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20
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Tyo KEJ, Liu Z, Petranovic D, Nielsen J. Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress. BMC Biol 2012; 10:16. [PMID: 22380681 PMCID: PMC3310788 DOI: 10.1186/1741-7007-10-16] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2012] [Accepted: 03/01/2012] [Indexed: 01/17/2023] Open
Abstract
Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase). Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
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Affiliation(s)
- Keith E J Tyo
- Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296 Göteborg, Sweden
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21
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Liu Z, Tyo KEJ, Martínez JL, Petranovic D, Nielsen J. Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae. Biotechnol Bioeng 2012; 109:1259-68. [PMID: 22179756 DOI: 10.1002/bit.24409] [Citation(s) in RCA: 111] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2011] [Revised: 11/28/2011] [Accepted: 12/08/2011] [Indexed: 11/06/2022]
Abstract
Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (µmol/L) of IP was found to be higher than that of α-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.
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Affiliation(s)
- Zihe Liu
- Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
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22
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Wu D, Chu J, Hao YY, Wang YH, Zhuang YP, Zhang SL. High efficient production of recombinant human consensus interferon mutant in high cell density culture of Pichia pastoris using two phases methanol control. Process Biochem 2011. [DOI: 10.1016/j.procbio.2011.05.015] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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23
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Idiris A, Tohda H, Kumagai H, Takegawa K. Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 2010; 86:403-17. [DOI: 10.1007/s00253-010-2447-0] [Citation(s) in RCA: 195] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2009] [Revised: 01/07/2010] [Accepted: 01/09/2010] [Indexed: 01/08/2023]
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24
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Carlage T, Hincapie M, Zang L, Lyubarskaya Y, Madden H, Mhatre R, Hancock WS. Proteomic profiling of a high-producing Chinese hamster ovary cell culture. Anal Chem 2009; 81:7357-62. [PMID: 19663468 DOI: 10.1021/ac900792z] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The productivity of mammalian cell culture expression systems is critically important to the production of biopharmaceuticals. In this study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-X(L) gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomarker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene that is known to have growth regulating properties in human cells. A total of 392 proteins were identified in this study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were downregulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-X(L). The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures.
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Affiliation(s)
- Tyler Carlage
- Biogen Idec, 15 Cambridge Center, Cambridge, Massachusetts 02142, USA
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25
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Xu P, Robinson AS. Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast. Biotechnol Bioeng 2009; 104:20-9. [PMID: 19415776 DOI: 10.1002/bit.22376] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the UPR. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties.
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Affiliation(s)
- Ping Xu
- Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
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26
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Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system. Biotechnol Appl Biochem 2009; 53:227-35. [PMID: 19531030 DOI: 10.1042/ba20090048] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well-characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high-level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein-expressing host systems are still required for the production of heterologous proteins involved in post-translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission-yeast strains.
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Abstract
The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial ("white") biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.
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Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 2008; 7:11. [PMID: 18394160 PMCID: PMC2322954 DOI: 10.1186/1475-2859-7-11] [Citation(s) in RCA: 232] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2007] [Accepted: 04/04/2008] [Indexed: 11/17/2022] Open
Abstract
Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.
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Affiliation(s)
- Brigitte Gasser
- University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Vienna, Austria
| | | | - Ursula Rinas
- Helmholtz Center for Infection Research, Braunschweig, Germany
| | - Martin Dragosits
- University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Vienna, Austria
| | - Escarlata Rodríguez-Carmona
- Autonomous University of Barcelona, Institute for Biotechnology and Biomedicine, Department of Genetics and Microbiology, and CIBER-BBN Network in Bioengineering, Biomaterials and Nanomedicine, Barcelona, Spain
| | - Kristin Baumann
- Autonomous University of Barcelona, Department of Chemical Engineering, Barcelona, Spain
| | - Maria Giuliani
- University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy
| | - Ermenegilda Parrilli
- University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy
| | - Paola Branduardi
- University of Milano-Bicocca, Department of Biotechnology and Bioscience, Milan, Italy
| | - Christine Lang
- Technical University Berlin, Faculty III, Institute for Microbiology and Genetics, Berlin, Germany
| | - Danilo Porro
- University of Milano-Bicocca, Department of Biotechnology and Bioscience, Milan, Italy
| | - Pau Ferrer
- Autonomous University of Barcelona, Department of Chemical Engineering, Barcelona, Spain
| | - Maria Luisa Tutino
- University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy
| | - Diethard Mattanovich
- University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Vienna, Austria
| | - Antonio Villaverde
- Autonomous University of Barcelona, Institute for Biotechnology and Biomedicine, Department of Genetics and Microbiology, and CIBER-BBN Network in Bioengineering, Biomaterials and Nanomedicine, Barcelona, Spain
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29
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Parallel analysis of tetramerization domain mutants of the human p53 protein using PCR colonies. Genomic Med 2007; 1:113-24. [PMID: 18923936 DOI: 10.1007/s11568-007-9011-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2006] [Accepted: 08/01/2007] [Indexed: 01/21/2023] Open
Abstract
A highly-parallel yeast functional assay, capable of screening approximately 100-1,000 mutants in parallel and designed to screen the activity of transcription activator proteins, was utilized to functionally characterize tetramerization domain mutants of the human p53 transcription factor and tumor suppressor protein. A library containing each of the 19 possible single amino acid substitutions (57 mutants) at three positions in the tetramerization domain of the human p53 protein, was functionally screened in Saccharomyces cerevisiae. Amino acids Leu330 and Ile332, whose side chains form a portion of a hydrophobic pocket that stabilizes the active p53 tetramer, were found to tolerate most hydrophobic amino acid substitutions while hydrophilic substitutions resulted in the inactivation of the protein. Amino acid Gln331 tolerated essentially all mutations. Importantly, highly parallel mutagenesis and cloning techniques were utilized which, in conjunction with recently reported highly parallel DNA sequencing methods, would be capable of increasing throughput an additional 2-3 orders of magnitude.
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Abstract
Although manipulation of the endoplasmic reticulum (ER) folding environment in the yeast Saccharomyces cerevisiae has been shown to increase the secretory productivity of recombinant proteins, the cellular interactions and processes of native enzymes and chaperones such as protein disulfide isomerase (PDI) are still unclear. Previously, we reported that overexpression of the ER chaperone PDI enabled up to a 3-fold increase in secretion levels of the Pyrococcus furiosus beta-glucosidase in the yeast S. cerevisiae. This result was surprising since beta-glucosidase contains only one cysteine per monomer and no disulfide bonds. Two possible mechanisms were proposed: PDI either forms a transient disulfide bond with the lone cysteine residue of the nascent beta-glucosidase during the folding and assembly process or acts as a chaperone to aid in proper folding. To discern between the two mechanisms, the single cysteine residue was mutated to serine, and the secretion of the two protein variants was determined. The serine mutant still showed increased secretion in vivo when PDI levels were elevated. When the folding bottleneck is removed by increasing expression temperatures to 37 degrees C rather than 30 degrees C, PDI no longer has an improvement on secretion. These results suggest that, unexpectedly, PDI acts in a chaperone-like capacity or possibly cooperates with the cell's folding or degradation mechanisms regardless of whether the protein is redox-active.
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Affiliation(s)
| | - Anne Skaja Robinson
- To whom correspondence should be addressed at: Tel: +1 302 831-0557, Fax: +1 302 831-1048,
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Wentz AE, Shusta EV. A novel high-throughput screen reveals yeast genes that increase secretion of heterologous proteins. Appl Environ Microbiol 2006; 73:1189-98. [PMID: 17189442 PMCID: PMC1828678 DOI: 10.1128/aem.02427-06] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The yeast Saccharomyces cerevisiae is an attractive host for the production of heterologous proteins. However, low-yield production of many proteins (from micrograms to milligrams/liter) leaves considerable room for optimization. By engineering the yeast cell via traceable genome-wide libraries, genes that can enhance protein expression level because of their roles in protein transcription, translation, folding, and trafficking processes can be readily identified. This report details a novel approach that combines yeast cDNA overexpression libraries with yeast surface display to allow the rapid flow cytometric screening of engineered yeast for gene products that improve the display of heterologous proteins. After optimization of the screening conditions, a genome-wide scan yielded five yeast gene products that promoted increased display levels of a single-chain T-cell receptor (scTCR). The display-enhancing genes included those coding for cell wall proteins (CCW12, CWP2, and SED1), a ribosomal subunit protein (RPP0), and an endoplasmic reticulum-resident protein (ERO1). Under the premise that yeast surface display levels could be used as a predictor of secretion efficiency, each display-enhancing gene product was tested for its ability to affect secretion levels of multiple scTCR and single-chain antibodies (scFv). All of the selected yeast gene products were shown to promote increased secretion of active protein (1.5-fold to 7.9-fold), with CCW12 and ERO1 being the most generalizable enhancers of scFv/scTCR secretion.
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Affiliation(s)
- Alane E Wentz
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
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Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, Stephanopoulos G. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol 2006; 72:5266-73. [PMID: 16885275 PMCID: PMC1538763 DOI: 10.1128/aem.00530-06] [Citation(s) in RCA: 173] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.
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Affiliation(s)
- Elke Nevoigt
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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Rakestraw A, Wittrup KD. Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae. Biotechnol Bioeng 2006; 93:896-905. [PMID: 16333864 DOI: 10.1002/bit.20780] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.
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Affiliation(s)
- Andy Rakestraw
- Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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Hackel BJ, Huang D, Bubolz JC, Wang XX, Shusta EV. Production of Soluble and Active Transferrin Receptor-Targeting Single-Chain Antibody using Saccharomyces cerevisiae. Pharm Res 2006; 23:790-7. [PMID: 16550469 DOI: 10.1007/s11095-006-9778-7] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2005] [Accepted: 12/16/2005] [Indexed: 11/25/2022]
Abstract
PURPOSE This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. METHODS The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv was purified using immobilized metal affinity chromatography, and tested for binding and internalization into the RBE4 rat brain endothelial cell line. RESULTS Secretion of OX26 scFv was optimal when expression was induced at 20 degrees C. Co-overexpression of heavy chain binding protein and protein disulfide isomerase elevated scFv expression levels by 10.4 +/- 0.3-fold. Optimization of scFv gene dosage increased secretion by 7.1 +/- 0.2-fold, but the overall benefits of binding protein and protein disulfide isomerase overexpression were diminished. Purified OX26 scFv yields of 0.5 mg/L secreted protein were achieved, and the scFv was actively internalized into RBE4 cells with a pattern similar to that observed with intact OX26 monoclonal antibody. CONCLUSIONS The optimized S. cerevisiae expression system is amenable to production of soluble and active brain targeting OX26 scFv, and the yeast-produced scFv has potential for the targeting and delivery of small molecules, proteins, or drug carriers across the blood-brain barrier (BBB).
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Affiliation(s)
- Benjamin J Hackel
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, USA
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35
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Li YY, Bendayan M. Alteration of chaperonin60 and pancreatic enzyme in pancreatic acinar cell under pathological condition. World J Gastroenterol 2006; 11:7359-63. [PMID: 16437643 PMCID: PMC4723395 DOI: 10.3748/wjg.v11.i46.7359] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the changes of chaperonin60 (Cpn60) and pancreatic enzymes in pancreatic acinar cells, and to explore their roles in the development of experimental diabetes and acute pancreatitis (AP). METHODS Two different pathological models were replicated in Sprague-Dawley rats: streptozotocin-induced diabetes and sodium deoxycholate-induced AP. The contents of Cpn60 and pancreatic enzymes in different compartments of the acinar cells were measured by quantitative immunocytochemistry. RESULTS The levels of Cpn60 significantly increased in diabetes, but decreased in AP, especially in the zymogen granules of the pancreatic acinar cells. The elevation of Cpn60 was accompanied with the increased levels of pancreatic lipase and chymotrypsinogen in diabetes. However, a decreased Cpn60 level was accompanied by high levels of lipase and chymotrypsinogen in AP. The amylase level was markedly reduced in both the pathological conditions. CONCLUSION The equilibrium between Cpn60 and pancreatic enzymes in the acinar cells breaks in AP, and Cpn60 content decreases, suggesting an insufficient chaperone capacity. This may promote the aggregation and autoactivation of the premature enzymes in the pancreatic acinar cells and play roles in the development of AP.
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Affiliation(s)
- Yong-Yu Li
- Department of Pathophysiology, Medical College of Tongji University, Shanghai 200092, China.
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36
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Alete DE, Racher AJ, Birch JR, Stansfield SH, James DC, Smales CM. Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities. Proteomics 2006; 5:4689-704. [PMID: 16247733 DOI: 10.1002/pmic.200500019] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high-level antibody secretion. We used GS-NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid-exponential growth phase. These were analysed by 2-D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up-regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell-engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.
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Affiliation(s)
- Daniel E Alete
- Protein Science Group, Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
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37
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Dinnis DM, James DC. Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnol Bioeng 2005; 91:180-9. [PMID: 15880827 DOI: 10.1002/bit.20499] [Citation(s) in RCA: 123] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
In this review we consider how cell specific recombinant monoclonal antibody (Mab) production by engineered mammalian cells can be improved. Whilst it is generally recognized that Mab production is limited post-transcriptionally at folding and assembly reactions, genetic engineering strategies based on overexpression of individual chaperones or foldases in mammalian cells have not reliably increased cell specific Mab production. Given that recent studies have established that chaperones and foldases themselves exist in a large multiprotein complex, which may coordinate the sequential processing of Mabs, we propose that global expansion of all components of the secretory pathway will likely be necessary to generically improve recombinant Mab production by mammalian cells. In this context, what can be learnt from nature? Important recent studies have delineated some of the main cellular pathways involved in the differentiation of B-cells into nature's own high level Mab producers, plasma cells. This is achieved by a dramatic re-programming of cellular function where the coordinated expansion of metabolic and secretory machinery precedes Ig production, then is maintained by induction of a key intracellular signaling pathway, the unfolded protein response (UPR). Here we review genetic engineering strategies to increase cell specific production rate and discuss whether manipulation of intracellular signaling systems such as the UPR will provide a novel means to engineer mammalian cells for high level recombinant Mab production.
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Affiliation(s)
- Diane M Dinnis
- School of Engineering, University of Queensland, St. Lucia, QLD 4072, Australia
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38
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Niebauer RT, Wedekind A, Robinson AS. Decreases in yeast expression yields of the human adenosine A2a receptor are a result of translational or post-translational events. Protein Expr Purif 2005; 37:134-43. [PMID: 15294291 DOI: 10.1016/j.pep.2004.06.001] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2004] [Revised: 06/01/2004] [Indexed: 11/21/2022]
Abstract
The human adenosine receptor (A2a), a G-protein-coupled receptor (GPCR), was C-terminally tagged with the green fluorescent protein (GFP) and expressed in the yeast Saccharomyces cerevisiae to gain an understanding of the expression limitations of this medically relevant class of membrane proteins. The A2a-GFP protein was able to bind adenosine analogs indicating that the GFP tag did not alter the ligand binding activity of the receptor. A screen based on whole cell fluorescence was developed and a library of clones with various gene copy numbers was screened via flow cytometry to isolate clones with the highest protein expression levels. All clones studied exhibited a decrease in the net A2a-GFP protein production rate over time as determined by whole cell fluorescence, Western blotting, confocal microscopy, and ligand binding. Quantitative PCR showed that A2a-GFP mRNA levels remained relatively high even as the protein production rate decreased. A cycloheximide chase experiment showed that the mature protein was stable over time and was not significantly degraded. Taken together, these results suggest that heterologous expression of GPCRs is limited by a translational or post-translational bottleneck that is unique from expression limitations seen for soluble proteins.
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Affiliation(s)
- Ronald T Niebauer
- Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
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39
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Smith JD, Richardson NE, Robinson AS. Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2005; 1752:18-25. [PMID: 16112628 DOI: 10.1016/j.bbapap.2005.07.016] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/02/2005] [Revised: 07/18/2005] [Accepted: 07/26/2005] [Indexed: 11/29/2022]
Abstract
Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.
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Affiliation(s)
- Jason D Smith
- Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, DE 19716, USA
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40
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Xu P, Raden D, Doyle FJ, Robinson AS. Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding. Metab Eng 2005; 7:269-79. [PMID: 15990348 DOI: 10.1016/j.ymben.2005.04.002] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2004] [Revised: 02/24/2005] [Accepted: 04/20/2005] [Indexed: 11/15/2022]
Abstract
The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20. These studies show that overexpression of BiP did not reduce the UPR activated by heterologous protein expression; however, overexpression of PDI or co-overexpression of BiP and PDI could reduce the UPR. We observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. We propose that BiP acts primarily to facilitate translocation and retain unfolded or partially folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and/or an isomerase, of disulfide bonds. Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR.
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Affiliation(s)
- Ping Xu
- 259 Colburn Laboratory, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
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41
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Ignowski JM, Schaffer DV. Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells. Biotechnol Bioeng 2005; 86:827-34. [PMID: 15162459 DOI: 10.1002/bit.20059] [Citation(s) in RCA: 128] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. However, the intracellular behavior and properties of the enzyme are not well characterized. Specifically, information on the intracellular kinetics and stability of luciferase activity is necessary for real-time luminescence counts from live cells to be quantitatively meaningful. Here, we report a dynamic analysis of luciferase activity in the context of living mammalian cells. We have determined the relative light units measured in living cells to be proportional to that found in cell lysate. We have also calculated the K(m) of luciferase in living cells to be approximately 1 mM, a value much higher than the 10 microM found for pure enzyme in vitro. In addition, a 2-hour half-life of luciferase activity in live cells was measured in real time. Finally, we have modeled luciferase activity in live cells for the purposes of understanding and translating the luciferase signal into a more effective metric of gene expression and cell behavior.
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Affiliation(s)
- Jolene M Ignowski
- Department of Chemical Engineering and The Helen Wills Neuroscience Institute, MC 1462, University of California-Berkeley, Berkeley, CA 94720-1462, USA
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42
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Merritt J, Butz JA, Ogunnaike BA, Edwards JS. Parallel analysis of mutant human glucose 6-phosphate dehydrogenase in yeast using PCR colonies. Biotechnol Bioeng 2005; 92:519-31. [PMID: 16193512 DOI: 10.1002/bit.20726] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We demonstrate a highly parallel strategy to analyze the impact of single nucleotide mutations on protein function. Using our method, it is possible to screen a population and quickly identify a subset of functionally interesting mutants. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools, and polymerase colonies. A defined mutant human glucose-6-phosphate-dehydrogenase library was constructed which contains all possible single nucleotide missense mutations in the eight-residue glucose-6-phosphate binding peptide of the enzyme. Mutant human enzymes were expressed in a zwf1 (gene encoding yeast homologue) deletion strain of Saccharomyces cerevisiae. Growth rates of the 54 mutant strains arising from this library were measured in parallel in conditions selective for active hG6PD. Several residues were identified which tolerated no mutations (Asp200, His201 and Lys205) and two (Ile199 and Leu203) tolerated several substitutions. Arg198, Tyr202, and Gly204 tolerated only 1-2 specific substitutions. Generalizing from the positions of tolerated and non-tolerated amino acid substitutions, hypotheses were generated about the functional role of specific residues, which could, potentially, be tested using higher resolution/lower throughput methods.
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Affiliation(s)
- Joshua Merritt
- Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
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43
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Lombraña M, Moralejo FJ, Pinto R, Martín JF. Modulation of Aspergillus awamori thaumatin secretion by modification of bipA gene expression. Appl Environ Microbiol 2004; 70:5145-52. [PMID: 15345393 PMCID: PMC520887 DOI: 10.1128/aem.70.9.5145-5152.2004] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Two different strains, Aspergillus awamori TGDTh-4 and A. awamori TGP-3 overexpressing a synthetic gene encoding the plant sweet protein thaumatin, showed an unfolded protein response. To facilitate protein secretion, the chaperone BiPA gene was expressed in A. awamori under control of the strong constitutive promoter of the gpdA gene. A good correlation was observed between the level of the bipA transcript in different strains and the amount of thaumatin secreted. Thaumatin secretion was increased 2- to 2.5-fold in transformants overexpressing the bipA gene compared with the parental strain. Secretion of the homologous proteins alpha-amylase and glucoamylase was not affected by the bipA gene overexpression. The requirement for BiPA for secretion of thaumatin was confirmed by attenuation of the endogenous bipA gene expression with an antisense RNA cassette. The decrease in bipA expression reduced the amount of secreted thaumatin up to 80% without affecting the secretion of the homologous alpha-amylase and glucoamylase proteins. The BiPA protein is, therefore, very important for secretion of some heterologous proteins, such as thaumatin in A. awamori.
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Affiliation(s)
- Marta Lombraña
- INBIOTEC, Faculad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain
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44
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Mattanovich D, Gasser B, Hohenblum H, Sauer M. Stress in recombinant protein producing yeasts. J Biotechnol 2004; 113:121-35. [PMID: 15380652 DOI: 10.1016/j.jbiotec.2004.04.035] [Citation(s) in RCA: 193] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2003] [Revised: 04/07/2004] [Accepted: 04/16/2004] [Indexed: 11/27/2022]
Abstract
It is well established today that heterologous overexpression of proteins is connected with different stress reactions. The expression of a foreign protein at a high level may either directly limit other cellular processes by competing for their substrates, or indirectly interfere with metabolism, if their manufacture is blocked, thus inducing a stress reaction of the cell. Especially the unfolded protein response (UPR) in Saccharomyces cerevisiae (as well as some other yeasts) is well documented, and its role for the limitation of expression levels is discussed. One potential consequence of endoplasmatic reticulum folding limitations is the ER associated protein degradation (ERAD) involving retrotranslocation and decay in the cytosol. High cell density fermentation, the typical process design for recombinant yeasts, exerts growth conditions that deviate far from the natural environment of the cells. Thus, different environmental stresses may be exerted on the host. High osmolarity, low pH and low temperature are typical stress factors. Whereas the molecular pathways of stress responses are well characterized, there is a lack of knowledge concerning the impact of stress responses on industrial production processes. Accordingly, most metabolic engineering approaches conducted so far target at the improvement of protein folding and secretion, whereas only few examples of cell engineering against general stress sensitivity were published. Apart from discussing well-documented stress reactions of yeasts in the context of heterologous protein production, some more speculative topics like quorum sensing and apoptosis are addressed.
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Affiliation(s)
- Diethard Mattanovich
- Institute of Applied Microbiology, BOKU--University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
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45
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Maréchal A, Tanguay PL, Callejo M, Guérin R, Boileau G, Rokeach LA. Cell viability and secretion of active proteins in Schizosaccharomyces pombe do not require the chaperone function of calnexin. Biochem J 2004; 380:441-8. [PMID: 14984369 PMCID: PMC1224181 DOI: 10.1042/bj20031546] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2003] [Revised: 02/20/2004] [Accepted: 02/25/2004] [Indexed: 11/17/2022]
Abstract
Folding of newly synthesized proteins within the ER (endoplasmic reticulum) is a rate-limiting step in protein secretion. Thus ER molecular chaperones and foldases have a major impact in determining the rate and yield of these crucial cellular processes. Calnexin is a key ER chaperone implicated in the folding, retention and targeting for degradation of proteins that go through the secretory pathway. Calnexin molecules contain a highly conserved central domain (hcd) that has been proposed to be involved in the interaction with folding substrates and other chaperones. To gain a better understanding of the roles played by calnexin in the secretory pathway, we examined the efficiency of fission yeast (Schizosaccharomyces pombe) strains expressing calnexin mutants to secrete different model proteins. Remarkably, calnexin hcd-deletion mutants, although devoid of detectable chaperone activity in vitro, confer viability and cause a considerable increase in the secretion of heterologous cellulase. Surprisingly the quality-control efficiency, measured as the activity/amount ratio of secreted model protein, was not severely reduced in these calnexin hcd-deletion mutant strains. Our results indicate that the essential function of calnexin does not reside in its role in the folding or in the retention of misfolded proteins. These observations suggest the existence of a highly stringent quality control mechanism in the ER of S. pombe that might reduce the secretion efficiency of endogenous proteins.
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Affiliation(s)
- Alexandre Maréchal
- Department of Biochemistry, Université de Montréal, C.P. 6128 succursale Centre-ville, Montréal, Québec H3C 3J7, Canada
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Smith JD, Tang BC, Robinson AS. Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast. Biotechnol Bioeng 2004; 85:340-50. [PMID: 14748090 DOI: 10.1002/bit.10853] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.
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Affiliation(s)
- Jason D Smith
- Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, Delaware 19716, USA
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Butz JA, Niebauer RT, Robinson AS. Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast. Biotechnol Bioeng 2003; 84:292-304. [PMID: 12968283 DOI: 10.1002/bit.10771] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The limitations to high-level expression of integral membrane proteins are not well understood. The human A(2)a adenosine receptor (A(2)a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks for G-protein coupled receptors. In the yeast system, A(2)a was not N-linked glycosylated but was functional and plasma membrane-localized. A(2)a also contained an intramolecular disulfide bond. Substance P receptor was also not N-linked glycosylated in yeast, but, unlike A(2)a, SPR was intracellularly retained, nonfunctional, and did not appear to contain an intramolecular disulfide bond. Since both receptors contain N-linked glycosylation and disulfide bonds in mammalian systems, machinery responsible for interacting with these modifications was investigated-specifically, the potential interactions between the nascent receptor and ER-resident proteins were explored. The chaperones calnexin and protein disulfide isomerase were co-overexpressed with the GPCRs to determine the effect on total and active yields of A(2)a and SPR, as well as on receptor trafficking. The effect of co-expressing the chaperone BiP on the total yields of A(2)a as well as intracellular fates of both receptors were determined. The co-expression of ER resident proteins did not improve A(2)a yields nor did they restore SPR activity or improve SPR cell surface expression. Taken together, these results indicate that an ER-folding bottleneck does not limit the expression of the mammalian receptors in yeast.
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Affiliation(s)
- James A Butz
- Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, DE 19716, USA
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Merritt J, DiTonno JR, Mitra RD, Church GM, Edwards JS. Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies. Nucleic Acids Res 2003; 31:e84. [PMID: 12888536 PMCID: PMC169973 DOI: 10.1093/nar/gng084] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
We describe a strategy to analyze the impact of single nucleotide mutations on protein function. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR. A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae. Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants. Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins. Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel. By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.
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Affiliation(s)
- Joshua Merritt
- Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
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Kim MD, Han KC, Kang HA, Rhee SK, Seo JH. Coexpression of BiP increased antithrombotic hirudin production in recombinant Saccharomyces cerevisiae. J Biotechnol 2003; 101:81-7. [PMID: 12523972 DOI: 10.1016/s0168-1656(02)00288-2] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
In order to increase a production level of antithrombotic hirudin, BiP was simultaneously expressed in recombinant Saccharomyces cerevisiae strains carrying ten and 15 copies of the hirudin expression cassette integrated in the chromosome. Coexpression of BiP greatly enhanced both cell growth and hirudin production in recombinant S. cerevisiae. Maximum hirudin concentration of 36 mg l(-1) was obtained from batch culture of the ten copy-number transformant concomitantly harboring an episomal copy of the BiP gene under the control of the GAL1 promoter, which is corresponding to a 2.5-fold increase compared with the control strain carrying the genomic BiP gene only. The mean size of the recombinant yeast cells expressing the BiP gene remained at a relatively constant level compared with the control strains of which size increased after the onset of hirudin expression by the GAL10 promoter.
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Affiliation(s)
- Myoung-Dong Kim
- Department of Food Science and Technology, Research Center for New Bio-Materials in Agriculture, Seoul National University, Suwon 441-744, South Korea
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Wang H, Entwistle J, Morlon E, Archer DB, Peberdy JF, Ward M, Jeenes DJ. Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger. Mol Genet Genomics 2003; 268:684-91. [PMID: 12589443 DOI: 10.1007/s00438-002-0790-2] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2002] [Accepted: 11/27/2002] [Indexed: 10/25/2022]
Abstract
We describe the isolation of a gene (clxA) encoding calnexin from laboratory and industrial strains of Aspergillus niger. Calnexin is a chaperone, which specifically recognises monoglucosylated glycoproteins in the endoplasmic reticulum, and is thus an essential component of the process that assesses the folded state of nascent secreted glycoproteins. Manipulation of chaperones has previously been adopted in attempts to overcome some of the problems associated with the secretion of heterologous proteins from filamentous fungi. The A. niger clxA gene encodes a 562-residue protein with strong homology to the calnexin of Schizosaccharomyces pombe. The clxAgene product complements a S. pombe cnx1 mutant. Motifs associated with genes controlled via the Unfolded Protein Response (UPR) were identified by sequence homology in the promoter of clxA. Steady-state levels of clxA mRNA were elevated in a strain expressing bovine prochymosin fused to the catalytic domain of glucoamylase. The ORF is punctuated by four introns, and contains two sets of four repeated peptide motifs that are characteristic of the calnexin family, together with a putative membrane-spanning domain. Deletion studies indicate that clxA is not an essential gene in A. niger.
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MESH Headings
- Animals
- Aspergillus niger/genetics
- Aspergillus niger/metabolism
- Base Sequence
- Calnexin/genetics
- Calnexin/metabolism
- Cattle
- Chymosin/biosynthesis
- Chymosin/genetics
- DNA, Fungal/genetics
- Enzyme Precursors/biosynthesis
- Enzyme Precursors/genetics
- Fungal Proteins/genetics
- Fungal Proteins/metabolism
- Gene Deletion
- Gene Expression
- Genes, Fungal
- Genetic Complementation Test
- Glucan 1,4-alpha-Glucosidase/biosynthesis
- Glucan 1,4-alpha-Glucosidase/genetics
- Molecular Sequence Data
- Mutation
- Promoter Regions, Genetic
- RNA, Fungal/genetics
- RNA, Fungal/metabolism
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Recombinant Fusion Proteins/biosynthesis
- Recombinant Fusion Proteins/genetics
- Sequence Homology, Amino Acid
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Affiliation(s)
- H Wang
- Genencor International Inc., Palo Alto, CA 94304, USA
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