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Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli

BACKGROUND: Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associate...

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Autores principales: Gawin, Agnieszka, Ertesvåg, Helga, Hansen, Sine Alise Hartvigsen, Malmo, Jostein, Brautaset, Trygve
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216727/
https://www.ncbi.nlm.nih.gov/pubmed/32393331
http://dx.doi.org/10.1186/s12896-020-00615-0
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author Gawin, Agnieszka
Ertesvåg, Helga
Hansen, Sine Alise Hartvigsen
Malmo, Jostein
Brautaset, Trygve
author_facet Gawin, Agnieszka
Ertesvåg, Helga
Hansen, Sine Alise Hartvigsen
Malmo, Jostein
Brautaset, Trygve
author_sort Gawin, Agnieszka
collection PubMed
description BACKGROUND: Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. RESULTS: We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173–2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. CONCLUSIONS: Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.
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spelling pubmed-72167272020-05-18 Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli Gawin, Agnieszka Ertesvåg, Helga Hansen, Sine Alise Hartvigsen Malmo, Jostein Brautaset, Trygve BMC Biotechnol Research Article BACKGROUND: Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. RESULTS: We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173–2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. CONCLUSIONS: Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli. BioMed Central 2020-05-11 /pmc/articles/PMC7216727/ /pubmed/32393331 http://dx.doi.org/10.1186/s12896-020-00615-0 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Article
Gawin, Agnieszka
Ertesvåg, Helga
Hansen, Sine Alise Hartvigsen
Malmo, Jostein
Brautaset, Trygve
Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
title Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
title_full Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
title_fullStr Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
title_full_unstemmed Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
title_short Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
title_sort translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in escherichia coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216727/
https://www.ncbi.nlm.nih.gov/pubmed/32393331
http://dx.doi.org/10.1186/s12896-020-00615-0
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