Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli
Abstract 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 a...
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| Format: | Article |
| Language: | English |
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BMC
2020-05-01
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| Series: | BMC Biotechnology |
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| Online Access: | http://link.springer.com/article/10.1186/s12896-020-00615-0 |
| _version_ | 1818060084390920192 |
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| author | Agnieszka Gawin Helga Ertesvåg Sine Alise Hartvigsen Hansen Jostein Malmo Trygve Brautaset |
| author_facet | Agnieszka Gawin Helga Ertesvåg Sine Alise Hartvigsen Hansen Jostein Malmo Trygve Brautaset |
| author_sort | Agnieszka Gawin |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-10T13:26:48Z |
| format | Article |
| id | doaj.art-667f7229511f48adbbd136236a9c6969 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-10T13:26:48Z |
| publishDate | 2020-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-667f7229511f48adbbd136236a9c69692022-12-22T01:47:08ZengBMCBMC Biotechnology1472-67502020-05-0120111110.1186/s12896-020-00615-0Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coliAgnieszka Gawin0Helga Ertesvåg1Sine Alise Hartvigsen Hansen2Jostein Malmo3Trygve Brautaset4Department of Biotechnology and Food Science, Norwegian University of Science and TechnologyDepartment of Biotechnology and Food Science, Norwegian University of Science and TechnologyDepartment of Biotechnology and Food Science, Norwegian University of Science and TechnologyVectron Biosolutions ASDepartment of Biotechnology and Food Science, Norwegian University of Science and TechnologyAbstract 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.http://link.springer.com/article/10.1186/s12896-020-00615-0Periplasmic translocationRecombinant protein productionTranslation initiation rateRibosome binding siteGenome editing |
| spellingShingle | Agnieszka Gawin Helga Ertesvåg Sine Alise Hartvigsen Hansen Jostein Malmo Trygve Brautaset Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli BMC Biotechnology Periplasmic translocation Recombinant protein production Translation initiation rate Ribosome binding site Genome editing |
| 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 | Periplasmic translocation Recombinant protein production Translation initiation rate Ribosome binding site Genome editing |
| url | http://link.springer.com/article/10.1186/s12896-020-00615-0 |
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