Characterizing and Improving pET Vectors for Cell-free Expression
Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2022.895069/full |
| _version_ | 1811333430966747136 |
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| author | Kara Jew Philip E. J. Smith Byungcheol So Jillian Kasman Javin P. Oza Michael W. Black |
| author_facet | Kara Jew Philip E. J. Smith Byungcheol So Jillian Kasman Javin P. Oza Michael W. Black |
| author_sort | Kara Jew |
| collection | DOAJ |
| description | Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system. |
| first_indexed | 2024-04-13T16:51:40Z |
| format | Article |
| id | doaj.art-025b3353de994855a5279911fdd54627 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-13T16:51:40Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-025b3353de994855a5279911fdd546272022-12-22T02:38:55ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-06-011010.3389/fbioe.2022.895069895069Characterizing and Improving pET Vectors for Cell-free ExpressionKara Jew0Philip E. J. Smith1Byungcheol So2Jillian Kasman3Javin P. Oza4Michael W. Black5Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United StatesChemistry & Biochemistry Department, California Polytechnic State University, San Luis Obispo, CA, United StatesChemistry & Biochemistry Department, California Polytechnic State University, San Luis Obispo, CA, United StatesChemistry & Biochemistry Department, California Polytechnic State University, San Luis Obispo, CA, United StatesChemistry & Biochemistry Department, California Polytechnic State University, San Luis Obispo, CA, United StatesBiological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United StatesCell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.https://www.frontiersin.org/articles/10.3389/fbioe.2022.895069/fullcell-freeprotein synthesispET30templatein vitrotranslation |
| spellingShingle | Kara Jew Philip E. J. Smith Byungcheol So Jillian Kasman Javin P. Oza Michael W. Black Characterizing and Improving pET Vectors for Cell-free Expression Frontiers in Bioengineering and Biotechnology cell-free protein synthesis pET30 template in vitro translation |
| title | Characterizing and Improving pET Vectors for Cell-free Expression |
| title_full | Characterizing and Improving pET Vectors for Cell-free Expression |
| title_fullStr | Characterizing and Improving pET Vectors for Cell-free Expression |
| title_full_unstemmed | Characterizing and Improving pET Vectors for Cell-free Expression |
| title_short | Characterizing and Improving pET Vectors for Cell-free Expression |
| title_sort | characterizing and improving pet vectors for cell free expression |
| topic | cell-free protein synthesis pET30 template in vitro translation |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2022.895069/full |
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