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Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-6425-5059
Number of Authors: 22021 (English)In: Bio-protocol, E-ISSN 2331-8325, Vol. 11, no 16, article id e4133Article in journal (Refereed) Published
Abstract [en]

pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used modern methods of DNA assembly and directed evolution to identify improved designs for these modules and demonstrated that these designs support higher protein production yields. Herein, we present two PCR protocols for implementing the designs and increasing protein production from existing pET expression plasmids.

Place, publisher, year, edition, pages
2021. Vol. 11, no 16, article id e4133
Keywords [en]
pET, Plasmid, T7/ac, Transcription initiation, Translation initiation region (TIR), Synthetic evolution, Bacterial cell factory, Recombinant protein
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-198434DOI: 10.21769/BioProtoc.4133ISI: 000687827100013OAI: oai:DiVA.org:su-198434DiVA, id: diva2:1610147
Available from: 2021-11-10 Created: 2021-11-10 Last updated: 2022-09-15Bibliographically approved

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Shilling, Patrick J.Daley, Daniel O.

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