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Synthetically evolving translation initiation regions for protein production
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bacteria are widely employed as cell factories to produce recombinant proteins that are used as biopharmaceuticals and industrial enzymes. Typically, a protein coding sequences is cloned into an expression vector that contains a number of genetic modules designed for high-level protein production, including those that allow efficient translation within the translation initiation region (TIR). Perplexingly, recombinant protein levels can vary in an unpredictable and context-dependent manner although all sequence features permitting maximum production levels are present. Here, we have taken a systematic approach to evaluate the efficiency of the TIR generated in the commonly used pET28a expression vector. By using a PCR-based randomisation approach that generates sequence variance covering the entire TIR, the most effective synthetically evolved TIRs within large TIR libraries were identified through a simple cell survival assay. This allowed us to determine which internal region of the TIR yields the most significant increase in protein production. Data presented in this study provide a framework for obtaining a synthetically evolved TIR that yields high protein production levels.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-158480OAI: oai:DiVA.org:su-158480DiVA, id: diva2:1236861
Available from: 2018-08-06 Created: 2018-08-06 Last updated: 2018-08-07Bibliographically approved
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