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Synthetically evolving translation initiation regions for protein production
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Daniel Daley)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Daniel Daley)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Daniel Daley)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Daniel Daley)
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(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: 2018-12-13Bibliographically approved
In thesis
1. Engineering microbial cell factories for protein production
Open this publication in new window or tab >>Engineering microbial cell factories for protein production
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are often produced using microbial cell factories for academic or industrial purposes. Protein production is however not an open-and-shut procedure. Production yields often vary in an unpredictable and context dependent manner, limiting the rational design of a straightforward production experiment.

This thesis gives an overview of how proteins are biosynthesised in bacterial cells and how this knowledge is used to produce proteins recombinantly in a host organism such as Escherichia coli. In the present investigation, we reason that unpredictable and poor protein production yields could result from incompatibility between the vector derived 5’ UTR and the 5’ end of the cloned CDS which leads to an unevolved translation initiation region (TIR). Data presented in this thesis show that an unevolved TIR could work more efficiently and yield more produced protein if subjected to synthetic evolution. Clones with an engineered synthetically evolved TIR showed enhanced protein production in both small- and large-scale production setups. This engineering method could lower production expenses, which in turn would result in increased functional determination of proteins and expanded availability of protein-based medicine to people globally. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 60
Keywords
Protein production, expression vector, recombinant DNA, Translation initiation region, Escherichia coli, mRNA secondary structure, Synthetic evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-158482 (URN)978-91-7797-366-9 (ISBN)978-91-7797-367-6 (ISBN)
Public defence
2018-09-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2018-08-29 Created: 2018-08-06 Last updated: 2018-08-29Bibliographically approved

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