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Efficient protein production requires selection at the vector: coding sequence junction
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Here we show that the junction formed when a coding sequence is cloned into an expression vector can affect protein production levels by up to a 1000-fold. Optimised junctions can be identified after a post-cloning optimisation step, which generates a library of sequence variants that differ only in ribosome-binding sites. The approach is simple, inexpensive and applicable to any experiment where efficient expression of a cloned coding sequence is sought.

Keyword [en]
protein production, mrna structure, coding sequence, efficient expression
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-116602OAI: oai:DiVA.org:su-116602DiVA: diva2:807047
Available from: 2015-04-22 Created: 2015-04-22 Last updated: 2016-01-29Bibliographically approved
In thesis
1. Engineering membrane proteins for production and topology
Open this publication in new window or tab >>Engineering membrane proteins for production and topology
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli.

For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 70 p.
Keyword
Membrane protein, Over-expression, Protein production, Codon optimization, Escherichia coli, AraH, NarK, mRNA secondary structure, coding sequence, ribosome binding site, RBS, topology, split-GFP
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-116598 (URN)978-91-7649-121-8 (ISBN)
Public defence
2015-05-28, Nordenskiöldsalen, Geovetenskapens Hus, Svante Arrhenius väg 12, Stockholm, 09:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2015-05-06 Created: 2015-04-22 Last updated: 2015-06-24Bibliographically approved

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