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Engineering membrane proteins for production and topology
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
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: urn:nbn:se:su:diva-116598ISBN: 978-91-7649-121-8 (print)OAI: oai:DiVA.org:su-116598DiVA: diva2:807050
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
List of papers
1. Improved production of membrane proteins in Escherichia coli by selective codon substitutions
Open this publication in new window or tab >>Improved production of membrane proteins in Escherichia coli by selective codon substitutions
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2013 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 15, 2352-2358 p.Article in journal (Refereed) Published
Abstract [en]

Membrane proteins are extremely challenging to produce in sufficient quantities for biochemical and structural analysis and there is a growing demand for solutions to this problem. In this study we attempted to improve expression of two difficult-to-express coding sequences (araH and narK) for membrane transporters. For both coding sequences, synonymous codon substitutions in the region adjacent to the AUG start led to significant improvements in expression, whereas multi-parameter sequence optimization of codons throughout the coding sequence failed. We conclude that coding sequences can be re-wired for high-level protein expression by selective engineering of the 5' coding sequence with synonymous codons, thus circumventing the need to consider whole sequence optimization. (C) 2013 Federation of European Biochemical Societies.

Keyword
Membrane protein, Transporter, Over-expression, Synthetic coding sequence, Codon optimization, AraH, NarK, Escherichia coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-93298 (URN)10.1016/j.febslet.2013.05.063 (DOI)000322606000010 ()
Funder
Swedish Research Council
Note

AuthorCount:6;

Available from: 2013-09-06 Created: 2013-09-06 Last updated: 2017-12-06Bibliographically approved
2. Efficient protein production requires selection at the vector: coding sequence junction
Open this publication in new window or tab >>Efficient protein production requires selection at the vector: coding sequence junction
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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
protein production, mrna structure, coding sequence, efficient expression
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-116602 (URN)
Available from: 2015-04-22 Created: 2015-04-22 Last updated: 2016-01-29Bibliographically approved
3. Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
Open this publication in new window or tab >>Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, 1848-1859 p.Article in journal (Refereed) Published
Abstract [en]

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

Keyword
Escherichia coli, cell envelope, proteome, membrane protein, protein complex, BN-PAGE, PpiD, YfgM
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-56162 (URN)10.1021/pr101105c (DOI)000288924000038 ()21210718 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
4. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
Open this publication in new window or tab >>Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
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2012 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 21, no 10, 1571-1576 p.Article in journal (Refereed) Published
Abstract [en]

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.

Keyword
topology, membrane protein, split-GFP, inner membrane
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-82120 (URN)10.1002/pro.2131 (DOI)000308994300015 ()
Note

AuthorCount:6;

Available from: 2012-11-09 Created: 2012-11-08 Last updated: 2017-12-07Bibliographically approved

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