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Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2012 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 423, no 4, 648-659 p.Article in journal (Refereed) Published
Abstract [en]

Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The 17 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the 17 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the 17 RNAP by the 17 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

Place, publisher, year, edition, pages
2012. Vol. 423, no 4, 648-659 p.
Keyword [en]
membrane protein production, optimization of protein expression, membrane protein biogenesis, 17 RNA polymerase-based overexpression, membrane protein functional/structural studies
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-83039DOI: 10.1016/j.jmb.2012.07.019ISI: 000310415800017OAI: oai:DiVA.org:su-83039DiVA: diva2:574201
Note

AuthorCount:9;

Available from: 2012-12-04 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically approved
In thesis
1. From protein production to genome evolution in Escherichia coli
Open this publication in new window or tab >>From protein production to genome evolution in Escherichia coli
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields.

To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 59 p.
Keyword
Escherichia coli, BL21(DE3), protein production, membrane proteins, secretory proteins, genome evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-94993 (URN)978-91-7447-786-3 (ISBN)
Public defence
2013-11-22, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2013-10-30 Created: 2013-10-20 Last updated: 2013-10-29Bibliographically approved
2. Optimizing membrane and secretory protein production in Gram-negative bacteria
Open this publication in new window or tab >>Optimizing membrane and secretory protein production in Gram-negative bacteria
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins fulfil a wide variety of essential functions in the cell. Recombinant protein production in the Gram-negative bacterium Escherichia coli (E. coli) facilitates structural and functional studies of proteins and it has been instrumental in biotechnology for the manufacturing of e.g. many protein-based drugs. However, to obtain sufficient amounts of active recombinant protein is not always a trivial task. The production of proteins that reside in membranes is limited by their complex biogenesis and their hydrophobic nature. Consequently, despite the importance of membrane proteins in health and disease (approx. 70% of today’s drugs act on membrane proteins), structures of only a very small fraction of the existing membrane proteins have yet been solved. Many soluble proteins are also difficult to produce, e.g. those containing disulfide bonds (e.g. antibody fragments and most hormones). Disulfide bond-containing proteins have to be produced in the periplasm of E. coli to be able to fold properly. To reach the periplasm, these proteins have to be ‘secreted’ across the inner membrane, which makes that also their biogenesis is complex. The aim of my PhD studies has been to improve E. coli-based production of recombinant membrane and secretory proteins. I have found that (i) the previously developed Lemo21(DE3) protein production strain can be used to set the expression intensity of a gene encoding a membrane protein such that the protein is optimally produced in the cytoplasmic membrane without causing any notable stress. Also, (ii) membrane protein production using the Lemo21(DE3) strain can be improved and simplified using carefully optimized culturing and induction conditions, demonstrated by the development of the ‘MemStar recipe’. Furthermore, I found that (iii) when using the standard BL21(DE3)/pT7 expression system for the production of membrane and secretory proteins, omitting the inducer IPTG leads to drastically improved yields as compared to when IPTG is added, owing to a lower initial target protein production rate. In the fourth study, I found that (iv) when using the PrhaBAD promoter for expression of the target gene, protein accumulation rates appear to be mostly unaffected by the inducer concentration. Using a strain-engineering approach, PrhaBAD-based protein production rates could be made constant and rhamnose concentration dependent. This dramatically improved production yields of both membrane and secretory proteins, using only very low amounts of inducer. Taken together, in accordance with previous studies, lowering production rates is an efficient strategy to increase production yields of both membrane- and secretory proteins. This is mostly due to alleviating saturation of the machinery involved in the biogenesis of these proteins. Finally, I also conducted a study (v) where, in both E. coli and Salmonella, I orchestrated the production of two membrane proteins (one that mediates the production of antigens on the surface of Gram-negative bacteria and another that makes defined pore-structures in the Gram-negative bacterial cell envelope) for the development of a safe (non-living) vaccine platform.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-123418 (URN)978-91-7649-284-0 (ISBN)
Public defence
2016-01-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
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: 2015-12-21 Created: 2015-11-25 Last updated: 2015-12-10Bibliographically approved

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