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Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
2013 (engelsk)Inngår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 12, s. 24-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Background: In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results: Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions: Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment.

sted, utgiver, år, opplag, sider
2013. Vol. 12, s. 24-
Emneord [en]
Recombinant protein, Protein production, Escherichia coli, Lemo21(DE3), Protein translocation, Periplasm, Sec-translocon
HSV kategori
Identifikatorer
URN: urn:nbn:se:su:diva-89590DOI: 10.1186/1475-2859-12-24ISI: 000316339500001OAI: oai:DiVA.org:su-89590DiVA, id: diva2:618958
Merknad

AuthorCount:6;

Tilgjengelig fra: 2013-04-30 Laget: 2013-04-30 Sist oppdatert: 2022-03-23bibliografisk kontrollert
Inngår i avhandling
1. From protein production to genome evolution in Escherichia coli
Åpne denne publikasjonen i ny fane eller vindu >>From protein production to genome evolution in Escherichia coli
2013 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. s. 59
Emneord
Escherichia coli, BL21(DE3), protein production, membrane proteins, secretory proteins, genome evolution
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-94993 (URN)978-91-7447-786-3 (ISBN)
Disputas
2013-11-22, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

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

Tilgjengelig fra: 2013-10-30 Laget: 2013-10-20 Sist oppdatert: 2022-02-24bibliografisk kontrollert

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