The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli

Microbial Cell Factories

Technical Notes

Zhe Zhang¹  Alexandros Karyolaimos¹  Jan-Willem de Gier¹  Grietje Kuipers²  Gianluca Trinco³  Dirk Jan Slotboom³  Nurzian Ismail⁴  David Vikström⁴ 

[1]Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91, Stockholm, Sweden

[2]Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91, Stockholm, Sweden

[3]Xbrane Biopharma AB, SE-111 45, Stockholm, Sweden

[4]University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, NL-9747 AG, Groningen, The Netherlands

[5]Xbrane Biopharma AB, SE-111 45, Stockholm, Sweden

关键词: Escherichia coli;    Protein production;    Membrane protein;    Secretory protein;    T7 RNA polymerase;    Lemo21(DE3);   

DOI  :  10.1186/s12934-017-0840-4

 received in 2017-09-20, accepted in 2017-12-05,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundTo optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector.ResultsBy incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein.ConclusionsHere, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).

【 授权许可】

CC BY   
© The Author(s) 2017