Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

Abstract

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PLacUV5), which produces more T7 RNAP than wild-type lac promoter (PLacWT) to promote the production of recombinant proteins. However, there is a resource allocated limitation between cell growth and protein production when producing autolytic proteins or membrane proteins. T7 RNAP is the key factor to solve this problem. Hence, we replaced respectively PLacUV5 with other inducible promoters: arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet) to optimize the production of recombinant protein by regulating the transcription level of T7 RNAP. Compared with BL21 (DE3), the constructed engineering strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the glucose dehydrogenase (GDH) production, the engineered strains BL21 (DE3::tet) exhibited great biomass, cell survival rate and foreign protein expression level. In addition, these engineered strains had been successfully applied to the production of other membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and E. coli F-ATPase subunit b (Ecb). The engineering strains constructed in this paper provided more host choices for the production of recombinant proteins.