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 (P_lacUV5), which is leakier and more active than wild-type lac promoter (P_lacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced P_lacUV5 with other inducible promoters respectively, including arabinose promoter (P_araBAD), rhamnose promoter (P_rhaBAD), tetracycline promoter (P_tet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

Keywords: BL21 (DE3); Inducible promoter; Recombinant protein; T7 RNA polymerase.

Fig. 1

Construction of three engineered strains and fluorescence intensity of EGFP fermentation for 18 h. A Genetic design of engineered T7 RNAP in E. coli strains BL21 (DE3). Three engineered T7 RNAP expression strains which containing different inducible promoters were constructed. B Fluorescence intensity of different strains containing EGFP plasmid with or without inducer and fluorescence diagram (C). The error bar denotes the standard deviation of the mean from the three replicates

Fig. 2

Expression of GDH-EGFP in different strains. A Fluorescence intensity of GDH-EGFP in the whole fermentation process. B The percentage of plasmid-carrying cells of BL21 (DE3), BL21 (DE3::ara), BL21 (DE3::rha) and BL21 (DE3::tet) was tested during the entire fermentation period. Values and error bars represent the means and the deviations from triplicate experiments. GDH glucose dehydrogenase

Fig. 3

Expression of three membrane proteins in three engineered strains and control strain BL21 (DE3). A Fluorescence intensity of Codb-EGFP of BL21 (DE3), BL21 (DE3::ara), BL21 (DE3::rha) and BL21 (DE3::tet) in the whole fermentation process and the percentage of plasmid-carrying cells in B. C Fluorescence intensity of Ecb-EGFP of BL21 (DE3), BL21 (DE3::ara), BL21 (DE3::rha), and BL21 (DE3::tet) in the whole fermentation process and the percentage of plasmid-carrying cells in D. E Fluorescence intensity of Yidc-EGFP of BL21 (DE3), BL21 (DE3::ara), BL21 (DE3::rha), and BL21 (DE3::tet) in the whole fermentation process and the percentage of plasmid-carrying cells in F. Values and error bars represent the means and the deviations from triplicate experiments. CodB, E. coli cytosine transporter protein. Ecb, E. coli F-ATPase subunit b. YidC, E. coli membrane protein insertase/foldase

Fig. 4

Tunable expression of GDH and three membrane proteins in three engineered strains and control strain BL21 (DE3) under different inducer concentrations at 36 h. A Tunable expression of GDH. B Tunable expression of Codb. C Tunable expression of Ecb. D Tunable expression of Yidc. Values and error bars represent the means and the deviations from triplicate experiments