Co-expression And Chromosome Expression Fold Adjustment Factor Of Exogenous Protein Expression Of Functional Impact Study

The E.coli strains were used widely in the research of gene engineering. However, many of the heterologous proteins are expressed in E.coli in form of inclusion body without biological activity. While the refolding process of inclusion body is very difficult and costly. Therefore, it's meaningful to study how to increase the functional expression of heterologous protein in E,coli.The properly folding of most heterologous proteins need the helper molecular chaperones. There are two kinds of molecular chaperones playing critical roles in the folding of protein in E.coli. One is the DnaK-DnaJ and the other is the GroEL-S. The folding of protein with multiple disulfide bonds, such as urokinase(UK) and human tissue plasminogen activitors vtPA are greatly assisted by Dsb(disulfide bond ) molecules. Urokinase, which contains 6 pairs of disulfide bonds, is often used as a model protein to study the folding of multiple disulfide bonds. In order to study the effects of such chaperones and Dsb molecules on the folding of heterologous proteins, the genes of DnaK, DnaJ GroEL , GroES, the signal sequenceless DsbA and DsbC were isolated from the chromosome of E.coli by PCR. According to a paper, the mutants of DsbA and DsbC were also constructed using spliced overlap extension PCR technology. The genes mentioned above were cloned into plasmids under the control of araB promoter and were transformed into E.coli BW25113 and HB101 together with a compatible vector for UK. The activity of UK was analyzed by fibrinolysis assay. The results show that the yield of active UK was not detected in BW25113 and HB101. The yield of active UK increased by co-expressing with DnaK, GroEL-S, DsbA, DsbC and their mutants in BW25113. However, in HB101, only GroEL-S and DsbC enhanced the yield of active UK. The highest level of active urokinase was achieved by co-expressing with GroEL-S in both strains.Co-expression with GroEL-S and DsbC resulted in an increase in the level of active UK. Thereby, GroEL-S and DsbC were integrated into galK site of HB101 by Red recombination. The strain integrated of dsbC was named HB-C(galK::dsbC), and the otherintegrated of groEL-S named HB-G(galK:groEL-S). The active UK was achieved in a modest increase in HB-C(galK::dsbC) compared to wild-type strain, but not in HB-G(galk::groEL-S). Co-expression of GroEL-S gave significantly higher accumulation of active UK in HB-C(galK::dsbC), 4-fold greater than co-expression of GroEL-S and 17-fold than co-expression of DsbC in wild-type HB101. Such effects were not observed in HB-G. These results demonstrated that the strain integrated of dsbC could increase the yield of active heterologous proteins with multiple disulfide bonds.In general, we constructed the method of gene integration into chromosome of E.coli by Red recombination. The effects of the folding modulators on the functional expression of heterologous protein with multiple disulfide bonds were observed by co-expression and integration into chromosome. We obtained a strain that increased the level of functional expression of heterologous protein with multiple bonds. There is a new option to construct E.coli strain for efficient expression of heterologous proteins.