Study Of DsbA-DsbA^mut As Molecular Chaperone In Prokaryotic Expression System

The E. coli has been widely used in gene engineering for producing recombinant protein with the advantages of short period, easy control and clear genetic background. There are several problems for prokaryotic expression which still remained unsatisfied solved, for example, the renaturation of inclusion body, relative low expression level, low bioactivity and easy degradation of the products. Due to the high reductive environment, disulfide bond and the correct quarternary structure formation of recombinant protein rarely occur in the cytoplasm of E. coli, it is difficult to produce proteins with proper natural structure and native biological activity. The disulfide bond of E. coli is formed under the oxidatic condition in periplasm. Some specialized thiol-disulfide exchanging enzymes with the sequence motif of Cys-X-X-Cys play important roles in the formation and reduction of disulfide bonds. Disulfide bond formation protein A (DsbA) is a powerful oxidative protein of the thioredoxin family. DsbA can help protein to fold correctly both in vivo and in vitro. DsbA^mut (DsbA mutant protein) is site-specific mutagenesis converting active site cysteine residues to serine residues and forming a constitution as Ser-Pro-His-Ser. Beyond these modifications, the oxidoreductase activity of DsbA^mut is abolished while the solubility or localization behavior of the mutant protein still remaimed.Base on the structure and bioactive characteristics of DsbA and DsbA^mut, we focused on researching their function in prokaryotic expression system. First, by using human nerve growth factor (hNGF) as target protein, we compared the functions of several proteins belong thioredoxin familys on rhe expression of hNGF in E. coli. The results showed that DsbA displayed a higher ability to improve the refolding effcicency and sustain the biological activity of hNGF compared with the Trx and DsbC protein. Then, we found that the combining form of DsbA and DsbA^mut, that is DsbA -DsbA^mut , can assist hNGF expressing in E. coli with better solubility and biological activity when hNGF fused to DsbA-DsbA^mut.Considering the feature and function of DsbA-DsbA^mut fusion protein, we futher successfully constructed a novel coexpression vector. With this vector, we have expressed several eukaryotic proteins in E. coli in soluble way. Our results suggested that DsbA-DsbA^mut fusion protein could facilitate target protein to fold into right tertiary structure and sustain its native biological activity. The vector we constructed is novel and universal vector for most eukaryotic protein expression in E. coli.For the merit that the fusion protein expressed with this vector can sustain target protein in native folding state, we exploited it to study the structure and function of hNGF. We constructed and expressed 9 mutatants of hNGF, and the bioactivity assay results demonstrated that the biological activity of the protein decreased significantly or lost when deleting the amino acids at the N terminus or C terminus of hNGF.In order to facilitate the partification and purification of the aimed protein in the future, we finaly try to co-express the target protein with the DsbA-DsbA^mut . But the results showed that rarely hNGF expressed in E. coli when coexpressed with DsbA-DsbA^mut. We then further fused the DsbA to another DsbA family member, DsbC, as a chaperone to coexpress with hNGF. DsbC is a disulfie isomase which can correted the wrong disfulfide folding and form a new disulfide bond. By a two-promoter vector, we successfully coexpressed DsbA-DsbC fusion protein with hNGF in procaryote cytoplasm and obtained the soluble hNGF protein, but the DsbA or DsbC alone do not show this function.In summary, we successfully constructed a fusion expression vector which can be used to express most of the eukaryotic protein.The advantage of this vector is that the target protein can be expressed in soluble way and with nearly native biological activity and can be used to study the structure and function of certain functional protein. We test the strategy to co-express hNGF with DsbA-DsbA^mut and DsbA-DsbC in E. coli. This may be of potential reference value for expressing proteins with several disulfie bonds within the molecules.