Bacterial Expression Of Eukaryotic Genes And Analysis

Prokaryotic cell expression of eukaryotic proteins is an important field, and is also one of the effective ways to obtain a large quantity of purified proteins that could be studied for their characteristics and functions. Escherichia coli expression system is well described, and has been widely used for over-expression of foreign genes. In this study, we chose Escherichia coli as expression system, and constructed 12 different genes and fusion genes into different bacterial expression vectors. They were transferred into bacteria that were induced to express proteins with IPTG. These proteins were purified by affinity chromatography for further analysis and identification. The main results are as follows:1. The amplification of target genes. Genes encoding chitinase, Fusarium graminearum-specific single chain antibodies E1 and E9, two chitinase-antibody (El or E9) fusion proteins (Chiel, Chie9), two cecropinA-melittin (MsrA1)-antibody (El or E9) related fusion proteins (MsrA1e1, MsraA1e9) were amplified by PCR. In addition, a gene sequence encoding Choler a toxin B subunit was fused with a polypeptide that is from heat shock protein 60, includiong its dimmer and trimer constructs; AN and UN genes from wheat that were induced by deoxynivalenol, were also amplified. The 12 genes above were digested by restriction enzymes and cloned into a prokaryotic expression vector pET-22b (+) individually, while AN and UN genes were cloned into vector pGEX-6p-1.2. The vectors above were transferred into Escherichia coli BL21 (DE3) and the ceels were then induced to express special proteins and optimized the expression conditions. The result showed that the addition of IPTG to a final concentration of 1 mmol/L while the OD600 values of bacteria reached 0.6. The optimum expression temperature and time were determined as follows:the optimum conditions for Chitinase, antibody El and E9, CTB-p277, CTB-2p277, CTB-3p277, AN, UN were 6 hours at 28?. For Chiel, Chie9, the expression level reached the peak at 6 hours. For MsrAlel, MsrA1e9, their expression levels reached the peak after only two hours at 37?.3. SDS-PAGE and Western blot analyese indicate that the molecular weitghts of the 12 proteins were consistent with the expectation. All of the proteins above were in inclusion body in E. coli, however, we could dissolve the proteins of inclusion body in 8 mol/L urea buffer and purify the proteins by Ni-NTA and finally obtain a large amount of soluble proteins. Through analysis of some proteins, we found that they had the ability of inhibiting the growth of F. graminearum. All these results provide materials and informations for further researches.