Prokaryotic expression, purification and identification of human mutant nm23-H1 protein / 重庆医科大学学报

ABSTRACT

Objective:To construct prokaryotic expression vector of mutant nm23-H1(S120G,S120G-P96S, S44A), and then express and purify the proteins. Methods: Mutant nm23-H1 fragments were amplified by PCR. The prokaryotic expression vectors of pET28a-nm23-H1 were constructed by gene recombination technique and verified by restriction enzyme analysis and sequencing. The positive clones were transformed into E. coli BL21(DE3) and soluble analysis of the expression was conducted in these systems. The proteins were purified by nickel column chromatography and identified by Western blot. Results: The sequences and open read frames of all the pET28a-nm23-H1 were completely correct. After transforming, these plasmids could express the target proteins. The protein production was very high. Among these mutant proteins, S44A was soluble expression and S120G was partly soluble. However, S120G-P96S was mostly in the inclusion bodies. The molecular weight of these mutant proteins was 20 kD detected by Western blot, which was as the same as the objective protein. Conclusion: We have succeeded in constructing the prokaryotic expression vectors of pET28a-nm23-H1(S120G, S120G-P96S, S44A), and the S120G and S44A proteins can be used in following studies. However, S120G-P96S protein which expressed in this system may not be suitable for the following studies.