[Construction, Expression and Purification of Wild and Mutant Type of nm23-H1 in Prokaryotic Expression System.]
Xueqin Yang1, Yu Fan, Jun Chen
1Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
Background:
Nm23-H1 is a metastasis-suppressor gene. However, its molecular mechanism of suppressing metastasis is unknown until now. The aim of this study is to construct prokaryotic expression vector of wild and mutant type of nm23-H1 (WT, P96S, H118F), and then express and purify the proteins.
Methods:
wild and mutant type of nm23-H1 fragments were amplified by PCR. The prokaryotic expression vectors of pET28anm23-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 this system. 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 plasmids were completely correct. After transforming, these plasmids can express the target proteins. The protein production was very high, and all the proteins were soluble expression. The molecular weight of wild and mutant type of nm23-H1 was 20 kDa detected by Western blot, which was as the same as the objective protein.
Conclusions:
We have succeeded in constructing the prokaryotic expression vectors of pET28a-nm23-H1 (WT, P96S, H118F) and the proteins which expressed can be used in following studies.
