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[Enzymatic activities of recombinant human NAMPT and NAMPT (H247A) proteins]
Feng Wang1, Peng Huang, Zhu Liu
1Department of Pharmacology, College of Medicine, Zhejiang University, Hangzhou 310058, China.
Objective:
To prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.
Methods:
Using pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) mutant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT (H247A) proteins were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities of recombinant proteins were assessed by solution NMR.
Result:
The DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased compared to wild-type NAMPT.
Conclusion:
The recombinant hNAMPT and hNAMPT (H247A) proteins have been successful prepared and purified. The H247A mutation dramatically decreases the enzymatic activity of NAMPT.
