Enzymatic activities of recombinant human NAMPT and NAMPT (H247A) proteins.
- Author:
Feng WANG
1
;
Peng HUANG
;
Zhu LIU
;
Yun-Bi LU
;
Er-Qing WEI
;
Wei-Ping ZHANG
;
Chun TANG
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Cytokines; genetics; isolation & purification; metabolism; Escherichia coli; genetics; Genetic Vectors; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nicotinamide Phosphoribosyltransferase; genetics; isolation & purification; metabolism; Plasmids; genetics; Recombinant Proteins; genetics; metabolism; Transformation, Bacterial
- From: Journal of Zhejiang University. Medical sciences 2011;40(2):156-162
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.
METHODSUsing 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.
RESULTThe 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.
CONCLUSIONThe recombinant hNAMPT and hNAMPT (H247A) proteins have been successful prepared and purified. The H247A mutation dramatically decreases the enzymatic activity of NAMPT.
