title: [Enzymatic activities of recombinant human NAMPT and NAMPT (H247A) proteins].
abstract: To prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.
  
  
  
  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.
  
  
  
  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.
  
  
  
  The recombinant hNAMPT and hNAMPT (H247A) proteins have been successful prepared and purified. The H247A mutation dramatically decreases the enzymatic activity of NAMPT.
authors.affiliation: Department of Pharmacology, College of Medicine, Zhejiang University, Hangzhou 310058, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.forename: Zhu
authors.initials: Z
authors.lastname: Liu
authors.forename: Yun-Bi
authors.initials: YB
authors.lastname: Lu
authors.forename: Er-Qing
authors.initials: EQ
authors.lastname: Wei
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Zhang
authors.forename: Chun
authors.initials: C
authors.lastname: Tang
mesh_terms: D001483:Base Sequence; D016207:Cytokines / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D054409:Nicotinamide Phosphoribosyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D016207:Cytokines; D011994:Recombinant Proteins; D054409:Nicotinamide Phosphoribosyltransferase; C085455:nicotinamide phosphoribosyltransferase, human