Site-directed Mutagenesis and Enzymatic Activity Assay of Gln49-Phospholipase A_2 Mutant
- VernacularTitle:Gln49-磷脂酶A_2基因工程定点突变及酶活性分析
- Author:
Jia DOU
;
He CAI
;
Fang-Ling JI
;
Wen-Ju CUI
;
Jing-Yun WANG
;
Yong-Ming BAO
;
Li-Jia AN
;
- Publication Type:Journal Article
- Keywords:
Phospholipase A2 PCR site-directed mutagenesis IMAC Inclusion body refolding Protein expression
- From:
China Biotechnology
2006;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2(Gln49-PLA2),site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR.Aspartic acid 49 phospholipase A2(Asp49-PLA2-Q49D-PLA2),the mutant of Gln49-PLA2 was expressed in E.coli with pET32a+ vector.The fusion protein,expressed as inclusion body,after being denatured,was on-column refolded and purified by immobilized metal affinity chromatography(IMAC),and then cleaved by Factor Xa.The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography,with the recovery rate of 1.3%,and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg.It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding,especially in rich disulfide bonds conditions.
