%0 Journal Article
%T Site-directed mutagenesis and enzymatic activity assay of Gln49-Phospholipase A2 mutant<br>Gln49-磷脂酶A2基因工程定点突变及酶活性分析
%A DOU Jia
%A CAI He
%A JI Fang-ling
%A CUI Wen-ju
%A WANG Jing-yun
%A BAO Yong-ming
%A AN Li-jia
%A <br>窦佳
%A 蔡河
%A 姬芳玲
%A 崔文举
%A 王静云
%A 包永明
%A 安利佳
%J 中国生物工程杂志
%D 2007
%I 
%X 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.
%K Phospholipase A2 PCR site-directed mutagenesis IMAC Inclusion body refolding Protein expression<br>磷脂酶A2
%K PCR
%K 定点突变
%K 固定化金属离子亲和层析
%K 包涵体复性
%K 蛋白表达
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=951380788B355DEA7D75520FA3B199C9&aid=0E73F1E6F2A0445B&yid=A732AF04DDA03BB3&vid=DB817633AA4F79B9&iid=94C357A881DFC066&sid=659D3B06EBF534A7&eid=DB817633AA4F79B9&journal_id=1671-8135&journal_name=中国生物工程杂志&referenced_num=0&reference_num=19