大鼠甘氨肽酰化单氧酶在CHO细胞中的活性表达及在体外酰胺化修饰中的应用

Functional Expression of Rat Peptidylglycine αamidating Monooxygenase in CHO Cells and Its Use in in vitro Amidation

将大鼠脑ｃＤＮＡ库来源的ＰＡＭ基因片段，经克隆重组，构建成真核表达质粒ｐＳＶＰＡＭ，并转染ＣＨＯ细胞，获得在ＣＨＯ中稳定表达活性型α酰胺化酶的细胞株ＤＧＡＥ。表达产物为双功能酶，分泌至培养基中的酶活力远高于胞内。体外酰胺化加工研究表明，以αＮａｃｅｔｙｌＴｙｒＶａｌＧｌｙ为底物，该酶催化反应的Ｋｍ为１２５μｍｏｌ／Ｌ，Ｖｍａｘ为１８０μｍｏｌ／ｍｇ／ｈ，而且催化反应中表现有最适铜离子浓度和ｐＨ值范围。表达的双功能酶可直接用于合成的多肽和基因工程表达产物的酰胺化加工修饰。

Rat peptidylglycine αamidatimg monooxygenase (PAM) was functionally expressed in stably transfected CHO cells by using eukaryotic expression plasmid pSVPAM,which was constructed with rPAM gene screened from rat brain cDNA library.The product mainly secreted in the medium is a bifunctional enzyme with the optimal pH value and concentration of copper ion.The kinetics studys of PAM show that the Km value is 12.5μmol/L and the Vmax is 180μmol/mg/h in the amidating reaction with αNacetylTyrVal Gly.Recombinant bifunctional PAM can be used in amidating process of synthesized polypeptides and proteins expressed by gene engineering.