血管抑制素功能结构域K1与PKA底物磷酸化基序的融合表达及磷酸化标记

Fusion Expression of Functional Domain K1 of Angiostatin with PKA Phosphorylating Motif and Labeling of the Protein by Phosphorylation

通过 RT- PCR,从人肝组织中扩增出血管形成抑制素 ( angiostatin) c DNA的 K1片段 ,经DNA序列分析证实其正确性 ;将 K1与 GST融合并带上 1 7个氨基酸的 PKA底物磷酸化基序 ,IPTG诱导表达 ,以还原型谷胱甘肽偶联的琼脂糖凝胶亲合层析直接从细菌裂解上清中纯化融合蛋白 ;以 PKA催化单位将 3 2 P通过磷酸化作用标记至纯化的蛋白 ,再用凝血酶切去 GST,进行SDS- PAGE.放射自显影结果显示 ,GSTag- K1和 Tag- K1分别在 40 k D和 1 7k D处有信号强而特异的显影条带 ,表明带有磷酸化序列的蛋白能够被

The first kringle domain of angiostatin was amplified from human liver tissue by RT PCR.Sequence analysis indicated that the amplified fragment was correct.K1 fragment was fused with GST and 17 amino acid sequence of cAMP response element binding protein which could be phosphorylated by PKA specifically.The fusion protein displayed high level soluble expression in E.coli by the induction of 0.1 mmol/L IPTG,and the expression protein were purified directly from supernatant of E.coli lysis by affinity chromatograph with reduced glutathione linked agarose. 32 P were labeled to purified protein through phosphorylation reaction mediated by PKA,and GST was cut off by thrombin.The labeled protein was assigned to 15% SDS PAGE and radioautograph.The results indicated that recombinant K1 fused with PKA phosphorylating motif could be labeled with 32 P by PKA specifically.