Expression and purification of NSH2 domain protein for NMR research
首发时间:2009-03-06
Abstract:SHP-2 protein, which has two SH2 domains, is essential for the embryonic development, haematopoiesis and signaling downstream of a variety of growth factors. SHP-2 proteins are related to many diseases. To facilitate fundamental studies, it is important that the proteins can be expressed in high quality and in a large quantity. In this work, the amino-terminal SH2 (NSH2) domain of SHP-2 protein which is important for the protein’s self-regulation was recombinated into the prokaryotic expression vector, pGEX-2T, and introduced into E. coli BL21 for a prokaryotic expression. Two recombinant vectors with different extra amino acid residues were designed on the basis of properties of NSH2 protein and the sequence of expression vector, pGEX-2T, to produce NSH2 protein with a high property. The effects of the two groups of different extra amino acid residues on solubility and stability were compared. The comparisons indicated that end extra amino acid residues may have strong effects on both solubility and stability. The higher soluble and stable NSH2 protein was selected and labeled with isotope 15N for NMR study. The high resolution of NMR demonstrated the correction folding of the protein and the interactions of the protein with phosphop-tyrosin peptides.
keywords: SHP-2 SH2 protein purification
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NSH2蛋白的原核表达纯化和同位素标记
摘要:SHP-2蛋白是一种含有两个SH2结构域的磷酸酶。SHP-2的N端SH2结构域(NSH2)是细胞因子或病毒因子激活该蛋白的分子内靶点。将NSH2以不同的方法重组到原核表达载体pGEX-2T中,在大肠杆菌BL21 (DE3)中表达融合蛋白并进行纯化,选择其中效率高的表达方案,对蛋白进行15N同位素标记和纯化。
关键词: SHP-2; SH2; 蛋白纯化
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No.3000739417712363****
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