融合蛋白
亲和层析
表达式向量
生物化学
大肠杆菌
等电聚焦
化学
肽
融合基因
分子生物学
聚丙烯酰胺凝胶电泳
羟胺
生物
色谱法
重组DNA
基因
酶
作者
Tomas Moks,Lars Abrahmsén,Erik Holmgren,M Bilich,Anders Olsson,Mathias Uhlén,Gunnar Pohl,Catharina Sterky,Hans Hultberg,Staffan Josephson
出处
期刊:Biochemistry
[American Chemical Society]
日期:1987-08-25
卷期号:26 (17): 5239-5244
被引量:137
摘要
Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.
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