Moks T, Abrahmsén L, Holmgren E, Bilich M, Olsson A, Uhlén M, Pohl G, Sterky C, Hultberg H, Josephson S
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Biochemistry. 1987 Aug 25;26(17):5239-44. doi: 10.1021/bi00391a005.
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.
构建了人胰岛素样生长因子I（IGF-I）基因与葡萄球菌蛋白A不同IgG结合片段基因之间的几种融合体，并就该肽类激素的表达、分泌和纯化进行了比较。从金黄色葡萄球菌或大肠杆菌的生长培养基中通过IgG亲和纯化融合蛋白后，用羟胺裂解天冬酰胺-甘氨酸肽键释放出天然IGF-I。基于对羟胺具有抗性的修饰合成IgG结合结构域（z）的优化表达系统，融合蛋白产量最高。裂解后，通过第二次过IgG亲和柱可将激素与IgG结合部分以及未裂解的融合蛋白分离。通过放射受体测定、N端序列分析、聚丙烯酰胺凝胶电泳、等电聚焦和高效液相色谱法证实了所得IGF-I的生物活性和纯度。
