Canova-Davis E, Eng M, Mukku V, Reifsnyder D H, Olson C V, Ling V T
Department of Medicinal and Analytical Chemistry, Genentech Inc., South San Francisco, CA 94080.
Biochem J. 1992 Jul 1;285 ( Pt 1)(Pt 1):207-13. doi: 10.1042/bj2850207.
Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.
重组DNA技术被用于生物合成人胰岛素样生长因子I（hIGF-I）作为融合蛋白，其中融合多肽是源自葡萄球菌蛋白A的IgG结合部分。这种融合蛋白在大肠杆菌中产生并分泌到发酵液中。为了释放成熟的重组来源的hIGF-I（rhIGF-I），融合蛋白用羟胺处理，羟胺可切割已被设计到融合蛋白基因中的敏感天冬酰胺-甘氨酸键。反相高效液相色谱法用于估计rhIGF-I制剂的纯度，特别是用于含甲硫氨酸亚砜变体的定量。已确定融合蛋白的羟胺切割作为副反应产生rhIGF-I中天冬酰胺和谷氨酰胺残基的异羟肟酸酯。虽然等电聚焦在检测方面有效，反相高效液相色谱法用于产生异羟肟酸酯变体的富集级分，但离子交换色谱法是更具确定性的方法，因为它允许对这些变体进行定量并易于去除。通过金黄色葡萄球菌V8蛋白酶消化，然后进行质谱分析确定变体为异羟肟酸酯，因为该修饰对氨基酸和N端序列分析是透明的。rhIGF-I的生物活性通过其将[3H]胸苷掺入BALB/c373细胞DNA的能力以及利用人胎盘膜的放射受体测定来确定。两种测定均表明天然、重组以及甲硫氨酸亚砜和异羟肟酸酯IGF-I变体基本具有同等效力。
