Basu M, Dharm E, Levine J F, Kramer R A, Crowl R M, Campbell R M
Department of Protein Biochemistry, Roche Research Center Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Arch Biochem Biophys. 1991 May 1;286(2):638-44. doi: 10.1016/0003-9861(91)90093-x.
A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.
已构建一种重组质粒，用于在大肠杆菌中指导Leu27GRF(1 - 44)OH的合成，作为一种融合蛋白，该融合蛋白含有一个六组氨酸尾，其后是干扰素-γ的1 - 99个氨基酸，且在N端有一个甲硫氨酸残基。通过异丙基硫代半乳糖苷处理诱导18 kDa融合蛋白（H6GAMGRF）的表达，该蛋白以不溶性聚集体的形式积累在包涵体中。将蛋白聚集体溶解在6 M盐酸胍中，并通过在镍螯合柱上的亲和层析直接纯化。通过溴化氰切割从融合蛋白中释放出生长激素释放因子（GRF）部分，并通过阴离子交换层析继以反相层析纯化至同质。通过将其保留时间与合成的Leu27GRF(1 - 44)OH的保留时间进行比较来确定GRF峰的身份。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、N端测序和氨基酸分析对纯化的物质进行进一步表征。重组衍生产物和合成化合物对抗GRF多克隆抗体显示出相同的反应性，并且通过生长激素释放活性的体外生物学测定确定它们基本等效。
