Gan Z R, Condra J H, Gould R J, Zivin R A, Bennett C D, Jacobs J W, Friedman P A, Polokoff M A
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Gene. 1989 Jun 30;79(1):159-66. doi: 10.1016/0378-1119(89)90101-7.
A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.
一种编码血小板聚集抑制剂echistatin（Ecs）的基因已被化学合成。天然蛋白第28位的甲硫氨酸在重组Ecs中被亮氨酸取代。为了在大肠杆菌中表达该合成基因，通过将大肠杆菌cheB和cheY基因复合体的部分片段插入质粒pUC13构建了表达载体pJC264。通过在表达载体中使其与大肠杆菌cheY基因融合，实现了合成的[Leu-28]Ecs的高水平表达。通过在插入CheY蛋白和[Leu-28]Ecs之间的甲硫氨酸处进行CNBr切割，从融合蛋白中释放出重组的[Leu-28]Ecs。通过反相高效液相色谱法将重组的[Leu-28]Ecs纯化至同质。重新折叠的[Leu-28]Ecs在抑制血小板聚集方面与天然Ecs相同，这表明第28位的甲硫氨酸对于这种血小板聚集抑制剂的生物活性并非必不可少。
