Fassina G, Merli S, Germani S, Ciliberto G, Cassani G
Protein Engineering Unit, TECNOGEN S.c.p.A., Piana di Monte Verna (CE), Italy.
Protein Expr Purif. 1994 Dec;5(6):559-68. doi: 10.1006/prep.1994.1077.
A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.
一种编码人 big 内皮素（Big ET）的 DNA 构建体，其前面带有因子 Xa 蛋白酶识别位点（Ile-Glu-Gly-Arg），与麦芽糖结合蛋白序列读框融合，已被导入 DH5-α 细胞中。融合产物（MBP-Big ET）以接近 100 微克/毫升培养液的浓度表达，约占总蛋白含量的 50%。含有融合产物的粗细胞提取物已在温和变性条件下直接用胰蛋白酶处理，以便从加合物中释放出 big 内皮素（1-37）。MBP-Big ET 加合物的切割产率接近 70%。通过亲和色谱将 Big ET(1-37)与细菌提取物胰蛋白酶消化产生的无关肽分离。亲和柱是通过固定一种对蛋白酶有抗性的肽配体制备的，该配体能够以足够的亲和力、选择性和特异性识别 Big ET。从亲和步骤（回收率 90%）获得了纯度接近 80%的重组 Big ET(1-37)。然后用α-胰凝乳蛋白酶消化亲和纯化的重组产物，以释放内皮素（1-21），然后通过反相高效液相色谱（RP-HPLC）进行纯化。采用这种两步纯化方案，从 1 毫升细菌培养液中回收了 3 微克内皮素，纯度接近 95%。
