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    • 2. 发明申请
    • METHOD OF REMOVING N-TERMINAL ALANINE RESIDUES FROM POLYPEPTIDES WITH <i>AEROMONAS</i> AMINOPEPTIDASE
    • 使用氨基酸钠从多糖中除去N-末端氨基酸残基的方法
    • WO0066761A3
    • 2001-03-15
    • PCT/US0008746
    • 2000-04-26
    • MONSANTO COTOU JACOB STAYLOR DOUGLAS W
    • TOU JACOB STAYLOR DOUGLAS W
    • C07K1/12C07K14/61C07K14/81C12P21/06C12N15/18C07K14/575
    • C12P21/06C07K14/61C07K14/8114
    • This invention describes a method of removing N-terminal alanine residues from polypeptides, preferably recombinant proteins, using an aminopeptidase derived from the marine bacterium Aeromonas proteolytica . Accordingly, Aeromonas aminopeptidase (AAP; E.C. 3.4.11.10) can be used to remove N-terminal alanyl residues from derivatives of human somatotropin (hST, human growth hormone, or hGH), porcine somatotropin (pST), and bovine somtotropin (bST), for example, to yield proteins having their native amino acid sequences. The enzyme reactions can be carried out in free solution, or the AAP can be immobilized on a solid support, for reactions carried out in vitro . An efficient method for converting Ala-hGH to hGH, for example, comprises expression of Ala-hGH in E. coli , recovery of inclusion bodies, solubilization and refolding in detergent, detergent removal by ultrafiltration, selective precipitation, enzyme cleavage, followed by two column chromatography steps.
    • 本发明描述了使用源自海洋细菌气单胞菌的氨基肽酶从多肽,优选重组蛋白中除去N-末端丙氨酸残基的方法。 因此,气单胞菌氨基肽酶(AAP; EC 3.4.11.10)可用于从人生长激素(hST,人生长激素或hGH),猪生长激素(pST)的衍生物中除去N-末端丙氨酰残基, ,以及牛嗜马托溶素(bST),例如产生具有天然氨基酸序列的蛋白质。 酶反应可以在游离溶液中进行,或者AAP可以固定在固体支持物上,用于在体外进行的反应。 例如,将Ala-hGH转化为hGH的有效方法包括在E中表达Ala-hGH。 大肠杆菌,包涵体的回收,去污剂中的溶解和重折叠,通过超滤去除洗涤剂,选择性沉淀,酶切,然后是两个柱层析步骤。