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    • 9. 发明申请
    • A PROCESS FOR PREPARING RIPE PROTEINS FROM FUSION PROTEINS, SYNTHETIZED IN PRO- OR EUKARYOTIC CELLS
    • 从融合蛋白制备RIPE蛋白的方法,在原核细胞中合成
    • WO1984002351A1
    • 1984-06-21
    • PCT/DK1983000118
    • 1983-12-09
    • NORDISK INSULINLABORATORIUMCHRISTENSEN, ThorkildBALSCHMIDT, PerDAHL, Hans-Henrik, MarstrandHEJNAES, Kim, Ry
    • NORDISK INSULINLABORATORIUM
    • C12P21/06
    • C12N9/2462C07K14/545C07K14/61C07K14/62C07K2319/00C07K2319/02C07K2319/50C07K2319/75C12N9/22C12N15/62C12P21/06
    • A fusion protein with the amino acid sequence: (Ym...Y2-Y1)-(Pro)p-(X1-X2...Xn) in which (Ym...Y2-Y1)-(Pro)p is the pro-sequence and the rest is the ripe protein, m is an integer greater than 2, and Y is an arbitrary amino acid, p is O ifX1 or X2 is Pro, and 1 if X1 or X2 is different from Pro, X is an arbitrary amino acid, and n is integer equal to or greater than 4, is converted into ripe proteins by enzymatic cleavage, which is carried out by stepwise cleavage off of the amino acid groups Ym...Y2 if p = 1 or X1 = Pro, or the groups Ym...Y2-Y1 if X2 = Pro, by means of an aminopeptidase, and then the two amino acids Y1-Pro if P = 1 are cleaved off enzymatically in one or two steps in a manner known per se, and similarly Y1 alone is cleaved off if X1 = Pro. The preferred aminopeptidase is leucine aminopeptidase. The process enables removal in vitro of the formyl methionine or the methionine group and optionally the pro-sequence from a protein synthetized in pro- or eukaryotic cells by genetic engineering, and thus achievement of a desired ripe protein, such as human growth hormone or human proinsulin.
    • 具有(Ym ... Y2-Y1) - (Pro)p-(X1-X2 ... Xn)的氨基酸序列的融合蛋白(Ym ... Y2-Y1) - (Pro)p为 原序列,其余为成熟蛋白,m为大于2的整数,Y为任意氨基酸,如果X1或X2为Pro,p为O,如果X1或X2与Pro不同,则为0,X为 任意氨基酸,n为4以上的整数,通过酶裂解转化为成熟蛋白质,其通过逐步裂解氨基酸基团Ym ... Y2进行,如果p = 1或X1 = Pro,或Ym ... Y2-Y1,如果X2 = Pro,通过氨基肽酶,然后如果P = 1,两个氨基酸Y1-Pro以一个或两个步骤以一种或两个步骤以已知的方式被切割 而且,如果X1 = Pro,则单独地将Y1切断。 优选的氨基肽酶是亮氨酸氨基肽酶。 该方法能够通过基因工程在体外除去甲酰甲硫氨酸或甲硫氨酸基团以及任选的来自在真核或真核细胞中合成的蛋白质的前序列,从而实现所需的成熟蛋白质,例如人生长激素或人 胰岛素原。