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    • 5. 发明公开
    • 재조합 인체 티로시나제 단백질을 대장균에서 대량생산하는 방법
    • 在大肠杆菌中生产重组人类酪氨酸酶的方法
    • KR1020040045961A
    • 2004-06-05
    • KR1020020073728
    • 2002-11-26
    • 공광훈
    • 공광훈
    • C12N9/48
    • C12N9/0059C12N15/70C12Y110/03001
    • PURPOSE: A method for mass-producing a recombinant human tyrosinase in E. coli is provided, thereby stably expressing the recombinant human tyrosinase having enzyme activity which is useful for studying on skin diseases from E. coli without forming inclusion body. CONSTITUTION: The method for mass-producing a recombinant human tyrosinase in E. coli comprises the steps of: (a) PCR amplifying the human tyrosinase gene having the nucleotide sequence set forth in SEQ IN NO: 1 using a pair of primers having the nucleotide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3; (b) inserting the amplified human tyrosinase gene into a vector pET-26b(+) to prepare a recombinant expression vector pET-hTyr; (c) transforming E. coli with the recombinant expression vector pET-hTyr; and (d) culturing the transformed E. coli, isolating purifying the recombinant human tyrosinase, and subjecting it to DEAE=Sephacel anion exchange chromatography and His-Bind resin chromatography.
    • 目的:提供大肠杆菌中大规模生产重组人酪氨酸酶的方法,从而稳定地表达具有酶活性的重组人酪氨酸酶,其可用于研究来自大肠杆菌的皮肤病,而不形成包涵体。 构成:在大肠杆菌中大规模生产重组人酪氨酸酶的方法包括以下步骤:(a)使用具有核苷酸的一对引物对具有SEQ ID NO:1所示核苷酸序列的人酪氨酸酶基因进行PCR扩增 SEQ ID NO:2和SEQ ID NO:3所示的序列; (b)将扩增的人酪氨酸酶基因插入载体pET-26b(+)中以制备重组表达载体pET-hTyr; (c)用重组表达载体pET-hTyr转化大肠杆菌; (d)培养转化的大肠杆菌,分离纯化重组人酪氨酸酶,并进行DEAE = Sephacel阴离子交换层析和His-Bind树脂色谱法。