PRODUCTION OF NATIVE CREATINE KINASE-B IN INSECT CELLS USING A BACULOVIRUS EXPRESSION VECTOR
PRODUCTION OF NATIVE CREATINE KINASE-B IN INSECT CELLS USING A BACULOVIRUS EXPRESSION VECTOR
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DOI：
10.1007/bf00925927
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发表时间：
1995-02-09
影响因子：
4.3
通讯作者：
WIERINGA, B
中科院分区：
文献类型：
--
作者：
DEKOK, YJM;GEURDS, MPA;WIERINGA, B
用全长人肌酸激酶B（B-CK）cDNA制备重组杆状病毒（AcDZ 1-BCK）。用该重组体感染的Sf 9细胞表达由43 kDa亚基组成的同源二聚体蛋白，其在最佳条件下形成高达30%的总可溶性细胞蛋白。经PAGE、酶谱分析和凝胶过滤层析分析，重组蛋白表现为真实的二聚体人BB-CK蛋白。用新生产的针对蛋白质N-末端表位的单克隆抗体（CK-BYK/2 1 E10）进行的研究证实了产物的身份。重组BB-CK蛋白质从AcDZl-CKB感染细胞的总蛋白质提取物中纯化至超过99%的均一性，该纯化在涉及FPLC中MonoQ上的阴离子交换柱色谱的一个单一步骤中进行。透析蛋白的比活性为239 U/mg蛋白。
A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZl-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/2 1E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZl-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.