THE PREPARATION OF CATALYTICALLY ACTIVE HUMAN CATHEPSIN-B FROM ITS PRECURSOR EXPRESSED IN ESCHERICHIA-COLI IN THE FORM OF INCLUSION-BODIES
THE PREPARATION OF CATALYTICALLY ACTIVE HUMAN CATHEPSIN-B FROM ITS PRECURSOR EXPRESSED IN ESCHERICHIA-COLI IN THE FORM OF INCLUSION-BODIES
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DOI：
10.1111/j.1432-1033.1995.0533k.x
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发表时间：
1995-04-15
期刊：
影响因子：
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通讯作者：
TURK, V
中科院分区：
文献类型：
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作者：
KUHELJ, R;DOLINAR, M;TURK, V
使用T7聚合酶表达系统在大肠杆菌中高水平表达编码人组织蛋白原B的cDNA克隆，导致形成不溶性胞质蛋白聚集体（包涵体）。重组蛋白经复性、氧化复性后溶解。随后用胃蛋白酶处理酶原以产生酶活性酶。通过系统地改变影响折叠、二硫键形成和将组织蛋白酶原B加工成催化活性成熟形式的参数，设计了一种简单的复性方法，可以生产约3 mg纯化的活性组织蛋白酶B/l E。大肠杆菌培养液。以这种方式获得的酶由单链组成，并且作为胃蛋白酶处理的结果，在其N末端具有三个氨基酸的延伸。该酶具有与天然人组织蛋白酶B相似的动力学和免疫学特性。
A cDNA clone encoding human procathepsin B was expressed at a high Level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/l E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.