Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization
Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization
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DOI：
10.1016/j.bbapap.2004.11.006
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发表时间：
2005-03-14
影响因子：
3.2
通讯作者：
Chang, LS
中科院分区：
文献类型：
--
作者：
Cheng, YC;Yan, FR;Chang, LS
从中华眼镜蛇（Naja atra Taiwan cobra）毒腺中提取细胞RNA，构建了编码糜蛋白酶抑制剂（Chymotrypsin inhibitor）的cDNA。氨基酸序列分析表明，该成熟蛋白由57个氨基酸残基和6个半胱氨酸残基组成。从大肠杆菌的包涵体中表达并分离了重组蛋白。大肠杆菌并在体外重新折叠成功能蛋白。删除前三个残基在其N-末端引起对胰凝乳蛋白酶的抑制常数（Ki）的适度增加。通过PCR扩增编码糜蛋白酶抑制剂的基因组DNA。该基因与银环蛇毒素B1链（一种蛇Kunitz/BPTI神经毒性同系物）基因的结构组织几乎相同。此外，N. atra糜蛋白酶抑制剂和β-银环蛇毒素B1链基因的检出率高达83%。这些发现强烈表明，蛇Kunitz/BPTI蛋白酶抑制剂和神经毒性同系物可能起源于一个共同的祖先。(C)2004 Elsevier B. V.保留所有权利。
A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (K-i) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and beta-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. (C) 2004 Elsevier B.V. All rights reserved.