Heparinase III from Flavobacterium heparinum: Cloning and recombinant expression in Escherichia coli
Heparinase III from Flavobacterium heparinum: Cloning and recombinant expression in Escherichia coli
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DOI：
10.1006/bbrc.1996.1246
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发表时间：
1996-08-23
影响因子：
3.1
通讯作者：
Sasisekharan, R
中科院分区：
文献类型：
--
作者：
Godavarti, R;Davis, M;Sasisekharan, R
肝素酶III(E.C.4.2.2.8)，前身为肝素酶I，由肝素黄杆菌产生，是一种特异性地裂解酸性多糖中富含硫酸肝素的区域的酶。在这项研究中，我们报道了用聚合酶链式反应(PCR)克隆肝素酶III基因。以黄杆菌基因组DNA为模板，以纯化的肝素酶III胰酶多肽的氨基酸序列为基础，利用两个简并的寡核苷酸，通过聚合酶链式反应(PCR)扩增出一条与1100碱基相似的探针。利用该探针对黄杆菌基因组DNA文库进行了筛选。肝素酶III基因的开放阅读框全长为1980个碱基，编码一个75,950 Da的前体蛋白，其中10个胰蛋白酶多肽定位在开放阅读框上，相当于该蛋白的18%。利用T7聚合酶pET表达系统在大肠杆菌中表达重组肝素酶III。这是首次克隆和重组表达一种主要降解硫酸乙酰肝素的酶。(C)1996年学术出版社。
Heparinase III (E.C. 4.2.2.8), formerly hepartinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a similar to 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to similar to 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate. (C) 1996 Academic Press, Inc.