CLONING AND EXPRESSION OF HEPARINASE-I GENE FROM FLAVOBACTERIUM-HEPARINUM
CLONING AND EXPRESSION OF HEPARINASE-I GENE FROM FLAVOBACTERIUM-HEPARINUM
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DOI：
10.1073/pnas.90.8.3660
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
LANGER, R
中科院分区：
文献类型：
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作者：
SASISEKHARAN, R;BULMER, M;LANGER, R
肝素酶是裂解肝素和硫酸乙酰肝素的酶，与从伤口愈合到肿瘤转移的生理和病理功能有关，可用于脱肝素治疗。我们报告了使用 PCR 从肝素黄杆菌中克隆肝素酶 I (EC 4.2.2.7) 基因。两种简并寡核苷酸基于纯化肝素酶胰蛋白酶肽的氨基酸序列，以黄杆菌基因组 DNA 为模板，通过 PCR 扩增产生 600 bp 探针。该探针用于筛选 pUC18 中的黄杆菌基因组 DNA 文库。肝素酶I的开放阅读框长度为1152 bp，编码43.8 kDa的前体蛋白。 11 个胰蛋白酶肽（几乎相当于总氨基酸的 35%）映射到开放阅读框上。氨基酸序列揭示了共有肝素结合域和具有特征性 Ala-(Xaa)-Ala 切割位点的 21 个残基前导肽。使用 T7 聚合酶 pET 表达系统，重组肝素酶在大肠杆菌中表达为可溶性蛋白。重组肝素酶对肝素的裂解与天然肝素酶的裂解相同。
Heparinases, enzymes that cleave heparin and heparan sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies. We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR. Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. This probe was used to screen a Flavobacterium genomic DNA library in pUC18. The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa. Eleven of the tryptic peptides (almost-equal-to 35% of the total amino acids) mapped onto the open reading frame. The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site. Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system. The recombinant heparinase cleavage of heparin was identical to that of native heparinase.