Ancylostoma caninum anticoagulant peptide: Cloning by PCR and expression of soluble, active protein E-coli
Ancylostoma caninum anticoagulant peptide: Cloning by PCR and expression of soluble, active protein E-coli
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DOI：
10.1016/0166-6851(96)02658-8
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发表时间：
1996-09-01
影响因子：
1.5
通讯作者：
Hotez, PJ
中科院分区：
文献类型：
--
作者：
Cappello, M;Hawdon, JM;Hotez, PJ
犬钩虫抗凝肽（AcAP）是存在于成年犬钩虫提取物中的主要抗凝活性物质。这种8.7 kDa的蛋白是一种有效的、特异性的人凝血因子Xa抑制剂。利用PCR技术，我们从成年犬a . caninum cDNA文库中分离到了一个编码AcAP的cDNA。利用a . caninum cDNA和线虫剪接引物对应的5′引物，采用反转录PCR （RT-PCR）方法鉴定了AcAP cDNA的5′端。利用原核表达载体在大肠杆菌中表达AcAP cDNA，利用镍树脂亲和层析和反相高效液相色谱法纯化重组融合蛋白（rAcAP）。纯化的rAcAP在抑制活性方面与天然蛋白相当，对265 +/- 71 pM的Xa因子的抑制具有明显的平衡抑制解离常数（K-i*）。纯化的蛋白还以剂量依赖的方式延长人血浆的凝血酶原和部分凝血活酶时间。
Ancylostoma caninum Anticoagulant Peptide (AcAP) is the major anticoagulant activity present in extracts of adult Ancylostoma caninum hookworms. This 8.7 kDa protein is a potent and specific inhibitor of human coagulation factor Xa. Using PCR, we have isolated a cDNA encoding for AcAP from an adult A. caninum cDNA library. The 5' end of the AcAP cDNA was identified by reverse transcription PCR (RT-PCR) using A. caninum cDNA and a 5' primer corresponding to a nematode spliced leader sequence. The AcAP cDNA was expressed in E. coli using a prokaryotic expression vector, and the recombinant fusion protein (rAcAP) was purified to homogeneity using nickel resin affinity chromatography and reverse phase HPLC. Purified rAcAP is comparable to the native protein in inhibitory activity, with an apparent equilibrium inhibitory dissociation constant (K-i*) for the inhibition of factor Xa of 265 +/- 71 pM. The purified protein also prolongs the prothrombin and partial thromboplastin times of human plasma in a dose dependent manner.