Expression, refolding, and in vitro activation of a recombinant snake venom pro-metalloprotease
Expression, refolding, and in vitro activation of a recombinant snake venom pro-metalloprotease
复制标题
重组蛇毒原金属蛋白酶的表达、重折叠和体外激活
DOI：
10.1016/s1046-5928(02)00644-7
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发表时间：
2003-03-01
影响因子：
1.6
通讯作者：
Selistre-de-Araujo, HS
中科院分区：
文献类型：
--
作者：
Ramos, OHP;Carmona, AK;Selistre-de-Araujo, HS
金属蛋白酶包括降解细胞外基质的大多数组分的Zn 2 +-内肽酶家族。蛇毒是金属蛋白酶的丰富来源，其也消化纤维蛋白原以及纤维蛋白，并且在某些情况下，诱发出血。一些低分子量蛇毒金属蛋白酶（svMP）被描述为没有出血活性，但它们具有很强的直接作用纤维蛋白溶解活性。这一特性可能对血栓治疗非常有帮助。ACLF是一种纤维蛋白溶解性，非出血性金属蛋白酶，来自北美蛇类Agkistrodon contoraciallaticinetus的毒液。我们已经开发了一种表达系统，用于从从毒腺cDNA文库中分离的克隆（ACLPREF）生产重组前ACLF。通过PCR扩增出包含原酶结构域和成熟蛋白结构域的编码区，亚克隆到pET 28a载体中，转化大肠杆菌BL21（DE 3）。在37 ℃培养转化体，在用1.0 mM IPTG诱导后，导致不溶性48 kDa蛋白质的过表达。用6 M缓冲尿素从包涵体中回收表达的蛋白，并在变性条件下通过亲和层析纯化。二硫苏糖醇处理后，通过透析逐渐去除变性剂进行蛋白质重折叠。前酶在重折叠过程中进行自激活，它对纤维蛋白原和合成底物具有活性。为了控制活化步骤，迅速除去变性剂以使蛋白质保持未加工的形式，随后加入Ca2+和Zn2+离子。这允许在需要时控制酶活化。(C)2002 Elsevier Science（美国）。All rights reserved.
Metalloproteases comprise a family of Zn2+-endopeptidases that degrade most components of the extracellular matrix. Snake venoms are rich sources of metalloproteases, which also digest fibrinogen as well as fibrin, and in some cases, induce hemorrhage. A few low-molecular weight snake venom metalloproteases (svMPs) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity. This property could be very helpful in thrombosis therapy. ACLF is a fibrinolytic, non-hemorrhagic metalloprotease from the venom of the North American snake Agkistrodon contortrix laticinetus. We have developed an expression system for production of a recombinant pro-ACLF from a clone (ACLPREF) isolated from a venom gland cDNA library. The coding region including both the pro-enzyme domain and the mature protein domain was amplified by PCR and subcloned into the pET28a vector and the new plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at 37 degreesC led to the overexpression of an insoluble 48 kDa protein after induction with 1.0 mM IPTG. The expressed protein was recovered from inclusion bodies with 6 M buffered urea and purified by affinity chromatography under denaturing conditions. After dithiothreitol treatment, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The pro-enzyme underwent auto-activation during refolding and it was active on fibrinogen and on a synthetic substrate. To control the activation step, the denaturing agent was rapidly removed to keep the protein in an unprocessed form, followed by later addition of Ca2+ and Zn2+ ions. This allowed controlling the enzyme activation, when it is needed. (C) 2002 Elsevier Science (USA). All rights reserved.