Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.
Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.
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Bothropstoxin-I（一种 Lys49-磷脂酶 A2 同源物）的重折叠和纯化，在大肠杆菌中表达为包涵体。
DOI：
10.1006/prep.2000.1353
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发表时间：
2001
影响因子：
1.6
通讯作者：
L. J. Greene
中科院分区：
文献类型：
--
作者：
R. Ward;A.H.C. de Oliveira;R. Bortoleto;J. Rosa;V. M. Faça;L. J. Greene
II 类磷脂酶 A2 酶对磷脂的水解涉及 His48 残基对 sn-2 酯键的亲核攻击以及与蛋白质活性位点区域中的 Asp49 残基结合的 Ca2+ 离子辅助因子对反应中间体的稳定作用。 Bothropstoxin-I (BthTX-I) 是一种存在于Bothrops jararacussu 蛇毒液中的PLA(2) 变体，其显示Asp49 被Lys 取代，缺乏水解活性，但通过非催化Ca2+ 独立机制损坏人工膜。为了更好地表征这种不寻常的膜损伤机制，我们在大肠杆菌中建立了 BthTX-I 的表达系统。 BthTX-I的DNA编码序列被亚克隆到载体pET11-d中，并且BthTX-I在大肠杆菌BL21(DE3)中表达为包涵体。天然 BthTX-I 含有七个二硫键，并且已经开发出一种简单的方案，可以使用尺寸排阻色谱基质在表面活性剂存在下以高蛋白浓度重新折叠重组蛋白。复性后，常规回收率为2.5%（相当于每升细菌培养物4-5mg复性重组BthTX-I）。重折叠后，与天然蛋白相比，重组 BthTX-I 获得了相同的荧光和圆二色光谱。此外，通过测量脂质体中捕获的荧光标记的释放来评估，天然和重折叠的重组蛋白表现出相同的膜损伤特性。
Hydrolysis of phospholipids by Group II phospholipase A2 enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region. Bothropstoxin-I (BthTX-I) is a PLA(2) variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E. coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes.
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分泌型磷脂酶A2的结构和催化机制。
影响因子：
--
作者：
Scott,DL;Sigler,PB
通讯作者：
Sigler,PB
影响因子：
13.8
作者：
Dennis, EA
通讯作者：
Dennis, EA
影响因子：
2.9
作者：
Sekar,K;Yu,BZ;Rogers,J;Lutton,J;Liu,X;Chen,X;Tsai,MD;Jain,MK;Sundaralingam,M
通讯作者：
Sundaralingam,M
来自 Agkistridon piscivorus piscivorus 毒液的（无活性）Lys-49 磷脂酶 A2 的晶体学和生化研究。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Scott,DL;Achari,A;Vidal,JC;Sigler,PB
通讯作者：
Sigler,PB
影响因子：
2.9
作者：
Li,Y;Yu,BZ;Zhu,H;Jain,MK;Tsai,MD
通讯作者：
Tsai,MD