Estrada Georgina, Garcia Blanca I, Schiavon Emanuele, Ortiz Ernesto, Cestele Sandrine, Wanke Enzo, Possani Lourival D, Corzo Gerardo
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM. Apartado Postal 510-3, Cuernavaca Morelos, 62250, Mexico.
Biochim Biophys Acta. 2007 Aug;1770(8):1161-8. doi: 10.1016/j.bbagen.2007.04.006. Epub 2007 May 1.
The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.
将四硫键桥联的苏氏正钳蝎毒素II基因克隆到含有6His标签和FXa蛋白酶切割区域的表达载体pQE30中。将该重组载体转染到大肠杆菌BL21细胞中，并用异丙基硫代半乳糖苷（IPTG）诱导表达。表达水平为每升培养基24.6毫克，发现His标签重组毒素（HisrCssII）仅存在于包涵体中。溶解后，通过亲和色谱和疏水相互作用色谱法纯化HisrCssII肽。从亲和色谱步骤获得的HisrCssII产物的反相HPLC图谱显示，几个肽段具有相同的9392.6 Da分子量，表明HisrCssII被氧化形成了几种不同的二硫键桥排列形式。还原后的多种形式的HisrCssII从柱上洗脱下来，成为一种单一的9400.6 Da蛋白质组分。同样，还原后的HisrCssII的体外折叠产生了一种单一的氧化形式的HisrCssII，其被蛋白酶FXa切割成重组CssII（rCssII）。rCssII的分子量如预期的那样为7538.6 Da。由于天然CssII（nCssII）在C末端残基处被酰胺化，而rCssII以游离羧基末端的形式异源表达，因此在比较这两种肽（天然的与异源表达的）时，两者相差1 Da。然而，当颅内注射到小鼠体内时，它们显示出相似的毒性，并且nCssII和rCssII在Na(v)1.6通道中均显示出β毒素的典型电生理特性，这在此首次得到证明。用放射性标记的CssII进行的结合和置换实验证实了电生理结果。讨论了与异源表达的含四个二硫键的毒素相关的几个问题。
