摘要
目的原核表达、纯化并筛选幽门螺旋菌胶原蛋白酶的结晶条件。方法通过基因重组构建幽门螺杆菌hp0169基因重组表达质粒,在大肠埃希菌进行重组蛋白的原核表达;经Ni 2+亲和层析、阴离子交换层析、凝胶过滤层析纯化蛋白;通过Hampton Research结晶试剂盒筛选蛋白结晶条件。结果扩增的幽门螺杆菌hp0169基因片段略大于1 000bp,构建重组质粒pET-28a-hp0169,转化至大肠埃希菌BL21(DE3)菌株后以IPTG诱导表达该U32家族蛋白酶HpPrtC;亲和层析,离子交换及凝胶过滤层析纯化的目标蛋白经SDS-PAGE鉴定为HpPrtC蛋白单体和二体,分子质量单位(Mr)为50×103和100×103,但以单体形式为主。使用结晶机器人筛选HpPrtC蛋白的初始结晶条件为0.2mol/L MgCl2,0.1mol/L Tris,pH 8.5,3.4mol/L己二醇。结论 HpPrtC可在大肠埃希菌中可溶性表达,并具有较好的可结晶性,为其结构与功能研究奠定了基础。
Objective To express,purify,and screen conditions for crystallization of Helicobacter pylori collagenase HpPrtC. Methods A recombinant plasmid was constructed to produce HpPrtC in Escherichia coli.The expressed protein was purified with a three-step chromatography strategy using an Ni 2＋affinity column,an anion exchange column,and a gel filtration column.Preliminary crystallization conditions were screened using Hampton Research crystallization kits. Results The hp0169 gene coding for HpPrtC,a member the U32 collagenase family,was amplified using PCR.A PCR product with an apparent molecular mass slightly bigger than 1 000 bp was subsequently ligated into pET-28 a.The resultant plasmid pET-28 a-hp0169 was then transformed into E.coli host strain BL21（DE3）for IPTG-induced protein expression.The destination protein was purified using KTA pure.According to SDS-PAGE,the recombinant HpPrtC protein was purified to 95% of purity with a yield of roughly 12 mg/L.An SDS-PAGE profile of the purified protein displayed two clearly visible bands,corresponding to an HpPrtC monomer and dimer.A protein crystallization robot was used to screen about 500 crystallization conditions,and the final crystallization conditions were 0.2 mol/L MgCl2,0.1 mol/L Tris,apH of 8.5,and 3.4 mol/L hexanediol. Conclusion Prokaryotic expression of HpPrtC led to soluble production of this protein,which could be crystallized.The current results have established a good starting point for further study of the structure and functions of HpPrtC.
作者
田新利
刘东
郭玲
崔丽伟
刘威
TIAN Xin-li;LIU Dong;GUO Ling;CUI Li-wei;LIU Wei(Department of Pathogenic Biology and Immunology,Xingtai Medical College,Xingtai 054000,Hebei,China;Institute of Immunology,Army Medi-cal University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2018年第7期719-723,共5页
Journal of Pathogen Biology
