摘要
目的通过建立大肠埃希菌BL21(DE3)原核表达系统,得到高纯度及构象均一的YaaA蛋白,筛选蛋白晶体,为其结构解析及相关功能研究奠定基础。方法利用生物信息学方法对YaaA蛋白的性质及结构进行预测和分析。使用PCR的方法从沙门菌基因组中扩增yaaA基因,将其克隆到原核表达载体pGl01上,获得重组质粒pGl01-YaaA。利用DE3培养后,IPTG合并氯霉素进行YaaA蛋白的可溶性诱导表达,通过镍离子亲和层析柱、阴离子交换柱以及分子筛凝胶层析柱得到高纯度的目标蛋白。纯化的YaaA蛋白采用悬滴气象扩散法获得其蛋白晶体。通过胰蛋白酶酶切试验测定YaaA蛋白核心区及稳定性。结果YaaA蛋白基因成功克隆到pGl01载体上,并在DE3中可溶性表达。通过层析纯化得到高纯度、形态均一的YaaA蛋白,相对分子质量约为30×10^(3),浓度为6mg/mL,纯度约为98%。YaaA蛋白的稳定性较好,晶体培养条件为:0.2 mol/L Ammonium sulfate,0.1 mol/L BIS-TRIS,pH 6.5,25%(w/v)Polyethyleneglycol3350。结论通过原核表达、亲和层析及凝胶过滤层析获得高纯度且构象单一的YaaA蛋白,该蛋白稳定性较好,可用于蛋白晶体的筛选,为YaaA的结构解析和功能验证奠定了基础。
Objective The high-purity and conformational YaaA protein was obtained by a prokaryotic expression system for Escherichia coli BL21(DE3).The crystal screening of the protein was further carried out to lay the foundation for its structural analysis and related functional research.Methods The basic properties and solubility of Salmonella YaaA protein were predicted by Expas.Then the homologous model of Salmonella YaaA structure was established by SWISS MODEL,which based on the structure of the E.coli YaaA protein(PDB code:5ACJ).The structure Analysis of YaaA was used Coot and PyMoL.The yaaA gene was amplified from the genome of Salmonella by PCR and then cloned into the prokaryotic expression vector pGlo1 to obtain the recombinant plasmidpGlo1-YaaA.Subsequently,E.coli BL21(DE3)was used to express the YaaA protein.The soluble expression of YaaA protein was induced by IPTG and chloramphenicol after being cultured with DE3.Then the high purity YaaA protein was purified by nickel ionaffinity chromatography,anion exchange column and molecular sieve gel chromatography column.YaaA protein crystals were obtained by hanging drop meteorological diffusion method.Furthermore,the stability of YaaA protein was determined by trypsin digestion test,and its core region was determined.Results The Salmonella YaaA protein consists of 257 amino acids,its the relative molecular weight is about 30 ku,its isoelectric point is 5.89,and there is no signal peptide.According to bioinformatics analysis,YaaA may be a DNA binding protein.The yaaA gene was successfully cloned into the pGlo1 vector andexpressed soluble in DE3.The YaaA protein with high purity and uniform morphology was obtained by chromatography,the relative molecular weight was about 30×10^(3),the concentration was 6 mg/mL,and the purity was about 98%.YaaA is stable when the concentration of trypsin is less than 2 mg/mL,and its crystal culture conditions are as follows:0.2 mol/L Ammonium sulfate,0.1 mol/L BIS-TRIS,pH 6.5 Polyethylene glycol 3350.Conclusion In this study,YaaA pro
作者
戴元吉
张敏
程龙云
许祥冬
华浩然
王玮玮
李冰清
DAIYuanji;ZHANG Min;CHENG Longyun;XU Xiangdong;HUA Haoran;WANG Weiwei;LI Bingqing(Department of Pathogen Biology,School of Clinical and Basic Medicine(Institute of Basic Medicine),Shandong First Medical University(Shandong Academy of Medical Sciences),Jinan 250000,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2023年第5期513-518,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.32170034,81902038)。
关键词
鼠伤寒沙门菌
YaaA蛋白
原核表达
晶体培养
蛋白稳定性
Salmonella typhimurium
YaaA protein
Prokaryotic expression
Crystal cultivation
Protein stability
