鼠伤寒沙门菌效应因子SteA蛋白的表达纯化与晶体培养

Purification and crystal culture of SteA protein,an effector of Salmonella typhimurium

目的对鼠伤寒沙门菌效应蛋白SteA进行原核表达与纯化,以期获得高质量蛋白和晶体,为SteA的活性检测及结构解析奠定基础。方法利用生物信息学分析方法,对SteA的氨基酸及核苷酸序列进行分析,了解蛋白性状与序列保守性;使用基因工程方法,将steA基因克隆到原核表达载体pGLO1上;使用大肠埃希菌BL21(DE3)进行SteA蛋白的外源表达;使用镍离子亲和层析,离子交换层析,凝胶过滤层析的方法对SteA蛋白进行体外纯化;使用蛋白结晶试剂盒对提纯的SteA蛋白进行晶体普筛,摸索适合的晶体生长条件并进行优化,培养高质量单晶用于结构解析。结果克隆出SteA全长及SteA 35-173 aa片段基因,并构建了SteA35-173 aa C58S突变体。SteA全长和SteA 35-173 aa C58S蛋白在大肠埃希菌感受态细胞BL21中成功表达,获得了无聚集的SteA蛋白,浓度为7.6 mg/mL。发现在1.2 mol/L Sodium phosphate monobasic/0.8 mol/L Potassium phosphate dibasic;0.1 mol/L CAPS/Sodium hydroxide pH 10.5;0.2 mol/L Lithium sulfate条件下能够获得晶体,条件优化获得可用于数据收集的高质量单晶。结论鼠伤寒沙门菌效应蛋白SteA可在原核表达系统中稳定表达,且可溶性良好,Cys58对SteA二聚体的形成起到重要作用。SteA 35-173 C58S蛋白在磷酸盐和硫酸盐环境中易结晶。

Objective This study aims to express and purify the effector protein SteA of Salmonellatyphimuriumin prokaryotic expression,in order to obtain high-quality protein and crystals,and lay the foundation for the activity detection and structural analysis of SteA.Methods Based on bioinformatics,the amino acid and nucleotide sequences of SteA were analyzed to understand protein traits and sequence conservation.The steA gene was cloned into a prokaryotic expression vector by genetic engineering and the SteA protein was stably expressed in E.coli BL21.The SteA protein was purified in vitro using nickel ion affinity chromatography,ion exchange chromatography,and molecular sieve chromatography in sequence.The purified SteA protein crystals were screened by protein crystallization kit,and the appropriate crystal growth conditions were explored and optimized to cultivate high-quality single crystals for structural analysis.Results The full-length and the 35-173aa fragment of SteA gene were cloned,and a C58S mutant was successfully constructed.The full length and the 35-173aa C58Sprotein of SteA were successfully expressed in E.coli receptive cell BL21.A non-aggregated form of SteA protein was obtained at a concentration of 7.6mg/mL.It was found that crystals could be obtained under the condition of 1.2mol/L Sodium phosphate monobasic/0.8 mol/L Potassium phosphate dibasic;0.1 mol/L CAPS/Sodium hydroxide pH 10.5;0.2 mol/L Lithium sulfate,and high-quality single crystals were optimized for data collection.Conclusion The effector protein SteA of S.typhimurium can be stably expressed in the prokaryotic expression system and has good solubility.Cys58plays an important role in the formation of SteA dimers.The SteA 35-173C58Sprotein is prone to crystallization in phosphate and sulfate environments.