摘要： 目的·采用生物化学和结构生物学的方法，制备和结晶高纯度的新型口腔细菌丝氨酸蛋白酶抑制剂Tannerpin。方法·通过氨基酸序列分析，在牙周疾病相关细菌中筛选得到新型丝氨酸蛋白酶抑制剂，并将其命名为Tannerpin；构建融合表达载体pSUMO3-Tannerpin，将其转入大肠埃希菌BL21（DE3）系统进行诱导表达；采用镍离子亲和层析、SUMO特异性蛋白酶2（SUMO-specific protease 2，SENP2）酶切和凝胶过滤层析组合的纯化方案对Tannerpin进行分离纯化，并经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行验证；而后，对该纯化蛋白的结晶条件进行筛选及优化，并对其作用底物进行筛选。结果·SDS-PAGE结果显示，SUMO3-Tannerpin融合蛋白在大肠埃希菌中成功表达。利用纯化方案去除该蛋白的融合标签后，获得了相对分子质量约为43 000的Tannerpin。通过结晶条件的初筛和优化，最终在20 ℃、pH=6.0、沉淀剂为28%聚乙二醇3350和8% Tacsimate的条件下获得了分辨率为1.7 ?的Tannerpin晶体。然而，未筛选到Tannerpin的作用底物。结论·成功制备了口腔细菌分泌的新型丝氨酸蛋白酶抑制剂Tannerpin及其晶体，或将为后续其结构分析及功能研究奠定基础。
关键词: 口腔细菌, 新型丝氨酸蛋白酶抑制剂, 原核表达, 纯化, 结晶 Abstract: Objective·To prepare and crystallize a novel oral bacterial serine protease inhibitor Tannerpin with high purity by means of biochemistry and structural biology.
Methods·A novel serine protease inhibitor was screened from periodontal disease-related bacteria by amino acid sequence analysis, and named as Tannerpin. The fusion expression vector pSUMO3-Tannerpin was constructed and transferred into E. coli BL21 (DE3) system for induction expression. Tannerpin was isolated and purified by a combination of nickel-affinity chromatography, SUMO-specific protease 2 (SENP2) digestion and gel filtration chromatography, and was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the crystallization conditions of the purified protein were screened and optimized, and its acting substrates were screened.
Results·SDS-PAGE showed that SUMO3-Tannerpin fusion protein was successfully expressed in E. coli. After removing the fusion label of the protein by purification scheme, Tannerpin with relative molecular weight of about 43 000 was obtained. After crystallization screening and optimization, Tannerpin crystals with a resolution of 1.7 ? were finally obtained under the conditions of 20 ℃, pH=6.0, precipitant of 28% polyethylene glycol 3350 and 8% Tacsimate. However, the acting substrate of Tannerpin was not obtained.
Conclusion·A novel serine protease inhibitor Tannerpin secreted by oral bacteria and its crystal are successfully prepared, which may lay a foundation for subsequent structural analysis and functional study.
Key words: oral bacteria, novel serine protease inhibitor, prokaryotic expression, purification, crystallization PDF全文下载地址:
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