新型口腔细菌丝氨酸蛋白酶抑制剂Tannerpin的制备与结晶

doi:10.3969/j.issn.1674-8115.2021.10.004

摘要/Abstract

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

中图分类号:

杨婷婷, 许佳伟, 周爱武, 叶玮. 新型口腔细菌丝氨酸蛋白酶抑制剂Tannerpin的制备与结晶[J]. 上海交通大学学报(医学版), 2021, 41(10): 1297-1302.

Ting-ting YANG, Jia-wei XU, Ai-wu ZHOU, Wei YE. Preparation and crystallization of a novel oral bacterial serine protease inhibitor Tannerpin[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(10): 1297-1302.

图/表 5

图1 Tannerpin、Miropin及α1-AT的RCL序列对比

Fig 1 Comparison of RCL sequences of Tannerpin, Miropin and α1-AT

图 2 pSUMO3-Tannerpin的质粒图谱Note：TANNERPIN is inserted between BamHⅠ(5'-GGATCC-3') and XhoⅠ(5'-CTCGAG-3').

Fig 2 Plasmid mapping of pSUMO3-Tannerpin

图 3 融合蛋白（SUMO3-Tannerpin）的诱导表达、纯化及验证Note：A. Expression of total bacterial protein and soluble protein in supernatant before and after induction. Lane1—Marker, Lane2—total bacterial protein sample before induction, Lane3—total bacterial protein sample 7 h after induction, Lane4—total bacterial protein sample 24 h after induction, Lane5—supernatant sample before induction, Lane6—supernatant sample 7 h after induction, Lane7—supernatant sample 24 h after induction. B. SDS-PAGE detection of nickel-affinity chromatography for the first time. Lane1—Marker, Lane2—supernatant, Lane3—flow-through solution after the nickel column, Lane4-11—eluate. C. SDS-PAGE detection of nickel-affinity chromatography for the second time. Lane1—Marker, Lane2—SENP2 before digestion, Lane3—SENP2 after digestion, Lane4—flow-through solution after the nickel column, Lane5—equilibrium solution, Lane6—eluate.

Fig 3 Induced expression, purification and validation of fusion protein (SUMO3-Tannerpin)

图 4 Tannerpin蛋白结晶条件的筛选、优化及晶体X射线衍射Note：A. Tannerpin crystal under primary screening. B. Tannerpin crystal under optimized conditions. C. X-ray diffraction result of optimized Tannerpin crystal.

Fig 4 Screening and optimization of crystallization conditions of Tannerpin protein and its crystal X-ray diffraction

图 5 Tannerpin作用底物的初步筛选结果Note：Lane1/10—Marker, Land2, 11—Tannerpin. The other lanes are mixed solutions of Tannerpin and substrate. The specific substrates are Land3—FⅡa, Land4—HLE, Land5—elastase, Land6—PC, Land7—t-PA, Land8—FⅨa, Land9—FⅪa, Land12—trypsin, Land13—CB, Land14—CG, Land15—KLK1, Land16—KLK7, Land17—PR3.

Fig 5 Preliminary screening results of Tannerpin substrates

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