上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 10: 1297 - 1302

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.10.004

论著 · 基础研究

新型口腔细菌丝氨酸蛋白酶抑制剂Tannerpin的制备与结晶

  • 杨婷婷 ,
  • 许佳伟 ,
  • 周爱武 ,
  • 叶玮
展开
  • 1.上海交通大学医学院附属第九人民医院口腔预防科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海 200011
    2.遵义医科大学珠海校区，珠海 519040
    3.上海交通大学医学院病理生理学系，上海 200025
杨婷婷（1994—），女，硕士生；电子信箱：1476459716@qq.com。
叶玮，电子信箱：jyyewei@163.com。

网络出版日期: 2021-09-13

收起

Preparation and crystallization of a novel oral bacterial serine protease inhibitor Tannerpin

  • Ting-ting YANG ,
  • Jia-wei XU ,
  • Ai-wu ZHOU ,
  • Wei YE
Expand
  • 1.Department of Preventive Dentistry, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
    2.Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China
    3.Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
YE Wei, E-mail: jyyewei@163.com.

Online published: 2021-09-13

Fold

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

本文引用格式

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 . DOI: 10.3969/j.issn.1674-8115.2021.10.004

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

参考文献

1 Law RH, Zhang Q, McGowan S, et al. An overview of the serpin superfamily[J]. Genome Biol, 2006, 7(5): 216.
2 Carrell R, Travis J. α1-Antitrypsin and the serpins: variation and countervariation[J]. Trends Biochem Sci, 1985, 10(1): 20-24.
3 Song J, Matthews AY, Reboul CF, et al. Predicting serpin/protease interactions[J]. Methods Enzymol, 2011, 501: 237-273.
4 Huntington JA. Serpin structure, function and dysfunction[J]. J Thromb Haemost, 2011, 9(): 26-34.
5 Mkaouar H, Akermi N, Kriaa A, et al. Serine protease inhibitors and human wellbeing interplay: new insights for old friends[J]. PeerJ, 2019, 7: e7224.
6 Irving JA, Shushanov SS, Pike RN, et al. Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation[J]. J Biol Chem, 2002, 277(15): 13192-13201.
7 Marijanovic EM, Fodor J, Riley BT, et al. Reactive centre loop dynamics and serpin specificity[J]. Sci Rep, 2019, 9(1): 3870.
8 Gettins PG. Serpin structure, mechanism, and function[J]. Chem Rev, 2002, 102(12): 4751-4804.
9 Ray CA, Black RA, Kronheim SR, et al. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1β converting enzyme[J]. Cell, 1992, 69(4): 597-604.
10 Zhou A, Wei Z, Stanley PL, et al. The S-to-R transition of corticosteroid-binding globulin and the mechanism of hormone release[J]. J Mol Biol, 2008, 380(1): 244-251.
11 Pemberton PA, Stein PE, Pepys MB, et al. Hormone binding globulins undergo serpin conformational change in inflammation[J]. Nature, 1988, 336(6196): 257-258.
12 Nagata K. Hsp47: a collagen-specific molecular chaperone[J]. Trends Biochem Sci, 1996, 21(1): 22-26.
13 Zou Z, Anisowicz A, Hendrix MJ, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells[J]. Science, 1994, 263(5146): 526-529.
14 Gaci N, Dobrijevic D, Boudebbouze S, et al. Patented biotechnological applications of serpin: an update[J]. Recent Pat DNA Gene Seq, 2013, 7(2): 137-143.
15 Irving JA, Steenbakkers PJ, Lesk AM, et al. Serpins in prokaryotes[J]. Mol Biol Evol, 2002, 19(11): 1881-1890.
16 Ksiazek M, Mizgalska D, Enghild JJ, et al. Miropin, a novel bacterial serpin from the periodontopathogen Tannerella forsythia, inhibits a broad range of proteases by using different peptide bonds within the reactive center loop[J]. J Biol Chem, 2015, 290(1): 658-670.
17 Goulas T, Ksiazek M, Garcia-Ferrer I, et al. A structure-derived snap-trap mechanism of a multispecific serpin from the dysbiotic human oral microbiome[J]. J Biol Chem, 2017, 292(26): 10883-10898.
18 Warinner C, Speller C, Collins MJ. A new era in palaeomicrobiology: prospects for ancient dental calculus as a long-term record of the human oral microbiome[J]. Philos Trans R Soc Lond B Biol Sci, 2015, 370(1660): 20130376.
19 Paster BJ, Boches SK, Galvin JL, et al. Bacterial diversity in human subgingival plaque[J]. J Bacteriol, 2001, 183(12): 3770-3783.
20 Petersen PE, Bourgeois D, Ogawa H, et al. The global burden of oral diseases and risks to oral health[J]. Bull World Health Organ, 2005, 83(9): 661-669.
21 Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the ? ?red complex' ', a prototype polybacterial pathogenic consortium in periodontitis[J]. Periodontol 2000, 2005, 38: 72-122.
22 Socransky SS, Haffajee AD, Cugini MA, et al. Microbial complexes in subgingival plaque[J]. J Clin Periodontol, 1998, 25(2): 134-144.
23 Dewhirst FE, Chen T, Izard J, et al. The human oral microbiome[J]. J Bacteriol, 2010, 192(19): 5002-5017.
24 Belaaouaj A. Neutrophil elastase-mediated killing of bacteria: lessons from targeted mutagenesis[J]. Microbes Infect, 2002, 4(12): 1259-1264.
25 Collins CB, Aherne CM, Ehrentraut SF, et al. α1-Antitrypsin therapy ameliorates acute colitis and chronic murine ileitis[J]. Inflamm Bowel Dis, 2013, 19(9): 1964-1973.
26 Teshigawara S, Wada J, Hida K, et al. Serum vaspin concentrations are closely related to insulin resistance, and rs77060950 at SERPINA12 genetically defines distinct group with higher serum levels in Japanese population[J]. J Clin Endocrinol Metab, 2012, 97(7): E1202-E1207.
27 Teoh SS, Vieusseux J, Prakash M, et al. Maspin is not required for embryonic development or tumour suppression[J]. Nat Commun, 2014, 5: 3164.
28 Wang WL, Xu SY, Ren ZG, et al. Application of metagenomics in the human gut microbiome[J]. World J Gastroenterol, 2015, 21(3): 803-814.
29 Cremonesi E, Governa V, Garzon JFG, et al. Gut microbiota modulate T cell trafficking into human colorectal cancer[J]. Gut, 2018, 67(11): 1984-1994.
30 Yu T, Guo F, Yu Y, et al. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy[J]. Cell, 2017, 170(3): 548-563.e16.
Options
文章导航

/

〈 〉