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

doi:10.3969/j.issn.1674-8115.2021.10.004

Abstract

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

CLC Number:

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.

Figures/Tables 5

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

Fig 2 Plasmid mapping of pSUMO3-Tannerpin

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

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

Fig 5 Preliminary screening results of Tannerpin substrates

TrendMD

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