胱天蛋白酶募集域蛋白9的可溶性表达及纯化

中国真菌学杂志　 2023, Vol. 18 　Issue (2): 140-145.

胱天蛋白酶募集域蛋白9的可溶性表达及纯化

彭敏¹, 张晨¹, 王珏², 陈浩², 孔庆涛^1,1

1. 南京大学医学院附属金陵医院皮肤科, 南京 210002;
2. 南京大学化学化工学院, 南京 210023

收稿日期:2022-11-18 出版日期:2023-04-28 发布日期:2023-05-26
通讯作者: 孔庆涛,E-mail:njukqt@163.com;桑红,E-mail:sanghong@nju.edu.com E-mail:njukqt@163.com;sanghong@nju.edu.com
作者简介:彭敏,女(汉族),硕士研究生在读.E-mail:pmbeidou@163.com
基金资助:
江苏省皮肤病学创新团队项目(CXTDA2017038)

Soluble expression and purification of caspase recruitment domain-containing protein 9

PENG Min¹, ZHANG Chen¹, WANG Jue², CHEN Hao², KONG Qingtao^1,1

1. Department of Dermatology, Affiliated Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China;
2. School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China

Received:2022-11-18 Online:2023-04-28 Published:2023-05-26

摘要/Abstract

摘要： 目的制备可溶性TrxA-CARD9蛋白及其临床突变型。方法将构建好的原核系统表达质粒转化至大肠杆菌感受态细胞后,通过不同诱导条件筛选合适的表达条件。使用精氨酸辅助目的蛋白进行非变性溶解,并通过聚乙烯亚胺和硫酸铵沉淀法去除核酸和杂蛋白,最终利用阴离子交换和肝素亲和色谱柱纯化融合蛋白。结果成功构建表达质粒并纯化融合蛋白,酶切去除TrxA标签后通过质谱鉴定其为可溶性CARD9蛋白。结论成功实现了全长CARD9序列在原核系统中的高可溶性表达,为研究CARD9蛋白生物结构和功能提供了基础,并为寻找治疗真菌感染的潜在靶点和治疗方法提供新思路。

关键词: 胱天蛋白酶募集域蛋白, 精氨酸, Trx-A标签, 蛋白表达, 可溶性蛋白

Abstract: Objective The caspase recruitment domain-containing protein 9 (CARD9) mediates intracellular signaling by Toll-like and C-type lectin receptors, and plays a critical role in the regulation of fungal infection. Since it is insoluble, the in vitro expression of the complete sequence of CARD9 is currently not feasible; hence, its structure and function remain unclear. In this study, we constructed a prokaryotic systemic expression plasmid using molecular cloning techniques to produce the TrxA-CARD9 protein and its clinical mutant phenotype in vitro. Methods Screening with different isopropyl β-d-1-thiogalactopyranoside concentrations and temperatures led to the successful expression of soluble full-length CARD9. To increase the solubility of the target protein, arginine was used to assist its non-denaturing solubilization. After the removal of nucleic acids and partial heteroprotein using polyethyleneimine and ammonium sulfate precipitation, respectively, the fusion protein was purified using an anion-exchange and heparin affinity chromatography column. Results We successfully achieved highly soluble expression of full-length CARD9 sequence in a prokaryotic system and provided an optimized purification protocol. Conclusion This study provides a basis for future in vitro experiments studying the biological structure and function of CARD9 protein. It proposes a new research idea to find potential targets for the treatment of fungal infections and the treatment of critical fungal infections.

Key words: caspase recruitment domain-containing protein 9, arginine, Trx-A tag, protein expression, insoluble protein

中图分类号:

彭敏, 张晨, 王珏, 陈浩, 孔庆涛, 桑红. 胱天蛋白酶募集域蛋白9的可溶性表达及纯化[J]. 中国真菌学杂志, 2023, 18(2): 140-145.

PENG Min, ZHANG Chen, WANG Jue, CHEN Hao, KONG Qingtao, SANG Hong. Soluble expression and purification of caspase recruitment domain-containing protein 9[J]. Chinese Journal of Mycology, 2023, 18(2): 140-145.

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