重组人酪氨酸-tRNA合成酶的表达、纯化及特性分析

Expression, purification, and characterization of rhTyrRS.

作者信息

Lang Yongjiang, Zhang Yanling, Zhan Ling, Feng Zhe, Zhou Xiushi, Yu Min, Mo Wei

机构信息

The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, P,R, China.

出版信息

BMC Biotechnol. 2014 Jul 15;14:64. doi: 10.1186/1472-6750-14-64.

DOI:10.1186/1472-6750-14-64

PMID:25027604

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4118627/

Abstract

BACKGROUND

Aminoacyl-tRNA synthetases (AARSs) catalyze the first step of protein synthesis. Emerging evidence indicates that AARSs may have additional functions, playing a role in signal transduction pathways regulating thrombopoiesis and inflammation. Recombinant human tyrosyl-tRNA synthetase (rhTyrRS) is engineered with a single amino acid substitution that unmasks its cytokine activity. An industrial production method that provides high yield as well as high purity, quality, and potency of this protein is required for preclinical research.

RESULTS

We expressed codon-optimized rhTyrRS in Escherichia coli under fermentation conditions. Soluble protein was purified by a three-step purification method using cation exchange chromatography, gel filtration chromatography, and anion exchange chromatography. We also established a method to test the biological activity of rhTyrRS by measuring aminoacylation and IL-8 release in rhTyrRS-treated HL-60 cells.

CONCLUSIONS

The characterization of purified rhTyrRS indicated that this protein can be used in pharmacodynamic and pharmacokinetic studies.

摘要

背景

氨酰-tRNA合成酶（AARSs）催化蛋白质合成的第一步。新出现的证据表明，AARSs可能具有其他功能，在调节血小板生成和炎症的信号转导途径中发挥作用。重组人酪氨酰-tRNA合成酶（rhTyrRS）经工程改造，有一个单氨基酸取代，从而揭示其细胞因子活性。临床前研究需要一种能提供高产量以及该蛋白高纯度、高质量和高效力的工业生产方法。