Expression, purification, and characterization of rhTyrRS

• Published in: BMC biotechnology 2014-07, Vol.14 (1), p.64-64, Article 64
• Main Authors: Lang, Yongjiang, Zhang, Yanling, Zhan, Ling, Feng, Zhe, Zhou, Xiushi, Yu, Min, Mo, Wei
• Format: Article
• Language: English
• Subjects: Acylation; Amino acids; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Codon; Cytokines; E coli; Enzymes; Escherichia coli; Escherichia coli - metabolism; Experiments; Fermentation; Gene Expression; Glucose; HL-60 Cells; Humans; Interleukin-8 - metabolism; Mass Spectrometry; Methods; Physiological aspects; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Studies; Trace elements; Tyrosine-tRNA Ligase - chemistry; Tyrosine-tRNA Ligase - genetics; Tyrosine-tRNA Ligase - metabolism
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-14-64

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