tailieunhanh - Expression, purification, and characterization of rhTyrRS

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. | Lang et al. BMC Biotechnology 2014 14 64 http 1472-6750 14 64 BMC Biotechnology RESEARCH ARTICLE Open Access Expression purification and characterization of rhTyrRS Yongjiang Lang1 2 Yanling Zhang1 2 Ling Zhan1 2 Zhe Feng1 2 Xiushi Zhou1 2 Min Yu1 2 and Wei Mo1 2 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. Keywords Recombinant human tyrosyl-tRNA synthetase Expression of rhTyrRS Protein purification Biological activity assay Background Aminoacyl-tRNA synthetases AARSs are enzymes that catalyze the essential first step of protein synthesis by covalently attaching each of the 20 standard amino acids to their cognate tRNA. Because the reactions require the capacity to recognize tRNAs as well as small chemicals such as amino acids and ATP the structures of AARSs are well equipped for interacting with diverse molecules that may be associated with their functional versatility 1 2 . AARSs such as .