Cloning, Expression And Purification Of Human Mitochondrion TRNA^Trp Synthetase

Aminoacyl-tRNA synthetases (aaRS) are the proteins earliestly emerged in the progression of organic evolution. AaRS can catalysis amino acids binding to cognate tRNAs, and also be involved in the process of gene expression and regulation, rRNA synthensis and RNA splicing, apoptosis according to recently researches. The objective of this study is to obtain the human mitochondria tryptophanyl-tRNA Synthetase (hmtTrpRS), and determine its kinetic parameters by the human mitochondria tRNATrp, which was transcripted in vitro. It provides a basis of the study on the structure, function and the species-specificity of the aminoacyl-tRNA synthetase hmtTrpRS.The total RNA was extracted from breast carcinoma cells (MDA-MB-435S), and the coding sequence of hmtTrpRS without signal peptides was amplified with reverse transcriptional (RT)-PCR. The PCR product was first cloned into the pMD18-T, then, pMD18-hmtTrpRS and pET24a(+) were both digested with restriction enzyme Ned I and EcoR I, and ligated to construct the expression plasmid which named pET24a(+)-hmtTrpRS. The positive clones were identified and selected by antibiotics resistance gene screening, molecular weight, the fragment after digesting and colony PCR.pET24a(+)-hmtTrpRS was transformed into BL21-CodonPlus(DE3)-RIL. The expression conditions were optimized by changing the IPTG concentration, induction time and temperature. The results indicated that the hmtTrpRS expression quantity was the most when ultima concentration of IPTG was O.lmmol/L and induced 4 h at 30℃, and the weight size is 38Kda conformited with the theory. The hmtTrpRS was then purified with Ni2+-NTA His-bind resin, and high specificity His-tagged hmtTrpRS protein was obtained.The human mitochondria tRNATrp was constructed by two steps PCR and transcripted in vitro. And it was tryptophanylated by hmtTrpRS. The Km is 1.08μmol/L, and kcat is 0.02 S-1, the specific activity of the purified soluble protein is 4411.18 U/mg.In conclusion, the prokaryotic expression vector for hmtTrpRS is successfully constructed and high specificity His-tagged hmtTrpRS protein was obtained. And the effienciency of aminoacylation was measured by tRNATrp.