High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.
Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.
Xu
F
F
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Life Sciences, Chinese Academy of Sciences, Shanghai, 200031, People's Republic of China.
Jia
J
J
Jin
Y
Y
Wang
D T
DT
D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D006639:Histidine; D006801:Humans; D007700:Kinetics; D009532:Nickel; D010957:Plasmids; D012364:RNA, Transfer, Trp / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014369:Tryptophan-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D012364:RNA, Transfer, Trp; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D014369:Tryptophan-tRNA Ligase