Cloning and expression of human phenylalanyl-tRNA synthetase in Escherichia coli: comparative study of purified recombinant enzymes.
Human phenylalanyl-tRNA synthetase (PheRS) was cloned and expressed in Escherichia coli. The cDNAs of the alpha and beta subunits were cloned into pET-21b(+) and pET-28b(+) vectors. The 6x histidine-tagged (HT) plasmids pET-21_HTbeta, pET-28_HTalpha, and pET-28_HTbeta were constructed. Three different types of (alphabeta)(2) heterodimers of human PheRS carrying HT at the N-terminus of either of two alpha or beta subunits or simultaneously on both of them were overproduced and purified. The heterodimeric protein with HT appended to the N-terminus of the beta subunit revealed no activity in the aminoacylation reaction as opposed to those with HT on the alpha subunit. It is known from the structure of the Thermus thermophilus Phe system that the N-terminal coiled-coil domain of the alpha subunit is involved in the binding of cognate tRNA(Phe). Our data demonstrate that a histidine-tagged N-terminal extension appended to the alpha subunit does not affect the kinetic parameters of tRNA(Phe) aminoacylation. Elimination of the HT from the alpha subunit by thrombin cleavage leads to nonspecific splitting of the enzyme that occurs in parallel to the main reaction. In addition to the tagged proteins the properly assembled heterodimer containing intact alpha and beta subunits free of HT was overproduced and purified. Aminoacylation activity of the overproduced human PheRS in the crude bacterial extract is two orders of magnitude higher than the corresponding activity in human placenta and the yield of the recombinant enzyme overproduced in E. coli is five times higher.
Moor
Nina
N
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Linshiz
Gregory
G
Safro
Mark
M
D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D010652:Phenylalanine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity
D021122:Protein Subunits; D011994:Recombinant Proteins; D010652:Phenylalanine-tRNA Ligase