Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis).
The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.
Institute of Biology, University of Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany.
Joachim
J
Hogg
Elfriede
E
Schiefermayr
Emile
E
Schiltz
Gabor L
GL
Igloi
D001126:Arginine-tRNA Ligase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D032449:Canavalia / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D001126:Arginine-tRNA Ligase