PMID:10024478
| Citation |
Chen, J, Li, Y, Wang, E and Wang, Y (1999) High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli. Protein Expr. Purif. 15:115-20 |
|---|---|
| Abstract |
A T7 promoter-based His6-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus. The vector allows overproduction and single-step purification of His6-fusion protein by immobilized metal (Ni2+) chelate affinity chromatography. The gene encoding leucyl-tRNA synthetase (leuS) was cloned into this vector and expressed in high level. The specific activity of the synthetase in the crude extract of E. coli JM109(DE3) transformant containing the His6-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector). The overproduced His6-fusion leucyl-tRNA synthetase can be purified to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55%. The His6-tag at the N terminus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure. |
| Links |
PubMed Online version:10.1006/prep.1998.0999 |
| Keywords |
Chromatography, Affinity/methods; Cloning, Molecular/methods; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Kinetics; Leucine-tRNA Ligase/biosynthesis; Leucine-tRNA Ligase/genetics; Leucine-tRNA Ligase/isolation & purification; Plasmids; Recombinant Proteins/biosynthesis; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Restriction Mapping |
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