The histidyl-tRNA synthetase from Streptococcus equisimilis: overexpression in Escherichia coli, purification, and characterization.
We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene (hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of approximately 18% of total cell protein (approximately 50 mg/liter of bacterial culture). The fusion HisRS was purified to > 99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the functional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M(r) = 53.7 kDa.
Menguito
C A
CA
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0647.
Papaconstantinou
J
J
Weigel
P H
PH
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006643:Histidine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012270:Ribosomes / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology*; D014169:Transformation, Bacterial; D014764:Viral Proteins
D046911:Macromolecular Substances; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D006643:Histidine-tRNA Ligase