TY - JOUR
T1 - Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis
AU - Menguito, Corazon A.
AU - Keherly, Michael J.
AU - Tang, Chuan ye
AU - Papaconstantinou, John
AU - Weigel, Paul H.
N1 - Funding Information:
We gratefully acknowledge Elizabeth Gerhardt, Shirley Chapman and Drs Lillian Chan, Richard Fritz and William Thompson for assistance with the DNA sequence analysis, Janet Oka for help preparing the figures, and Lisa Raney for help preparing the manuscript. This research was supported by grant # 004952 -1 1 from the Texas Higher Education Coordinating Board (to PW and JP). Cora Menguito was supported by a predoctoral fellowship from the James W.McLaughlin Fellowship Fund.
PY - 1993/2/11
Y1 - 1993/2/11
N2 - The histidyl-tRNA synthetase gene (hisS) from Streptococcus equisimilis was cloned and sequenced. The gene for this aminoacyl-tRNA synthetase has an open reading frame of 1278 nucleotides. The deduced amino acid sequence encodes a protein of 426 amino acids with MW = 47,932. The protein is predicted to be soluble with a pl = 5.27. The protein sequence has extensive overall identity/similarity with the Escherichla coli and the yeast histidyl-tRNA synthetases (̃58% and ̃20%, respectively). A putative promoter for gene transcription lies within two hundred nucleotides of the polypeptide start codon. The enzyme was overexpressed, to a level of about 18% of total cellular protein, as a fusion protein (containing an additional 15 amino acids) in E.coli using the pT7 expression system containing the T7 RNA polymerase/promoter (Tabor and Richardson, Proc. Nati. Aced. Sci. U.S.A. 82:1074-1078, 1985). The predicted MW for the hisS gene product Is in good agreement with the size of the fusion protein determined by SDS-PAGE (Mr = 53,700). Amino acid sequencing of the intact fusion protein and proteolytic fragments confirmed the deduced sequence of the synthetase at many positions throughout the protein. The expressed protein catalyzed the specific aminoacylation of tRNAHis in vitro.
AB - The histidyl-tRNA synthetase gene (hisS) from Streptococcus equisimilis was cloned and sequenced. The gene for this aminoacyl-tRNA synthetase has an open reading frame of 1278 nucleotides. The deduced amino acid sequence encodes a protein of 426 amino acids with MW = 47,932. The protein is predicted to be soluble with a pl = 5.27. The protein sequence has extensive overall identity/similarity with the Escherichla coli and the yeast histidyl-tRNA synthetases (̃58% and ̃20%, respectively). A putative promoter for gene transcription lies within two hundred nucleotides of the polypeptide start codon. The enzyme was overexpressed, to a level of about 18% of total cellular protein, as a fusion protein (containing an additional 15 amino acids) in E.coli using the pT7 expression system containing the T7 RNA polymerase/promoter (Tabor and Richardson, Proc. Nati. Aced. Sci. U.S.A. 82:1074-1078, 1985). The predicted MW for the hisS gene product Is in good agreement with the size of the fusion protein determined by SDS-PAGE (Mr = 53,700). Amino acid sequencing of the intact fusion protein and proteolytic fragments confirmed the deduced sequence of the synthetase at many positions throughout the protein. The expressed protein catalyzed the specific aminoacylation of tRNAHis in vitro.
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U2 - 10.1093/nar/21.3.615
DO - 10.1093/nar/21.3.615
M3 - Article
C2 - 8441673
AN - SCOPUS:0027230395
SN - 0305-1048
VL - 21
SP - 615
EP - 620
JO - Nucleic acids research
JF - Nucleic acids research
IS - 3
ER -