Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.
The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.
Williams
J
J
Immunology Research Centre, St Vincents Hospital, Fitzroy, Victoria, Australia.
Osvath
S
S
Khong
T F
TF
Pearse
M
M
Power
D
D
D000215:Acylation; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006032:Glycine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D006032:Glycine-tRNA Ligase