Cloning, sequencing and bacterial expression of human glycine tRNA synthetase

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Abstract
The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains alarge open reading frame (ORF) encoding 685 amino acids with predicted Mr=77 507 Da. The protein sequence has ∼60% identity with B.moriGlyRS and 45% identity with S.cerevisiaeGlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids isfound 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 Mr = 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 extractand confirms that the cDNA encodes human GlyRS.