Genomic Organization, cDNA Sequence, Bacterial Expression, and Purification of Human Seryl‐tRNA Synthase

Abstract

In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryltRNA synthetase, and its expression in Escherichia coli. Two human brain cDNA clones of different origin, containing overlapping fragments coding for human sery-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5' region of the cDNA was missing. This 5' region was obtained via PCR methods using a human brain 5' RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the introd exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gene structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-I1 aminoacyl-tRNA synthetases. The human seryl-tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl-tRNA synthetase sequence (5 14 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E. coli (25 %), Saccharomyces cerevisiae (40 %), Arabidopsis thaliana (41 %) and Caenorhabditis elegans (60 %). The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively). Human seryl-tRNA synthetase, similar to several other class-I and class-I1 human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA.

Antibodies against c‐Jun N‐terminal peptide cross‐react with neo‐epitopes emerging after caspase‐mediated proteolysis during apoptosis

“…To explore this problem we used recombinant seryl‐tRNA synthetase expressed in E. coli cells. Expression was induced by adding IPTG to E. coli HMS (DE3) cells carrying the plasmid pET16–5SerRS (Vincent et al . 1997).…”

“…This was achieved using the plasmid pET16–5 SerRS carrying the gene for SerRS (Vincent et al . 1997) once transformed into E. coli strain HMS174 (DE3).…”

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

“…Human SerRS was expressed and purified as described previously and dialyzed against 20 mM HEPES buffer, pH 7.2, 100 mM NaCl, 5 mM MgCl 2 and 10% glycerol (29). Recombinant human LysRS (his-tagged) and AspRS (biotin-ubiquitin tagged) were expressed in bacteria and purified by affinity chromatography (unpublished data).…”

Validation of an Integrated Genetic‐Epigenetic Test for the Assessment of Coronary Heart Disease

“…Mutational analyses conducted in zebrafish demonstrated the essential role of this gene in normal vascular development. 52 , 53 Demethylation at this locus is associated with obesity, coronary artery calcification, and cardiometabolic syndrome. 54 , 55 , 56 Lower serine levels are not only associated with diabetes, but also supplementation of the diets of diabetic mice with serine can reverse some of the pathology associated with diabetes.…”