cDNA, genomic sequence cloning and overexpression of ribosomal protein s20 gene (RPS20) from the Giant Panda (Ailuropoda melanoleuca)
AbstractRPS20 is a component of the 40S small ribosomal subunit encoded by RPS20 gene, which is conserved between eukaryotes, prokaryotes and archaebacteria. The cDNA and the genomic sequence of RPS20
were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively. Both sequences were analyzed preliminarily and the cDNA of the
RPS20 gene was also overexpressed in Escherichia coli BL21. The cDNA of the RPS20 cloned from Giant Panda is 392 bp in size, containing an open reading frame of 360 bp encoding 119 amino acids. The length of the genomic sequence is 1205 bp, which was found to possess 4 exons and 3 introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Pongo abelii, Macaca fascicularis, Mus musculus, Bos taurus and Rattus norvegicus are 93.1, 92.5, 92.2, 91.1, 90.6 and 90.0% respectively. The amino acid sequence encoded by RPS20 gene of the Giant Panda shared a high homology (100%) with those of H. sapiens, Mac. fascicularis, Mus musculus, B. taurus and R. norvegicus, except for P. abelii (99.88%). Primary structure analysis revealed that the molecular weight of the putative RPS20 protein is 13.373 kD with a theoretical pI 9.95. Topology prediction showed there is one ATP/GTP-binding site motif A, one
ribosomal protein S10 signature site, 5 protein kinase C phosphorylation sites and three Casein kinase II phosphorylation sites in the RPS20 protein of the Giant Panda. The RPS20 gene can be really expressed in E. coli and the RPS20 protein fusioned with the N-terminally GST-tagged form gave rise to the accumulation of an expected 39 kDa polypeptide.