CDNA, Genomic sequence cloning and over-expression of ribosomal protein L15 gene (RPL15) from the giant panda

Abstract

RPL15 is a component of the 60S large ribosomal subunit encoded by RPL15 gene and belongs to the L15e family of ribosomal proteins, which is located in the cytoplasm. The cDNA and genomic sequence of RPL15 was cloned successfully from the giant panda using RT-PCR and Touchdown-PCR technology, respectively. These two sequences were analyzed preliminarily and the cDNA of the RPL15 gene was also over expressed inEscherichia coli BL21. The length of fragment cloned is 669 bp, containing an open-reading frame (ORF) of 615 bp encoding 204 amino acids. The length of the genomic sequence is 1,835 bp, with three exons and two introns. Primary structure analysis revealed that, the molecular weight of the putative RPL15 protein is 24.142 kDa with a theoretical pI 12.11. Topology prediction shows that there are two cAMP- and cGMP-dependent protein kinase phosphorylation sites, four N-myristoylation site, two Protein kinase C phosphorylation site, two Casein kinase II phosphorylation sites, two Amidation site and one Ribosomal protein L15e signature in the RPL15 protein of the giant panda. Alignment analysis indicates that, the nucleotide sequence of the coding sequence shows a high homology to other knownRPL15 sequences of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Canis familiaris and Danio rerio; as determined by Blast analysis, 94.31, 89.92, 91.54, 91.22, 96.59 and 79.02%, respectively. The homologies for deduced amino acid sequence are all 100% compared with the first five animals and share a high homology with that of D. rerio by 95.59%. The cDNA of RPL15 was cloned successfully from the giant panda in this study. It provides scientific material for enriching and improving the RPL15 gene database. TheRPL15 gene can be really expressed in E. coli and the RPL15 protein fusioned with the N-terminally His-tagged protein gave rise to the accumulation of an expected 30 KDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained could be used for purification and study of its function further. Key words: Giant panda, RT-PCR, RPL15, genomic sequences, cloning, over-express.