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African Journal of Biotechnology

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cDNA, genomic sequence cloning and overexpression of ribosomal protein S16 gene (RPS16) from the Giant Panda

W Hou, Y Hou, Y Hao, T Zhang, Z Peng, Y Wu, G Wu

Abstract


RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain reaction (RT-PCR) technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily. The cDNA of the RPS16 gene was overexpressed in Escherichia coli BL21. The length of cDNA fragment cloned is 448 bp containing an open reading frame of 441 bp encoding 146 amino acids and the length of the genomic sequence is 2510 bp, containing five exons and four introns. Alignment analysis indicates that the nucleotide sequence share a high homology with those of Bos taurus, Homo sapiens, Mus musculus, Rattus norvegicus and Danio rerio by 95.46, 92.97, 89.80, 89.80 and 82.54%, respectively. The deduced amino acid sequence is entirely identical compared with the first four animals and share a high homology with that of D. rerio by 96.58%. Topology prediction shows that there is one cAMP- and cGMP-dependent protein kinase phosphorylation site, three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, two N-myristoylation sites, one amidation site and one ribosomal protein S9 signature in the RPS16 protein of the Giant Panda . The RPS16 gene can be readily expressed in E. coli and it fused with the N-terminally GST-tagged protein which gave rise to the accumulation of an expected 20.095 kDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained could be used for purification and further study of its function.



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