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African Journal of Biotechnology

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Sequence analysis and over-expression of ribosomal protein S28 gene (RPS28) from the Giant Panda

W Hou, Y Hou, Y Hao, Y Du, T Zhang, Z Peng

Abstract


RPS28 is a component of the 40S small ribosomal subunit encoded by RPS28 gene, which is specific to eukaryotes. The cDNA and the genomic sequence of RPS28 were cloned successfully from the Giant
Panda using RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily and the cDNA of the RPS28 gene was also overexpressed in Escherichia coli ER2566 and
immunoblotted. The cDNA of the RPS28 cloned from Giant Panda is 216 bp in size, containing an open reading frame of 210 bp encoding 69 amino acids. The length of the genomic sequence is 790 bp, which
was found to possess three exons and two introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos Taurus, Mus musculus, Rattus norvegicus and Sus scrofa (92.4, 92.4, 87.1, 86.7 and 89.5%, respectively) as determined by Blast analysis.. The amino acid sequence encoded by RPS28 gene of the Giant Panda shared a high homology (100%) with those of Homo sapiens, B. Taurus, M. musculus, R. norvegicus and S. scrofa. Primary structure analysis revealed that the molecular weight of the putative RPS28 protein is 7.841 kD with a theoretical pI 10.70. Topology prediction showed there is one ribosomal protein S28e signature site, three protein kinase C phosphorylation site and two casein kinase II phosphorylation site in the RPS28 protein of the Giant Panda. The RPS28 gene can be really expressed in Escherichia coli and the RPS28 protein fusioned with the N-terminally GST -tagged protein gave rise to the accumulation of an expected 34 KDa polypeptide. The expression product obtained could be used for purification and study of its function further.



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