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African Journal of Biotechnology

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cDNA, genomic sequence cloning and analysis of the ribosomal protein L37A gene (RPL37A) from the giant panda (Ailuropoda melanoleuca) and its overexpression

C Wu, J Sun, W Hou

Abstract


Ribosomal protein L37A (RPL37A) is a component of 60S large ribosomal subunit encoded by the RPL37A gene, which belongs to the family of ribosomal L37AE proteins, located in the cytoplasm. The complementary deoxyribonucleic acid (cDNA) and the genomic sequence of RPL37A were cloned successfully from giant panda using reverse transcription- polymerase chain reaction (RT-PCR) and touch-down PCR technology, respectively. Both sequences were analyzed preliminarily. The cDNA of RPL37A was 298 bp in length, containing an open-reading frame (ORF) of 279 bp encoding 92 amino acids with an estimated protein molecular weight of 10.23 kDa, and a theoretical isoelectric point (pI) of 11.11. The length of genomic sequence was 2587 bp, possessing four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence showed a high homology with previously reported L37A sequences for Homo sapiens, Pongo abelii, Mus musculus and Bos taurus. The amino acid sequence encoded by the RPL37A gene of giant panda shared a high homology with the four animals above. Topology prediction showed that there were one N-glycosylation site, three protein kinase C phosphorylation site and three N-myristoylation sites in the RPL37A protein of giant panda. The RPL37A gene could be readily expressed in Escherichia coli because it was fused with the N-terminally His-tagged protein which gave rise to accumulation of an expected 14-kD polypeptide, in good agreement with the predicted molecular weight. The over-expression product obtained could be purified for studies of its function. The cDNA of RPL37A was cloned successfully for the first time from the giant panda in this study. The result provide scientific material to enrich and improve the RPL37A gene database.

Key word: Giant panda, ribosomal protein L37A (RPL37A), cDNA cloning, sequence analysis, over-expression




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