cDNA, genomic sequence cloning and over-expression of ribosomal protein P2 gene ( RPLP2 ) from the Giant Panda (Ailuropoda melanoleuca )

Abstract

RPLP2 is a component of the 60S large ribosomal subunit encoded by RPLP2 gene and directly participate in protein synthesis, which is located in the cytoplasm. The cDNA and genomic sequence of RPLP2 were cloned successfully from the Giant Panda using RT-PCR and Touchdown-PCR technology, respectively. The results showed that the cDNA fragment cloned is 394 bp in size, containing an open reading frame of 348 bp. The length of the genomic sequence is 1838 bp, with four exons and three introns. The deduced protein is composed of 115 amino acids with 11.66 KD of estimated molecular weight and 4.14 of pI. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species reported, including Homo sapiens , Bos taurus , Mus musculus and Rattus norvegicus . The homologies for nucleotide sequences of Giant Panda PRLP2 are 90.80, 87.64, 88.79 and 87.64% to those of the four species, while the homologies for amino acid sequences are 88.27, 85.20, 86.31 and 85.20%. The RPLP2 gene was overexpressed in Escherichia coli BL21, and the result indicated that fusing RPLP2 with the N-terminally His-tagged form gave rise to the accumulation of an expected 17.5 KD polypeptide, in accordance with the predicted protein, which could be used to purify and investigate the function of this protein.