Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the Giant Panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E. coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.