Cloning, expression, and bioinformatics analysis of the sheep CARP gene

The cardiac ankyrin repeat protein (CARP) is a multifunctional protein that is expressed specifically in mammalian cardiac muscle and plays important roles in stress responses, transcriptional regulation, myofibrillar assembly, and the development of cardiac and skeletal muscle. In this study, the sheep homolog of the CARP gene was cloned and characterized. The coding region of the gene consists of 960 bp and encodes 319 amino acids with molecular weight 36.2 KD. Bioinformatics analysis demonstrated that the 3' untranslated region (3'-UTR) of the gene contains many AU-rich elements that are associated with mRNA stability and a potential regulatory site for miRNA binding. The protein was predicted to contain 14 potential phosphorylation sites and an O-GlcNAc glycosylation site and to be expressed in both the nucleus and cytoplasm. The evolutionary analysis revealed that the sheep CARP exhibited a high level of homology with the mammalian counterparts; however, the protein exhibited an increased evolutionary distance from the chicken, frog, and fish homologs. RT-PCR revealed that in addition to its high mRNA expression level in cardiac muscle, trace amounts of the sheep CARP mRNA were expressed in the skeletal muscle, stomach, and small intestine. However, western blot analysis demonstrated that the CARP protein was expressed only in cardiac muscle. The coding sequence was cloned into the pET30a-TEV-LIC vector, and the soluble CARP-MBP (maltose-binding protein) fusion protein was expressed in a prokaryotic host and purified by affinity chromatography. Our data provide the basis for future studies of the structure and function of sheep CARP.