Cell Journal (Jan 2009)
Mouse Peroxisomal Protein cDNA Cloning and Characterization of its Intraclleular Localization
Abstract
Objective: The aim of this study was to clone peroxisomal protein (PEP) cDNA in a mammalianexpression vector in a chimeric cDNA type, with enhanced green fluorescent protein(EGFP) cDNA. To investigate the intracellular localization of PEP protein linked to EGFPmarker, the constructed plasmid was used for transfection into the chinese hamster ovary(CHO) cells.Materials and Methods: Total RNA was extracted from the heart tissue of an adult mouse.PEP cDNA was constructed using reverse transcriptase and was amplified with specific primerscovering the entire length of ORF. RT-PCR products containing PEP cDNA were treatedby enzymatic digestion and inserted into the pEGFP-C1 downstream of EGFP cDNA and wereused for transformation into bacterial competent cells. The positive colonies which showedinserted PEP cDNA were selected for plasmid preparations and additional analysis was performedto ensure that PEP cDNA was inserted properly. Finally, to confirm the intracellularlocalization of EGFP-PEP, CHO cells were transfected with the constructed plasmid.Results: Our results confirmed amplification and cloning of the expected product. PEP cDNAencompasses 630 bp which encodes 209 amino acid residues. Bioinformatics analyses haveshown the presence of a fibronectin type III domain (31-114 a.a.) and two hydrophobic domains(12-32 a.a. and 152-169 a.a., respectively). Because of the presence of serine, Lysine,leucine (SKI) in the C-terminal of the related protein, transfection data showed peroxisomallocalization of PEP as was similar to the catalase.Conclusion: Taken together these data showed that PEP is a peroxisomal protein. Howeverthe importance of its fibronectin type III and two hydrophobic domains should be assessedby further experiments.
