Document Type : Original Article
Islamic Azad University (IAU), Science and Research Branch, Tehran, Iran
Cell and Molecular Biology Department, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran
Developmental Biology Department, University of Science and Culture, ACECR, Tehran, Iran
Stem Cells and Developmental Biology Department, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Objective: Amino acid alignment analysis of deduced amino acid residues revealed a tripeptide (SKI) at the carboxy terminus of peroxisomal protein cDNA. In order to see the importance of the above sorting signal, we have performed a site-directed mutagenesis to delete SKI tripeptide and its transfection into CHO-K1 and P19 cells.
Materials and Methods: In order to create the appropriate site-directed mutant, PCR was done with specific primers. Amplified PeP cDNAs either containing SKI or deleted ones were constructed downstream of EGFP cDNA under regulation of the cytomegalovirus (CMV) promoter in a pEGFP-C1 vector. Transfection of P19 and CHO cells was done with lipofectamine2000. Results: Gradient PCR showed that the best annealing temperature was 71.6 ºC. Transfection of plasmids containing chimera of EGFP-PEP cDNAs into the CHO-K1 and P19 cells showed several punctuate structures, presumably peroxisomes, while SKI deletion showed a cytosolic mislocalization of the EGFP pattern.
Conclusion: Taken together, these data strongly suggest that SKI, which is located at the C- terminus of the protein, is required for sorting this protein.