Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

Abstract

Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results: Bands on Western Blots with 6×His-tag monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) showed the EGFP polyarginine and EGFP proteins were expressed in Saccharomyces cerevisiae INVSc1 at relatively low levels. The lyticase incubation time modification and administration of 3-5 kDa microfilter to concentrate increased the yield of isolated protein. Conclusions: The sufficient amount of protein isolation in S. cerevisiae can be achieved by using lyticase and sonicators combination for the lysis process.