Volume 13(2)/2021 OPTIMIZATION OF PROTEIN PURIFICATION PROTOCOL FOR ARTIFICIAL TRANSCRIPTION FACTORS LEC2 Authors Rahman M. Z. A., Jainuddin H. and Kasran R. ABSTRACT Transcription factors (TFs) are essential for the expression of all proteins, including those involved in the plant regeneration and totipotency. LEAFY COTYLEDON 2 (LEC2) exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network and is sufficient to induce somatic embryo development in vegetative cells. Previously, five new TcLEC2 artificial transcription factors (ATFs) were successfully constructed and expressed using a prokaryotic system (pET100/D-TOPO). The recombinant proteins were separately purified by his-tagged protein purification using the ÄKTA pure protein purification system. However, the expressed ATFs were co-purified with the host's chromosomal DNA causing problems for subsequent application. Although this is the common issue for the case of DNA binding protein, an optimization of purification protocol should be conducted in order to obtain DNA-free purified protein samples. Optimization of binding and elution buffer's composition for his-tagged affinity chromatography had been conducted with additional washing steps to remove the bound DNA from the recombinant protein samples. Introduction of high salt washing step (20mM phosphate, 2M NaCl, 50 mM Imidazole, pH 7.4) before protein elution step successfully dissociated and removed the bound DNA from the targeted recombinant proteins. VIEW PDF DOWNLOAD