Recombinant Production and Purification of Green Fluorescent Protein (GFP)-Fused Metal Binding Protein for Palladium Nanoparticle Synthesis

In lieu of chemical and physical methods, biologically guided synthesis is increasingly used as a cost-effective medium for the fabrication of nanoparticles (NP). Recently, a palladium metal binding sequence Pd4 (TSNAVHPTLRHL) has been demonstrated to be instrumental in the production of palladium (Pd) nanoparticles. Although, by eliminating the additional cost of purification of the protein, the crude lysate of E. coli containing Pd specific protein has been proven to be a viable cost-effective means for the synthesis of Pd NP, studies have not be done to ascertain the comparative catalytic activity of nanoparticles synthesized from both clarified lysate and pure protein sample. This thesis aims to provide a comparative catalytic analysis of the turnover frequency of NP derived from pure Pd43-GFPuv and turnover ratio derived from NP synthesized by Mosleh et al. An E. coli expression system is employed for production of three copies of palladium binding sequence fused with green fluorescence protein [(Pd4)3-GFP] as a tag. This protein was purified with a Fast Protein Liquid Chromatography (FPLC) apparatus coupled with Immobilized Metal Affinity Column (IMAC). Transmission electron microscopy (TEM) was utilized to characterize the synthesized NPs from clarified lysate and pure protein sample. The catalytic property of fabricated NP from the purified protein was analyzed by a model Suzuki-Miyaura coupling reactions. It was observed that the purified metal binding protein had are three times higher turnover frequency compared to the crude lysate previously published.