Abstract
The construction of a fusion protein often involves the connecting of two domains or proteins by a peptide linker, which should be designed not to disturb the structures and functions of each domain. Many natural enzymes often consist of two or more distinct modules, which are also connected by a linker peptide. Among which glucoamylase (GA) from Rhizopus oryzae is known to contain a linker of 36 amino acids between an N-terminal starch-binding domain (SBD) and a C-terminal catalytic domain (CD). The SBD possesses a high binding affinity towards raw starch so it was applied in recombinant protein engineering to facilitate the purification of fusion proteins. In this study, enhanced green fluorescent protein (eGFP) was fused with SBD and a few linker variants, including the original linker from R. oryzae GA, in order to investigate the effects of different linker peptides on the expression and purification of fusion proteins, and the SBD and eGFP modules were shuffled between the N- and C- termini while constructing the fusion proteins. According to the results of binding assays, when the linker was located at the N-terminus of the SBD, the starch-binding capacity of SBD was reduced as the Bmax values of the fusion proteins containing an SBD at the C-terminus were lower than those of the fusion proteins containing an SBD at the N-terminus, thus it is more favorable to apply this SBD as an N-terminal affinity tag. On the contrary, the Kd values of all fusion proteins were quite similar, as they were determined to be in the same range (10-6 M level). Furthermore, two purification procedures using corn starch were developed: stirring method and starch column chromatography. Based on the comparison of the purification analyses of both methods, although the starch adsorption rate was promoted in the former, the yield was significantly improved in the latter. As a result, it was indicated that using starch column chromatography was more effective, and it is prospective to be developed into a commercial purification technique due to its convenience, efficiency and economy.