maedeh faraji

High efficiency and purity protein purification is a serious challenge in the production of recombinant proteins. This study is proposing an easy and new strategy for fabricating and describing magnetic microspheres consisting of a silica-coated magnetic core and a polyvinyl acetate shell modified with iminodiastic-acetic acid and complexed with Ni2+, (Fe3O4@SiO2@PVA@IDA-Ni2+) for the separation and purification of organophosphorus hydrolase (OPH) protein and histidine-containing outer membrane protein (OmpW).

The nanoparticles were synthesized in the following four steps: 1) synthesis of nanoparticles with vinyl groups on their surface, 2) grafting of polyvinyl alcohol (PVA) to these particles, 3) conversion of the hydroxyl groups of PVA to iminodiacetic acid, and 4) charging with nickel ions to form chelate groups. Structural properties and physicochemical parameters of nanoparticles were determined by FTIR, DLS, SEM, TEM, EDX, TGA and VSM analyzes. Recombinant protein subunits OPH and OmpW were expressed and purified as recombinant and their structure was confirmed. In this study, the genes encoding the recombinant proteins OPH and OmpW were cloned into the expression vector pet-26b (+) and expressed in E. coli BL21 (DE3) as a host and secreted into bacterial culture medium. Gene expression and purification were assessed by sodium dodecylsulfate-polyacrylamide gel (SDS-Page and Bradford test).

The results confirm that uniform and spherical magnetic polymer nanoparticles with high magnetization and superparamagnetic properties were successfully synthesized. According to the SEM images, the average diameter of the nanostructure was observed in the range of 24.56-62.19 nm. The formation of Fe3O4@SiO2@PVA/IDA-Ni2+ nanoparticles was confirmed by the peaks identified at 1405 and 1600 Cm-1 corresponding to the symmetric and asymmetric stretching vibration of the O=C−O− group and the removal of the peak at 1740 Cm-1. These microspheres showed a high degree of recovery of protein bands regardless of the nature of the protein. OmpW and OPH proteins as a His-tagged recombinant protein were expressed and purified using the final nanoparticles.

According to the SDS-PAGE results, there was a high degree of resolution in protein separation. The synthesized nanoparticles have a high protein binding capacity of about 1.25 mg of protein per mg of nanoparticles.