Lin Dongqiang

Protein adsorption on DEAE ion‐exchange resins with different ligand densities and pore sizes

Ion exchange chromatography (IEC) is a common and powerful technique for the purification of proteins. The ligand density and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In the present work, the adsorption isotherms of bovine serum albumin (BSA) and human serum albumin (HSA) were investigated systematically with series of diethylaminoethyl (DEAE) ion-exchange resins, which have different ligand densities and pore sizes. The Langmuir equation was used to fit the experimental data and the influences of ligand density and pore size on the saturated adsorption capacity and the dissociation constant were discussed. The zeta potentials and hydrodynamic diameters of proteins at different pHs were also measured, and the surface charge characteristics of proteins and the adsorption mechanism were discussed. The results demonstrated that the ligand density, pore size, and protein properties affect the protein adsorption capacities in an integrative way. An integrative parameter was introduced to describe the complicated effects of ligand density and pore size on the protein adsorption. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.

Purification of Cytochrome P450 BM-3 as a Monooxygenase

After investigating two anion-exchange resins, the purification factor and activity yields of P450 BM-3 were higher with Resource Q than with DEAE-Sepharose FF. Screening of HIC media showed that Source 15ISO was the most suitable for purification of P450 BM-3. An effective isolation and purification procedure of P450 BM-3 was developed and included three steps: 35%-70% saturation (NH(4))(2)SO(4) precipitation, Source 15ISO hydrophobic interaction chromatograph and Sephacryl S-200 gel filtration chromatography. Using this protocol, the purification factor and P450 BM-3 activity recovery was 13.5 and 13.7%, respectively.