Immobilized metal affinity cryogel-based high-throughput platform for screening bioprocess and chromatographic parameters of His sub(6)-GTPase

Among various tools of product monitoring, chromatography is of vital importance as it also extends to the purification of product. Immobilized metal affinity cryogel (Cu(II)-iminodiacetic acid- and Ni(II)-nitrilotriacetic acid-polyacrylamide) minicolumns (diameter 8 mm, height 4 mm, void volume 250 mu l) were inserted in open-ended 96-well plate and different chromatographic parameters and bioprocess conditions were analysed. The platform was first validated with lysozyme. Optimum binding of lysozyme (90%) was achieved when 50 mu g of protein in 20 mM Tris, pH 8.0 was applied to the minicolumns with maximum recovery (90%) upon elution with 300 mM imidazole. Thereafter, the platform was screened for chromatographic conditions of His sub(6)-GTPase. Since cryogels have large pore size, they can easily process non-clarified samples containing debris and particulate matters. The bound enzymes on the gel retain its activity and therefore can be assayed on-column by adding substrate and then displacing the product. Highest binding of His sub(6)-GTPase was achieved when 50 mu l of non-clarified cell lysate was applied to the cryogel and subsequently washed with 50 mM Tris, 150 mM NaCl, 5 mM MgCl sub(2), 10 mM imidazole, pH 8.0 with dynamic and static binding capacities of 1.5 and 3 activity units. Maximum recovery was obtained upon elution with 300 mM imidazole with a purification fold of 10; the purity was also analysed by SDS-PAGE. The platform showed reproducible results which were validated by Bland-Altman plot. The minicolumn was also scaled up for chromatographic capture and recovery of His sub(6)-GTPase. The bioprocess conditions were monitored which displayed that optimum production of His sub(6)-GTPase was attained by induction with 200 mu M isopropyl- beta -D-thiogalactoside at 25 degree C for 12 h. It was concluded that immobilized metal affinity cryogel-based platform can be successfully used as a high-throughput platform for screening of bioprocess and chromatographic parameters. [Figure not available: see fulltext.]