Immobilized metal affinity cryogel-based high-throughput platform for screening bioprocess and chromatographic parameters of His6-GTPase.

Abstract

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 8mm, height 4mm, void volume 250μl) 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 50μg of protein in 20mM Tris, pH8.0 was applied to the minicolumns with maximum recovery (∼90%) upon elution with 300mM imidazole. Thereafter, the platform was screened for chromatographic conditions of His6-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 His6-GTPase was achieved when 50μl of non-clarified cell lysate was applied to the cryogel and subsequently washed with 50mM Tris, 150mM NaCl, 5mM MgCl2, 10mM imidazole, pH8.0 with dynamic and static binding capacities of ∼1.5 and 3 activity units. Maximum recovery was obtained upon elution with 300mM 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 His6-GTPase. The bioprocess conditions were monitored which displayed that optimum production of His6-GTPase was attained by induction with 200μM isopropyl-β-D-thiogalactoside at 25°C for 12h. 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. Graphical abstract Capture and on-column analysis of bound enzyme from non-clarified cell lysate on immobilized metal affinity cryogel minicolumn-based high-throughput platform.