title: 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 8 mm, height 4 mm, void volume 250 μl) 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 μg 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 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 50 μl of non-clarified cell lysate was applied to the cryogel and subsequently washed with 50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 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 His6-GTPase. The bioprocess conditions were monitored which displayed that optimum production of His6-GTPase was attained by induction with 200 μM isopropyl-β-D-thiogalactoside at 25 °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. 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.
authors.affiliation: Department of Biological Sciences and Bioengineering and Centre for Environmental Sciences and Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, Uttar Pradesh, India.
authors.forename: Joyita
authors.initials: J
authors.lastname: Sarkar
authors.affiliation: Department of Biological Sciences and Bioengineering and Centre for Environmental Sciences and Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, Uttar Pradesh, India. ashokkum@iitk.ac.in.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
mesh_terms: D000327:Adsorption; D000345:Affinity Labels / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D059985:Cryogels / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000737:chemistry*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006639:Histidine / Q000032:analysis / Q000737:chemistry*; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000345:Affinity Labels; D059985:Cryogels; D004800:Enzymes, Immobilized; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D006639:Histidine; D003300:Copper; D020558:GTP Phosphohydrolases
keywords: Bioprocess; Cryogel; High-throughput screening; His6-GTPase; Immobilized metal affinity chromatography; Lysozyme