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

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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.

Capture and on-column analysis of bound enzyme from non-clarified cell lysate on immobilized metal affinity cryogel minicolumn-based high-throughput platform

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Acknowledgements

JS would like to acknowledge Council for Scientific and Industrial Research, India, for providing research fellowship. AK would like to acknowledge TATA Innovation fellowship from the Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India.

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  1. Department of Biological Sciences and Bioengineering and Centre for Environmental Sciences and Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, Uttar Pradesh, India

    Joyita Sarkar & Ashok Kumar

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  1. Joyita Sarkar
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Correspondence to Ashok Kumar.

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Sarkar, J., Kumar, A. Immobilized metal affinity cryogel-based high-throughput platform for screening bioprocess and chromatographic parameters of His6-GTPase. Anal Bioanal Chem 409, 2951–2965 (2017). https://doi.org/10.1007/s00216-017-0242-9

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  • DOI: https://doi.org/10.1007/s00216-017-0242-9

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