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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bgbsjsne

Authors: Hossein Tarrahimofrad, Amir Meimandipour, Sareh Arjmand, Mohammadtaghi Beigi Nassiri, Ehsan Jahangirian, Hossein Tavana, Javad Zamani, Somayyeh Rahimnahal, Saeed Aminzadeh

Summary: After the cultivation period, cells were collected by centrifugation at 3500 × g for 30 min at 4 °C and resuspended in 4 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole and 0.05% Tween 20 at a pH of 7). The mixture was sonicated with 4 × 40 sec pulses followed by 20 sec rest between cycles at 4 °C. The crude extracts were then centrifuged at 9000 × g for 30 min at 4 °C, and the resulting supernatant was heat-treated in a hot water bath at 90 °C for 15 min. Insoluble material was separated by centrifugation at 13000 × g for 10 min, and the supernatant, containing the enzyme, was analyzed on a 12% SDS–polyacrylamide. The purified elution fractions were dialysed overnight in a 50 mM potassium-phosphate buffer (pH=7) at 4 °C.

Associated Publications: Tarrahimofrad H, Meimandipour A, Arjmand S, Nassiri MB, Jahangirian E, Tavana H, Zamani J, Rahimnahal S, Aminzadeh S (2020) Structural and biochemical characterization of a novel thermophilic Coh01147 protease. PLoS ONE 15(6): e0234958. doi: 10.1371/journal.pone.0234958

Affiliations: Bioprocess engineering group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Bioprocess engineering group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran, Department of Animal Science and Food Technology, Agriculture Science and Natural Resources University Khouzestan, Ahwaz, Iran, Bioprocess engineering group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Department of Biomedical Engineering, The University of Akron, Akron, OH 44325, USA, Bioprocess engineering group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Bioprocess engineering group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Bioprocess engineering group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

External URL: https://doi.org/10.1371/journal.pone.0234958

Version: 1

Publication Date: 2020

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