Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Autor: Patil AB; Plant Virology Lab, Jain R&D, Jain Hills, Jain Irrigation Systems Limited, Shirsoli Road, Post Box No # 72, Jalgaon, 425001, Maharashtra State, India. Electronic address: atulpatil9096@gmail.com., Dalvi VS; Plant Virology Lab, Jain R&D, Jain Hills, Jain Irrigation Systems Limited, Shirsoli Road, Post Box No # 72, Jalgaon, 425001, Maharashtra State, India. Electronic address: virology1@jains.com., Azeez A; College of Forest Resources and Environmental Science, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, 906-487-1885, USA. Electronic address: aazeez@mtu.edu., Krishna B; Plant Molecular Biology Lab, Jain R&D, Jain Hills, Jain Irrigation Systems Limited, Shirsoli Road, Post Box No# 72, Jalgaon, 425001, Maharashtra State, India. Electronic address: balkrishna.yadav@jains.com., Mishra AA; Plant Virology Lab, Jain R&D, Jain Hills, Jain Irrigation Systems Limited, Shirsoli Road, Post Box No # 72, Jalgaon, 425001, Maharashtra State, India. Electronic address: dr.mishra.akhilesh@jains.com., Sane PV; Plant Virology Lab, Jain R&D, Jain Hills, Jain Irrigation Systems Limited, Shirsoli Road, Post Box No # 72, Jalgaon, 425001, Maharashtra State, India. Electronic address: rajsane@hotmail.com.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2021 Apr; Vol. 180, pp. 105811. Date of Electronic Publication: 2020 Dec 18.
DOI: 10.1016/j.pep.2020.105811
Abstrakt: Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl β-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.
(Copyright © 2020 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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