Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system
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Published:2023
Issue:4
Volume:68
Page:473-487
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ISSN:1450-8109
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Container-title:Journal of Agricultural Sciences, Belgrade
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language:en
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Short-container-title:J AGR SCI BELGRADE
Author:
Soleimani Faranak1, Motallebi Mostafa1, Zamani Mohammadreza1, Jourabchi Esmat1
Affiliation:
1. Plant Molecular Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract
Chimeric chitinase42 (Chit42 containing ChBD) has great potential as a
candidate for digesting and recycling chitin as a beneficial nutrient, which
can be produced in bioreactors. The plant is one of the most efficient
bioreactors that can produce the eukaryotic proteins in active forms. With
the plant hairy root system, it is possible to express a variety of
recombinant proteins cost-effectively, easily, and quickly. Due to the huge
amount of proteins in plants, protein purification can be facilitated by the
use of the His-tag. In this research, different computer programs were used
for the three-dimensional structural analysis of Chimeric chitinase42
containing His-tag. The results showed that these comparative modeling
approaches had a remarkable degree of accuracy in predicting the fused
protein structure. The Z-score of -9.38 and -3.64 predicted for Chit42 and
ChBD by ProSA represents the good quality of the model. In addition,
bioinformatic observations showed that the His-tag was exposed and can be
used to purify the Chimeric chitinase42. The Chimeric chitinase42 containing
a His-tag was expressed in Nicotiana tabacum hairy roots, and the role of
the His-tag in the detection by Western blot and purification using a Ni-NTA
column was investigated. The presence of the Chimeric chitinase42 was
confirmed by analyzing root extracts using SDS-PAGE and Western blot. The
purification step was achieved using the His-tag and the Ni-NTA column. The
plant-derived Chimeric chitinase42 was confirmed to be biologically active
by measuring the chitinase activity of the purified protein on a media plate
containing colloidal chitin.
Publisher
National Library of Serbia
Subject
Plant Science,Soil Science,Agronomy and Crop Science,Animal Science and Zoology
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