STRUCTURAL MODELING, EXPRESSION AND PURIFICATION OF CHIMERIC CHITINASE42 CONTAINING HIS-TAG IN NICOTIANA TABACUM HAIRY ROOT SYSTEM:

Mostafa Motallebi; Faranak   Soleimani; Mohammadreza  Zamani; Esmat  Jourabchi

Mostafa Motallebi Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology
Faranak Soleimani National Institute of Genetic Engineering and Biotechnology
Mohammadreza Zamani National Institute of Genetic Engineering and Biotechnology https://orcid.org/0000-0002-7165-2343
Esmat Jourabchi National Institute of Genetic Engineering and Biotechnology https://orcid.org/0000-0003-3439-0863

Keywords: chimeric chitinase42, His-tag, tobacco hairy root, structural modeling

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 efficient bioreactors which can produce the eukaryotic proteins in active forms. A suitable platform for easy, cost-effective, and fast expression of different recombinant proteins is provided by the plant hairy roots system. Due to the huge amount of proteins in plants, His-tag can be used to facilitate the purification of recombinant proteins. In this research, different computer programs were used for the analysis of the three-dimensional structure of chimeric chitinase42 containing His-tag. The results showed that these comparative modelings have 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 represent the good quality of the model. Also, bioinformatics observations showed that His-tag was exposed and can be used to purify chimeric chitinase42. The chimeric chitinase42 containing His-tag was expressed in Nicotiana tabacum hairy roots, and the role of His-tag in the detection by western blot and purification using Ni-NTA column was investigated. The presence of chimeric chitinase42 was verified by SDS-PAGE and western blot analysis of root extracts. The purification step was achieved using His-tag and Ni-NTA column. The biological activity of the plant-derived chimeric chitinase42 was confirmed by investigating the chitinase activity of purified protein on the media plate containing the colloidal chitin.

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