Original scientific paper
https://doi.org/10.5513/JCEA01/17.4.1818
Optimalisation of expression conditions for production of round-leaf sundew chitinase (Drosera rotundifolia L.) in three E. coli expression strains
Miroslav RAJNINEC
; Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, x, 950 07 Nitra, Slovak Republic
Jana LIBANTOVÁ
; Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, x, 950 07 Nitra, Slovak Republic
Martin JOPČÍK
; Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, x, 950 07 Nitra, Slovak Republic
Abstract
Round-leaf sundew (Drosera rotundifolia L.), family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3)-RIPL, E. coli ArcticExpress (DE3)RIL and E. coli SHuffle® T7) for production and isolation of heterologous round-leaf sundew chitinase (DrChit). Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.
Keywords
Drosera rotundifolia L.; Escherichia coli expression; protein solubility
Hrčak ID:
170850
URI
Publication date:
16.12.2016.
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