International Journal on Advanced Science, Engineering and Information Technology, Vol. 12 (2022) No. 2, pages: 719-725, DOI:10.18517/ijaseit.12.2.15022

Expression and Purification cry1Aa Gene

Enny Rimita Sembiring, Agus Rachmat, Wien Kusharyoto, Puspo Edi Giriwono, Satya Nugroho, Maggy Thenawidjaja Suhartono

Abstract

The cry gene encodes a crystal protein of Bacillus thuringiensis known as Cry toxin, which is toxic to major Lepidopteran insect pest. The effectiveness of Cry toxin against the rice yellow stem borer (YSB, Scirpophaga incertulas Wlk) has been reported. Transgenic Bacillus thuringiensis (Bt) rice has been developed by introducing the fusion gene cry1B::cry1Aa gene driven by the CaMV35S promoter to Javanica rice cv. Rojolele to improve its resistance against rice YSB. Resistance to YSB at a greenhouse and multi-site scales have been conducted and reported. Food and feed safety studies for the release and commercialization of genetically modified crops require a sufficient amount of purified protein sample. Therefore, a strategy to produce Cry1Aa protein in a short time with high productivity needs to be developed. In this study, the cry1Aa gene (1857 bp) was cloned into pRHA-SDM expression vector under the control of the pRHA promoter fused to a 6xHis tag on the C-terminal to produce pRHA-SDM-cry1Aa. The expression host used was Escherichia coli strain NiCo21, and protein purification was performed using IMAC Co2+ on the TALONĀ® matrix. The results showed that the recombinant Cry1Aa protein, approximately 69 kDa, was detected using Western blot performed using anti-rabbit Cry1Aa polyclonal antibody and His detector nickel-NTA conjugated with HRP reporter enzyme. Expression and purification protocols that have been developed can be used to produce Cry1Aa protein which can be utilized for further protein studies.

Keywords:

Bacillus thuringiensis; cloning; cry1Aa; expression; purification.

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