Heterologous Expression and Purification of Cry1Ac22 Toxin from Bacillus thuringiensis W015-1
1. College of Life and Technology Science, Guangxi University, Nanning, 530004, China
2. Haide Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025, China
3. Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry Uinversity, Harbin, 150040, China
* These authors contributed equally to this work
Bioscience Methods, 2010, Vol. 1, No. 2 doi: 10.5376/bm.2010.01.0002
Received: 01 Oct., 2010 Accepted: 24 Nov., 2010 Published: 27 Dec., 2010
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Preferred citation for this article:
Liu et al., 2010, Expression and Purification of Bacillus thuringiensis Cry1Ac22 protein in Escherichia coli, Bioscience Methods, 2010, Vol. 1, No. 2
A cry1Ac22 gene was amplified by PCR from Bacillus thuringiensis strain W015-1 isolated from diapausing larvae of silkworm (Bombyx mori). The full-length gene was ligated into the prokaryotic expression vector pQE30 to construct the recombinant plasmid pQE30-Cry1Ac22. The pQE30-Cry1Ac22 was transformed into competent cell of E.coli host strain M15 and then induced by IPTG to express His-tag-Cry1Ac22 fusion protein. The results showed that the His-tag-Cry1Ac22 was highly heterologous expressed in the presence of inclusion bodies in E.coli cell. Inducing experiments with different IPTG concentrations and temperatures revealed that the optimum condition for the expression of the fusion protein was 1 mmol/L IPTG and 28â„ƒ. SDS-PAGE analysis demonstrated that the host with pQE30-Cry1Ac22 generated a 133 kD His-tag-Cry1Ac22 fusion protein. The His-tag-Cry1Ac22 fusion protein was purified with affinity chromatography on a Ni2+-NTA resin column. Larvacidal assays were performed and showed that the engineered bacterial lysate and purified protein exhibited high insecticidal activity against second instar larvae of Plutella xylostella. This study might provide a basis for the preparation of antibody and for the determination of insecticidal activity using heterologous Cry1Ac22 protein.
Bacillus thuringiensis W015-1; Cry1Ac22; Fusion protein; Heterologous expression; in vitro Purification; Larvacidal assay