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Bioscience Methods
BM 2010, Vol.1, No.2 http://bm.sophiapublisher.com
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Research Article Open Access
Heterologous Expression and Purification of Cry1Ac22 Toxin from
Bacillus thuringiensis W015-1
Zhuo ming Liu 1,2* , Shenkui Liu 3* , Youzhi Li 1 , Xuanjun Fang 1,2,3
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
Corresponding author email: xuanjunfang@hitar.org; Authors
* These authors contributed equally to this work
Bioscience Methods 2010, Vol.1 No.2 DOI:10.5376/bm.2010.01.0002
Received: 1 Aug., 2010
Accepted: 24 Nov., 2010
Published: 27 Dec., 2010
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Liu et al., 2010, Heterologous Expression and Purification of Cry1Ac22 Toxin from Bacillus thuringiensis W015-1, Bioscience Methods (online), Vol.1 No.2
(DOI: 10.5376/bm.2010.01.0002)
Abstract 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 28℃.
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.
Keywords Bacillus thuringiensis W015-1, Cry1Ac22, Fusion protein, Heterologous expression, in vitro Purification, Larvacidal
assay
Background
Bacillus thuringiensis can generate insecticidal crystal
protein during sporulation. Since Schnep and
Whiteley isolated the first Bt insecticidal crystal
protein in 1981 (Schnepf et al., 1981), nearly five
hundred cry genes have been cloned in the world, with
the majority belonging to the Cry1 family of genes.
The Cry1A genes are listed in nine subfamilies
including cry1Aa、cry1Ab 、cry1Ac、cry1Ad、cry1Ae、
cry1Af、cry1Ag、cry1Ah、and cry1Ai. These contain a
total 93 hylotypes, while there are 34 genes in the
cry1Ac subfamily (Crickmore et al., 2011,
http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/ Bt/).
Cry1Ac insecticidal crystal protein was first found in
Bacillus thuringiensis spp Kurstaki strain HD-73,
which produces bipyramidal crystal proteins during
sporulation with molecular weights between 129 kD
to 138 kD. Bt HD-73 is recognized as an important
standard strain due to well documented researches.
The extensive application of transgenic plants with the
Cry1Ac gene for lepidopteran control has lead to
acquired resistance in Lepidoptera to the Cry1Ac
crystal protein and has attracted global attention.
Therefore, it becomes a more urgent task to look for
novel Bt strains and diverse insecticidal crystal
proteins to avoid the development resistance.
Bt W015-1 strains were isolated from the guts of
diapausing silkworm (Bombyx mori) by Haide
Institute of Tropical Agricultural Resources (HITAR),