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Expression, purification and refolding of recombinant Cry1Ab/Ac obtained in Escherichia coli as inclusion bodies
Author(s) -
Cao Sishuo,
Xu Wentao,
Luo Yunbo,
He Xiaoyun,
Guo Xing,
Yuan Yanfang,
Yuan Changmei,
Huang Kunlun
Publication year - 2009
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.3515
Subject(s) - escherichia coli , inclusion bodies , biology , bacillus thuringiensis , western blot , affinity chromatography , recombinant dna , biochemistry , chromatography , genetically modified organism , protein purification , microbiology and biotechnology , gene , chemistry , bacteria , enzyme , genetics
BACKGROUND: The Cry toxins are already a useful alternative or supplement to synthetic chemical pesticide application in commercial agriculture and forest management. RESULTS: The Cry1ab/ac gene from Bacillus thuringiensis was cloned from the genome of genetically modified rice by polymerase chain reaction (PCR). Owing to the large number of Escherichia coli low‐usage codons in the Cry1ab/ac gene, the first 20 codons were optimised by PCR to improve the expression of the Cry1ab/ac gene in E. coli. The Cry1Ab/Ac protein was highly expressed in E. coli as inclusion bodies that could be dissolved in 8 mol L −1 urea and purified on a His Trap ™ FF crude column under denaturing conditions. The purified Cry1Ab/Ac protein was dialysed in refolding buffers to obtain a soluble and biologically active protein. To achieve better biological activity, the His‐tag was digested from the Cry1Ab/Ac protein with enterokinase, and the Cry1Ab/Ac protein was further purified by gel filtration on a fast performance liquid chromatography Superdex 75 HR 10/30 column using an AKTA purifier. The identity of the purified Cry1Ab/Ac protein sequence was confirmed by western blot and matrix‐assisted laser desorption ionisation time‐of‐flight mass spectrometry. The final purified Cry1Ab/Ac protein was 99.2% pure and retained its biological activity, as determined in a growth inhibition assay of Chilo suppressalis . CONCLUSION: The purified Cry1Ab/Ac protein could be used to evaluate the food safety of transgenic plants containing the Cry1ab/ac gene and to produce antibodies for immune‐based methods employed in the detection of genetically modified organisms containing the Cry1ab or Cry1ac gene. It might also serve as a new biological insecticide to reduce the use of broad‐spectrum insecticides. Copyright © 2009 Society of Chemical Industry