Cloning，Expression and Purification of FK506 Binding Protein Gene from Helicoverpa armigera

Abstract

Abstract: In order to better understand the role of FK506-binding protein gene and its regulatory mechanism in the CYP6B6 expression of Helicoverpa armigera, the FK506-binding protein cDNA sequence from midgut of H. armigera by RT-PCR was cloned on the basis of result of yeast one-hybrid. The fragment digested by double enzymes was linked to a prokaryotic expression vector pET32a to construct the recombinant expression plasmid pET32a-FK506BP, and then converted into competent Escherichia coli BL21 cells. The fusion protein was induced to express by isopropyl-3-D-thiogalactoside(IPTG), and purified by immobilized metal-chelating affinity chromatography(IMAC)using a Ni²⁺ matrix column. SDS-polyaerylamide gel electrophoresis(SDS-PAGE)and Western blotting analysis were used to examine the fusion protein. The results of sequencing and sequence analysis showed that the open reading frame of the FK506 binding protein gene was 327 bp, encoding 108 amino acid residues with the predicted molecular weight and isoelectric point of 11.78 kD and 7.87, respectively. The predicted protein had no signal peptide. The recombinant containing recombinant pET32a-FK506BP expressed a soluble protein after being induced by IPTG. SDS-PAGE and Western blot analysis indicated that the fusion protein, purified using Ni²⁺ affinity chromatography, had the predicted size and higher purity.

Key words: Helicoverpa armigera, FK506-binding protein, Yeast one-hybrid

Liu Xiaoning, Li Fen, Zhao Wenbo, Zhu Yan, Zhao Jie. Cloning，Expression and Purification of FK506 Binding Protein Gene from Helicoverpa armigera[J]. Biotechnology Bulletin, 2014, 0(4): 115-120.

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