Functional expression of a Drosophila antifungal peptide in Escherichia coli.
Drosomycin is a key effector molecule involved in Drosophila innate immunity against fungal infection. This peptide is composed of 44 residues stabilized by four disulfide bridges. As the first step towards the understanding of the molecular basis for its specific antifungal activity, rapid and efficient production of the wild-type peptide and its mutants is needed. Here, we report a pGEX system for high-level expression of recombinant Drosomycin. The fusion Drosomycin protein with a carrier of Glutathione S-transferase (GST) was initially purified by affinity chromatography followed by Enterokinase cleavage. The digested product was separated by gel filtration and reverse phase HPLC. Mass spectrometry and circular dichroism spectroscopy analysis revealed that the recombinant peptide has identical molecular weight and correct structural conformation to native Drosomycin. Classical inhibition assay showed clear antifungal activity against Neurospora crassa with the IC(50) of 1.0muM. Successful expression of the CSalphabeta-type antifungal peptide in E. coli offers a basis for further studying its functional surface by alanine scanning mutagenesis strategy. Also, our work should be helpful in developing this peptide to an antifungal drug.
Yuan
Yuzhe
Y
Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, PR China.
Gao
Bin
B
Zhu
Shunyi
S
D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D004330:Drosophila / Q000737:chemistry*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000935:Antifungal Agents; D029721:Drosophila Proteins; D011994:Recombinant Proteins; C091227:DRS protein, Drosophila