Expression and Purification of the VpDef Defensin in Escherichia coli using the Small Metal-Binding Proteins CusF3H+ and SmbP.

Protein and Peptide Letters
Jorge M Montfort-GardeazabalXristo Zarate

Abstract

The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purif...Continue Reading

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