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

[ Vol. 28 , Issue. 1 ]

Author(s):

Jorge M. Montfort-Gardeazabal, Pilar C. Morales-San Claudio, Nestor G. Casillas-Vega and Xristo Zarate*   Pages 108 - 114 ( 7 )

Abstract:


Background: 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.

Objectives: 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.

Methods: 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.

Results: 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 purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative.

Conclusion: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.

Keywords:

SmbP, CusF3H+, antimicrobial peptides, protein expression and purification, VpDef, Escherichia coli.

Affiliation:

Facultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, San Nicolas de los Garza, NL 66455, Facultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, San Nicolas de los Garza, NL 66455, Departamento de Patologia Clinica, Universidad Autonoma de Nuevo Leon, Hospital Dr. Jose Eleuterio Gonzalez, Monterrey, NL 64460, Facultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, San Nicolas de los Garza, NL 66455



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