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Downstream processing of Cry4AaCter-induced inclusion bodies containing insect-derived antimicrobial peptides produced in Escherichia coli.

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The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to… Click to show full abstract

The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to create a platform for the production of antimicrobial peptides (AMPs) in Escherichia coli featuring a uniform resolubilization process independent of the peptide fused to the pull-down tag. The Cry4AaCter tag conserves the bioactivity of fusion proteins and thus allows the purification of simple AMPs and more complex AMPs stabilized by disulfide bonds. We developed a downstream process (DSP) for the purification of IBs containing the mutated Galleria mellonella insect metalloprotease inhibitor IMPI(I38V), which has a globular structure stabilized by five disulfide bonds. IMPI(I38V) is a potent inhibitor of the M4 metalloproteases used as virulence factors by several human pathogens. We used a single crossflow filtration for the washing and resolubilization of the Cry4AaCter-induced IBs and obtained bioactive IMPI(I38V) after tag removal. We achieved a 68-fold higher protein yield using our IB system compared to an alternative DSP approach in which a GST-fusion strategy was used to produce soluble IMPI(I38V). The Cry4AaCter-based process was transferable to gloverin (another G. mellonella AMP) and the visible marker green fluorescent protein, which accumulated in fluorescent IBs, confirming it is a broadly applicable strategy for the recovery of functional proteins.

Keywords: inclusion bodies; escherichia coli; impi i38v; antimicrobial peptides; tag; cry4aacter

Journal Title: Protein expression and purification
Year Published: 2019

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