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

 
: Hoffmann, D.; Eckhardt, D.; Gerlach, D.; Vilcinskas, A.; Czermak, P.

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Fulltext ()

Protein expression and purification 155 (2019), pp.120-129
ISSN: 1046-5928
Bundesministerium für Bildung und Forschung BMBF (Deutschland)
4-IN
English
Journal Article, Electronic Publication
Fraunhofer IME ()

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.

: http://publica.fraunhofer.de/documents/N-549371.html