Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes and/or modulating host immunity HDPs have great therapeutic potential against antibiotic-resistant bacteria viruses and even parasites but there are substantial roadblocks to their therapeutic application High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis Conversely the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective Here we have developed methodologies appropriate for large-scale industrial production of HDPs in particular we describe (I) a method using fusions to SUMO for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (II) a simplified 2-step purification method appropriate for industrial use We have used this method to produce seven HDPs to date (IDR1 MX226 LL37 CRAMP HHC-10 E5 and E6) Using this technology pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides Together these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans (C) 2010 Elsevier Inc All rights reserved