Expression of a cGMP compatible Lucilia sericata insect serine proteinase debridement enzyme.
Previously, we demonstrated the effectiveness of a research grade recombinant chymotrypsin, derived from the larvae of Lucilia sericata, in "debriding" slough/eschar from venous leg ulcers ex vivo. Furthermore, we were able to formulate this enzyme for successful delivery to in vitro wound healing assays, from a prototype hydrogel wound dressing, and showed that enzyme delivered in this way could degrade wound tissue ex vivo. Recently, to progress biotechnological development of the enzyme as a potential therapeutic product, we explored expression using current good manufacturing practice (cGMP) guidelines, and now report that a recombinant chymotrypsin I zymogen from L. sericata can be expressed in the cGMP acceptable strain of Escherichia coli (BLR-DE3). In addition, the conditions required for purification, refolding and activation of the chymotrypsinogen have been determined. The activated enzyme was stable, and effective in digesting wound slough/eschar tissue. To summarise, we have successfully initiated the production and characterisation of a novel cGMP compatible product for use in future clinical trials.
Immune Modulation Research Group, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, United Kingdom. david.pritchard@nottingham.ac.uk
David I
DI
Pritchard
Gary
G
Telford
Miliza
M
Diab
Walter
W
Low
D000595:Amino Acid Sequence; D000818:Animals; D003646:Debridement / Q000379:methods*; D004175:Diptera / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014647:Varicose Ulcer / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D014945:Wound Healing
D019476:Insect Proteins; D057057:Serine Proteases