Expression of a cGMP compatible Lucilia sericata insect serine proteinase debridement enzyme

Biotechnol Prog. Mar-Apr 2012;28(2):567-72. doi: 10.1002/btpr.1516. Epub 2012 Feb 17.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Debridement / methods*
  • Diptera / enzymology*
  • Enzyme Stability
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression*
  • Humans
  • Insect Proteins / chemistry
  • Insect Proteins / genetics
  • Insect Proteins / isolation & purification
  • Insect Proteins / metabolism*
  • Molecular Sequence Data
  • Serine Proteases / chemistry
  • Serine Proteases / genetics
  • Serine Proteases / isolation & purification
  • Serine Proteases / metabolism*
  • Varicose Ulcer / metabolism
  • Varicose Ulcer / physiopathology
  • Varicose Ulcer / therapy*
  • Wound Healing

Substances

  • Insect Proteins
  • Serine Proteases