title: Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy.
abstract: Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.
authors.affiliation: Celiac Sprue Research Foundation, P.O. Box 61193, Palo Alto, California 94306-1193, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Gass
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ehren
authors.forename: Gregg
authors.initials: G
authors.lastname: Strohmeier
authors.forename: Indu
authors.initials: I
authors.lastname: Isaacs
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D002446:Celiac Disease / Q000188:drug therapy* / Q000201:enzymology; D002626:Chemistry, Pharmaceutical; D045247:Chryseobacterium / Q000201:enzymology; D004063:Digestion / Q000187:drug effects; D004795:Enzyme Stability; D005285:Fermentation; D005983:Glutens / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D016941:Myxococcus xanthus / Q000201:enzymology*; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000494:pharmacology / Q000627:therapeutic use*; D013379:Substrate Specificity; D013608:Tablets, Enteric-Coated
chemical_list: D013608:Tablets, Enteric-Coated; D005983:Glutens; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases