A scaleable manufacturing process for pro-EP-B2, a cysteine protease from barley indicated for celiac sprue.
Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that EP-B2 (endoprotease B, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-EP-B2 (zymogen form of EP-B2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-EP-B2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-EP-B2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.
Vora
Harmit
H
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
McIntire
James
J
Kumar
Pawan
P
Deshpande
Milind
M
Khosla
Chaitan
C
D002446:Celiac Disease / Q000188:drug therapy; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D004345:Drug Industry / Q000379:methods; D004355:Drug Stability; D004789:Enzyme Activation; D004795:Enzyme Stability; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016941:Myxococcus xanthus / Q000201:enzymology* / Q000235:genetics; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases