title: Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production.
abstract: To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.
authors.affiliation: Division of Cellular, Molecular and Microbial Biology, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Sau-Ching
authors.initials: SC
authors.lastname: Wu
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Yeung
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Duan
authors.forename: Ruiqiong
authors.initials: R
authors.lastname: Ye
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Szarka
authors.forename: Hamid R
authors.initials: HR
authors.lastname: Habibi
authors.forename: Sui-Lam
authors.initials: SL
authors.lastname: Wong
mesh_terms: D000918:Antibody Specificity; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001709:Biotechnology; D002473:Cell Wall / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005337:Fibrin / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007128:Immunoglobulin Fragments; D018832:Molecular Chaperones; D005337:Fibrin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C104015:WprA protein, Bacillus subtilis