This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, S.-C.
Right arrow Articles by Wong, S.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, S.-C.
Right arrow Articles by Wong, S.-L.
Agricola
Right arrow Articles by Wu, S.-C.
Right arrow Articles by Wong, S.-L.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, July 2002, p. 3261-3269, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3261-3269.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Sau-Ching Wu,1 Jonathan C. Yeung,1 Yanjun Duan,1 Ruiqiong Ye,1 Steven J. Szarka,1,{dagger} Hamid R. Habibi,2 and Sui-Lam Wong1*

Division of Cellular, Molecular and Microbial Biology,1 Division of Zoology, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada2

Received 29 November 2001/ Accepted 9 April 2002

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.

* Corresponding author. Mailing address: Sui-Lam Wong, Department of Biological Sciences, University of Calgary, 2500 University Dr., N.W., Calgary, Alberta T2N 1N4, Canada. Phone: (403) 220-5721. Fax: (403) 289-9311. E-mail: slwong{at}ucalgary.ca.

{dagger} Present address: SemBioSys Genetics, Calgary, Alberta T1Y 7L3, Canada.

Applied and Environmental Microbiology, July 2002, p. 3261-3269, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3261-3269.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Berger, E., du Plessis, E., Gerber, I., Crampton, M., Nxumalo, N., Louw, M. (2009). Impact of Inactivated Extracellular Proteases on the Modified Flagellin Type III Secretion Pathway of Bacillus halodurans. Appl. Environ. Microbiol. 75: 271-274 [Abstract] [Full Text]  
  • Matsuoka, S., Yukawa, H., Inui, M., Doi, R. H. (2007). Synergistic Interaction of Clostridium cellulovorans Cellulosomal Cellulases and HbpA. J. Bacteriol. 189: 7190-7194 [Abstract] [Full Text]  
  • Nijland, R., Heerlien, R., Hamoen, L. W., Kuipers, O. P. (2007). Changing a Single Amino Acid in Clostridium perfringens {beta}-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis. Appl. Environ. Microbiol. 73: 1586-1593 [Abstract] [Full Text]  
  • Arai, T., Matsuoka, S., Cho, H.-Y., Yukawa, H., Inui, M., Wong, S.-L., Doi, R. H. (2007). Inaugural Article: Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation. Proc. Natl. Acad. Sci. USA 104: 1456-1460 [Abstract] [Full Text]  
  • Zhang, X.-Z., Cui, Z.-L., Hong, Q., Li, S.-P. (2005). High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800. Appl. Environ. Microbiol. 71: 4101-4103 [Abstract] [Full Text]  
  • Westers, H., Braun, P. G., Westers, L., Antelmann, H., Hecker, M., Jongbloed, J. D. H., Yoshikawa, H., Tanaka, T., van Dijl, J. M., Quax, W. J. (2005). Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis. Appl. Environ. Microbiol. 71: 1899-1908 [Abstract] [Full Text]  
  • Brans, A., Filee, P., Chevigne, A., Claessens, A., Joris, B. (2004). New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers. Appl. Environ. Microbiol. 70: 7241-7250 [Abstract] [Full Text]  
  • Cho, H.-Y., Yukawa, H., Inui, M., Doi, R. H., Wong, S.-L. (2004). Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Appl. Environ. Microbiol. 70: 5704-5707 [Abstract] [Full Text]  
  • Tjalsma, H., Antelmann, H., Jongbloed, J. D.H., Braun, P. G., Darmon, E., Dorenbos, R., Dubois, J.-Y. F., Westers, H., Zanen, G., Quax, W. J., Kuipers, O. P., Bron, S., Hecker, M., van Dijl, J. M. (2004). Proteomics of Protein Secretion by Bacillus subtilis: Separating the "Secrets" of the Secretome. Microbiol. Mol. Biol. Rev. 68: 207-233 [Abstract] [Full Text]  
  • Lian, Q., Szarka, S. J., Ng, K. K. S., Wong, S.-L. (2003). Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots. J. Biol. Chem. 278: 26677-26686 [Abstract] [Full Text]  
  • Wu, S.-C., Castellino, F. J., Wong, S.-L. (2003). A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy. J. Biol. Chem. 278: 18199-18206 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»