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J Bacteriol, June 1998, p. 2830-2835, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Enhanced Secretory Production of a Single-Chain Antibody Fragment from Bacillus subtilis by Coproduction of Molecular Chaperones

Sau-Ching Wu,1 Ruiqiong Ye,1 Xu-Chu Wu,1 Shi-Chung Ng,2 and Sui-Lam Wong1,*

Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada,1 and Cancer Research, Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60064-35002

Received 7 January 1998/Accepted 8 March 1998

Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from Bacillus subtilis. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.

* Corresponding author. Mailing address: Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, 2500 University Dr., NW, Calgary, Alberta T2N 1N4, Canada. Phone: (403) 220-5721. Fax: (403) 289-9311. E-mail: slwong{at}acs.ucalgary.ca.

J Bacteriol, June 1998, p. 2830-2835, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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