Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones.
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.
Wu
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SC
Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Ye
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Wu
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XC
Ng
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Wong
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D000937:Antigen-Antibody Reactions; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D018835:Chaperonin 10 / Q000096:biosynthesis / Q000235:genetics; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics; D018833:Chaperonins; D004268:DNA-Binding Proteins; D004077:Digoxin / Q000276:immunology*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D008074:Lipoproteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D012097:Repressor Proteins; C082160:prsA protein, bacteria; D004077:Digoxin; D018833:Chaperonins; C063842:dnaK protein, E coli