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 fro m Bacillus subtilis, To address this problem, three new strains with e nhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones i n an appropriate ratio without any heat shock treatment. This strain r educed the formation of insoluble SCA by 45% and increased the secreto ry production yield by 60%, The second strain, WB600B[pEPP], overprodu ces an extracytoplasmic molecular chaperone, PrsA, An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], cop roduces 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 secrete d by this strain were biologically active and showed affinity to digox in comparable to the affinity of those secreted by strains without ove rproduction 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 intermediat es in the process of achieving their final configuration.