BACKGROUND: Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX(R)) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production.RESULTS: The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX(R) system using the conventional C. glutamicum strain YDK010, but... More
BACKGROUND: Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX(R)) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production.RESULTS: The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX(R) system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Deltapbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of DeltacspB mutation on Fab secretion from ATCC13869. The Deltapbp1a mutation showed a positive effect on Fab secretion only in combination with the DeltacspB mutation. The DeltacspBDeltapbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion.
