Abstract
BackgroundAmong 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®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production.ResultsThe Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® 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 Δpbp1a 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 ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a 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.ConclusionThere are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system.
Highlights
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 Immunoglobulin G (IgG)
Bacteria were transformed with the pPKStrastFabHL plasmid containing heavy chain (HC) and light chain (LC) genes of the fragment antigen-binding (Fab) region fused with the signal peptide derived from the cspA gene of C. ammoniagenes under the control of the cspB promoter of C. glutamicum (Figure 1 and Additional file 1: Figure S1)
The transformant was cultured in 4 ml of MMTG medium at 30°C for 96 h and the culture supernatant was analyzed by non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-human IgG(H+L) antibody
Summary
Recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Using a strong cspB promoter and signal peptides derived from a corynebacterial cell surface protein and the Escherichia coli twin-arginine translocation pathway, C. glutamicum ATCC13869 exhibited great potential as a host for industrial-scale production of recombinant proteins [18,19,20,21,22,23,24]. This protein expression system has been awarded trademark registration as CORYNEX®
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