초록
<P><B>Background</B></P><P>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 <I>Corynebacterium glutamicum</I> protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production.</P><P><B>Results</B></P><P>The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional <I>C. glutamicum</I> 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 <I>pbp1a</I>, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this <I>Δpbp1a</I> mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the <I>cspB</I> gene encoding a surface (S)-layer protein, we evaluated the effect of <I>ΔcspB</I> mutation on Fab secretion from ATCC13869. The <I>Δpbp1a</I> mutation showed a positive effect on Fab secretion only in combination with the <I>ΔcspB</I> mutation. The <I>ΔcspBΔpbp1a</I> 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.</P><P><B>Conclusion</B></P><P>There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of <I>C. glutamicum</I>, the PG layer, and the S-layer. The <I>ΔcspBΔpbp1a</I> double mutant allows efficient Fab production using the CORYNEX® system.</P>