title: Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.
abstract: 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®) 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® 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.
  
  
  CONCLUSIONS
  There 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.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Matsuda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Itaya
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kitahara
authors.forename: Natalia Maria
authors.initials: NM
authors.lastname: Theresia
authors.forename: Ekaterina Aleksandrovna
authors.initials: EA
authors.lastname: Kutukova
authors.forename: Yurgis Antanas Vladovich
authors.initials: YA
authors.lastname: Yomantas
authors.forename: Masayo
authors.initials: M
authors.lastname: Date
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Kikuchi
authors.affiliation: Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. mwachi@bio.titech.ac.jp.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Wachi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D046928:Peptidoglycan Glycosyltransferase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D018719:Erb-b2 Receptor Tyrosine Kinases; C077188:cold-shock protein CspB, Bacteria; C575488:penicillin-binding protein 1A, E coli; C508053:ERBB2 protein, human
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