Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

Microbial Cell Factories

Research

Yurgis Antanas Vladovich Yomantas¹  Ekaterina Aleksandrovna Kutukova¹  Yuki Kitahara²  Natalia Maria Theresia²  Masaaki Wachi²  Yoshimi Kikuchi³  Hiroshi Itaya³  Masayo Date³  Yoshihiko Matsuda⁴ 

[1] Ajinomoto-Genetika Research Institute, 1st Dorozhny pr. 1, 113545, Moscow, Russia;Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8501, Midori-ku, Japan;Institute for Innovation, Ajinomoto Co, Inc, 1-1 Suzuki-cho, Kawasaki, 210-8681, Kawasaki-ku, Japan;Institute for Innovation, Ajinomoto Co, Inc, 1-1 Suzuki-cho, Kawasaki, 210-8681, Kawasaki-ku, Japan;Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8501, Midori-ku, Japan;

关键词: CORYNEX®;    Corynebacterium glutamicum;    Protein secretion;    Fab fragment;    CspB;    PBP1a;   

DOI  :  10.1186/1475-2859-13-56

 received in 2013-10-01, accepted in 2014-03-17,  发布年份 2014

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Matsuda et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.