Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

Microbial Cell Factories

Research

Ronan Crepin¹  Aurelie Schneider¹  Anne Beugnet¹  Selma Djender¹  Klervi Even Desrumeaux¹  Chiara Romani²  Ario de Marco³  Franck Perez⁴  Sandrine Moutel⁵ 

[1] Tumor Target and Therapeutic Antibody - Identification Platform (TAb-IP), Institut Curie, 3-5 Impasse Reille, 75014, Paris, France;Tumor Target and Therapeutic Antibody - Identification Platform (TAb-IP), Institut Curie, 3-5 Impasse Reille, 75014, Paris, France;Division of Gynecologic Oncology, Angelo Nocivelli Institute of Molecular Medicine, University of Brescia, Brescia, Italy;Tumor Target and Therapeutic Antibody - Identification Platform (TAb-IP), Institut Curie, 3-5 Impasse Reille, 75014, Paris, France;SIRIC INCa-DGOS-4654, Paris, France;CIC IGR Curie, 1428, Paris, France;Department of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9, SI-5261, Vipava, Slovenia;UMR144, Institut Curie, 12 Lohmond, 75005, Paris, France;UMR144, Institut Curie, 12 Lohmond, 75005, Paris, France;Translational Research Department, Institut Curie, 26 rue d'Ulm, Cedex 05, F75248, Paris, France;

关键词: Affinity purification;    Avidity effect;    Disulfide bonds;    Immune diagnostics;    Single-chain antibodies;    Single-domain antibodies;    Sortase;    Sulfhydryl oxidase;   

DOI  :  10.1186/s12934-014-0140-1

 received in 2014-07-10, accepted in 2014-09-08,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space.ResultsWe demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF).ConclusionsThe collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.

【 授权许可】

Unknown   
© Djender 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/4.0), 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.