title: Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  We 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).
  
  
  CONCLUSIONS
  The 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.
authors.forename: Selma
authors.initials: S
authors.lastname: Djender
authors.forename: Aurelie
authors.initials: A
authors.lastname: Schneider
authors.forename: Anne
authors.initials: A
authors.lastname: Beugnet
authors.forename: Ronan
authors.initials: R
authors.lastname: Crepin
authors.forename: Klervi Even
authors.initials: KE
authors.lastname: Desrumeaux
authors.forename: Chiara
authors.initials: C
authors.lastname: Romani
authors.forename: Sandrine
authors.initials: S
authors.lastname: Moutel
authors.forename: Franck
authors.initials: F
authors.lastname: Perez
authors.forename: Ario
authors.initials: A
authors.lastname: de Marco
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D002162:Camelus / Q000378:metabolism*; D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D051379:Mice; D019897:Periplasm / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015203:Reproducibility of Results; D057127:Single-Chain Antibodies / Q000096:biosynthesis*; D023041:Xenograft Model Antitumor Assays
chemical_list: D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies