title: High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli.
abstract: BACKGROUND
  For their application in the area of diagnosis and therapy, single-domain antibodies (sdAbs) offer multiple advantages over conventional antibodies and fragments thereof in terms of size, stability, solubility, immunogenicity, production costs as well as tumor uptake and blood clearance. Thus, sdAbs have been identified as valuable next-generation targeting moieties for molecular imaging and drug delivery in the past years. Since these probes are much less complex than conventional antibody fragments, bacterial expression represents a facile method in order to produce sdAbs in large amounts as soluble and functional proteins.
  
  
  RESULTS
  By the combined use of high cell density cultivation media with a genetically engineered E. coli mutant strain designed for the cytoplasmic formation of proper disulfide bonds, we achieved high level of intracellular sdAb production (up to 200 mg/L). Due to a carboxyterminal hexahistidine epitope, the soluble recombinant sdAbs could be purified by one-step immobilized metal affinity chromatography to apparent homogeneity and easily radiolabeled with 99mTc within 1 h. The intradomain disulfide bridge being critical for the stability and functionality of the sdAb molecule was shown to be properly formed in ~96% of the purified proteins. In vitro binding studies confirmed the high affinity and specificity of the expressed sdAb 7C12 towards its molecular target.
  
  
  CONCLUSIONS
  Our study demonstrates an efficient cultivation and expression strategy for the production of substantial amounts of soluble and functional sdAbs, which may be adopted for high-yield production of other more complex proteins with multiple disulfides as well.
authors.affiliation: Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstraße 400, 01328 Dresden, Germany. k.zarschler@hzdr.de.
authors.forename: Kristof
authors.initials: K
authors.lastname: Zarschler
authors.forename: Stefanie
authors.initials: S
authors.lastname: Witecy
authors.forename: Franz
authors.initials: F
authors.lastname: Kapplusch
authors.forename: Christian
authors.initials: C
authors.lastname: Foerster
authors.forename: Holger
authors.initials: H
authors.lastname: Stephan
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D061905:Single-Domain Antibodies / Q000235:genetics / Q000378:metabolism*
chemical_list: D061905:Single-Domain Antibodies