High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2013-12

AUTHORS

Kristof Zarschler, Stefanie Witecy, Franz Kapplusch, Christian Foerster, Holger Stephan

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. More... »

PAGES

97

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1186/1475-2859-12-97

    DOI

    http://dx.doi.org/10.1186/1475-2859-12-97

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1043609199

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/24161153


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