title: Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.
abstract: Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes.
authors.affiliation: Department of Molecular Medicine, Unit of Immunology and General Pathology, Via Ferrata, 9, University of Pavia, 27100, Pavia, Italy. Electronic address: maristella.maggi01@universitadipavia.it.
authors.forename: Maristella
authors.initials: M
authors.lastname: Maggi
authors.affiliation: Department of Molecular Medicine, Unit of Immunology and General Pathology, Via Ferrata, 9, University of Pavia, 27100, Pavia, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Scotti
mesh_terms: D000818:Animals; D002161:Camelids, New World; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D007074:Immunoglobulin G; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Inclusion bodies; Protein expression; Purification optimization; Single domain antibody; VHH; sdAb