title: Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.
abstract: Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein.
authors.affiliation: Department of Pharmacology, Institutes for Advanced Studies in Multidisciplinary Science and Technology, Shanghai 200062, PR China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Bu
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Zhou
authors.forename: Jian
authors.initials: J
authors.lastname: Tang
authors.forename: Fang
authors.initials: F
authors.lastname: Jing
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D020097:Natriuretic Peptide, Brain / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D020097:Natriuretic Peptide, Brain
keywords: BNP; Inclusion bodies; Protein expression; Refolding; scFv Antibody