title: Rapid, high-yield recovery of a recombinant digoxin binding single chain Fv from Escherichia coli.
abstract: We have isolated milligram quantities of active single chain antibody from the insoluble fraction of Escherichia coli cultures. The system relies on high-level expression from a T7 RNA polymerase-directed gene construct, 8 M urea to dissolve the desired protein out of the insoluble fraction, presumably inclusion bodies, isolation and concentration of the desired protein by nickel chelate [IDA-Ni(II)] immobilized metal-ion affinity chromatography (IMAC), and removal of urea from column fractions by dialysis directly into storage buffer. Routinely, about 50% of the protein loaded onto an IMAC column is recovered as single chain Fv at a concentration of approximately 0.7 mg/mL. As little as 3 days are required to obtain 10 mg of final product when starting with an overnight inoculum.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
authors.forename: E A
authors.initials: EA
authors.lastname: Burks
authors.forename: B L
authors.initials: BL
authors.lastname: Iverson
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004077:Digoxin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017931:DNA Primers; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D004077:Digoxin