RAPID, HIGH-YIELD RECOVERY OF A RECOMBINANT DIGOXIN BINDING SINGLE-CHAIN FV FROM ESCHERICHIA-COLI

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.