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.
Burks
E A
EA
Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Iverson
B L
BL
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
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D004077:Digoxin