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Rapid, High‐Yield Recovery of a Recombinant Digoxin Binding Single Chain Fv from Escherichia coli
Author(s) -
Burks Elizabeth A.,
Iverson Brent L.
Publication year - 1995
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp00031a017
Subject(s) - escherichia coli , urea , chemistry , chromatography , recombinant dna , fraction (chemistry) , yield (engineering) , chelation , affinity chromatography , inclusion bodies , biochemistry , enzyme , gene , organic chemistry , materials science , metallurgy
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.