Rapid refolding and polishing of single-chain antibodies from Escherichia coli inclusion bodies.
An inexpensive and fast-folding strategy for single-chain antibody (scFv) recovered from Escherichia coli inclusion bodies has been developed. Two anti-fluorescein single-chain antibodies, 4-4-20 and 4M5.3, were expressed as inclusion bodies in E. coli for use in a comparative refolding study. Active protein yields as well as degree of aggregation were evaluated for scFv produced by stepwise dialysis, redox dialysis, and a newly developed controlled dilution and filtration strategy. Although all three methods produced active protein for both 4-4-20 and 4M5.3, the extent of aggregation differed greatly among the methods. For 4-4-20, the controlled dilution and filtration strategy reduced aggregation by half, allowed batch processing times of 8h (an 18-fold improvement), and significantly reduced denaturant usage while increasing active yields by 150%. A hydroxyapatite resin polishing step was used to remove completely the aggregate species and inactive monomeric scFv from active scFv.
Sinacola
Jessica R
JR
Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, Delaware 19716, USA.
Robinson
Anne S
AS
D004926:Escherichia coli / Q000276:immunology*; D002479:Inclusion Bodies / Q000378:metabolism*; D017510:Protein Folding