Background:
The focus of this study was to prepare and characterize the single-chain variable
fragment antibody (scFv)-coupled immunoaffinity column for purification of subtilisin BRC.
Methods:
The scFv against subtilisin BRC was immobilized onto CNBr-activated Sepharose 4B. Adsorption
isotherm for subtilisin BRC on scFv-BRC-coupled Sepharose 4B was obtained and calculated
the maximum binding capacity. The extraction conditions, including eluting solution, the concentration
of eluting solution and flow rate, were optimized. Under the optimized eluting conditions, the dynamic
binding capacity of the immunoaffinity column was determined.
Results:
The scFv-BRC-coupled Sepharose 4B for immunoaffinity purification of subtilisin BRC was
prepared. The coupling efficiency was about 78.4%, e.g. about 8 mg of scFv-BRC was covalently coupled
to 1 g CNBr-activated Sepharose 4B. The maximum equilibrium binding capacity (qm) and dissociation
constant (Kd) of the immunoaffinity column for subtilisin BRC were 3.01 mg/mL and 0.465
mg/mL, respectively. The immunoaffinity chromatography conditions were optimized and the subtilisin
BRC was purified 3.29-fold with 55.6%.
Conclusion:
The subtilisin BRC was effectively purified with high purity using scFv-BRC-coupled
Sepharose 4B and the dynamic binding capacity of the column was determined. These results suggested
that scFv-BRC can be used as a ligand for affinity purification of subtilisin BRC.