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.

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.