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
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.