Preparation and characterization of scFv for affinity purification of reteplase.
The work was to explore the feasibility of protein affinity purification using ligand isolated from phage library. Reteplase was used as the model protein and a humanized semi-synthetic single chain fragment variable phage library as the source of ligand. After four rounds of biopanning, reteplase-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted to pET-29a and expressed in E. coli Rosseta. After purification by nickel-affinity and refolding, this scFv protein was proven to recognize reteplase specifically and sensitively in ELISA and dot-blotting. Its binding constant to reteplase was 1.84x10(-8) M, measured by surface plasmon resonance. After immobilized on Sepharose 4B, the scFv was used for the affinity purification of reteplase from milk. It was found that reteplase was highly purified from the starting material. In conclusion, it has been demonstrated that humanized scFv prepared with this approach could be used as a practical affinity ligand for efficient and cost-effective purification of reteplase, as well as other therapeutic proteins.
Guo
Zhigang
Z
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, China.
Bi
Feng
F
Tang
Yanchun
Y
Zhang
Jing
J
Yuan
Dawen
D
Xia
Zhinan
Z
Liu
Jian-Ning
JN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005240:Feasibility Studies; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D008024:Ligands; D008892:Milk / Q000201:enzymology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D010959:Tissue Plasminogen Activator / Q000276:immunology* / Q000302:isolation & purification
D007135:Immunoglobulin Variable Region; D008024:Ligands; D019151:Peptide Library; D011994:Recombinant Proteins; C087896:reteplase; D010959:Tissue Plasminogen Activator