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Recombinant β-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv
R L Anisimov1, O A Ershova1, A V Ershov1
1Generium LLC International Biotechnology Center, Volginsky, Vladimirskaya oblast, 601125, Russia.
Protein Expression and Purification
|January 26, 2020
Summary
Researchers developed highly stable antibodies for β-Glucocerebrosidase purification. These antibodies enable efficient recovery of active enzyme from cell culture media, offering a promising tool for bioprocessing and therapeutic applications.
Area of Science:
- Biochemistry
- Immunology
- Biotechnology
Background:
- β-Glucocerebrosidase is crucial for cellular function.
- Efficient purification methods are needed for therapeutic and research applications.
- Phage display technology offers a powerful platform for antibody discovery.
Purpose of the Study:
- To develop novel antibodies specific to human β-Glucocerebrosidase.
- To utilize these antibodies for the purification of active β-Glucocerebrosidase.
- To assess the efficacy of antibody-based affinity chromatography for enzyme recovery.
Main Methods:
- Selection of single-chain variable fragment (scFv) antibodies using phage display against immobilized β-Glucocerebrosidase.
- Conversion of scFv binders to full-size human IgG1 antibodies.
- Application of generated antibodies as ligands in affinity chromatography for enzyme purification.
- Characterization of purified enzyme activity and sorbent properties.
Main Results:
- Identification of highly stable antibodies with dissociation constants (Kd) in the 10-40 nM range.
- Demonstration of efficient and selective recovery of biologically active β-Glucocerebrosidase from Chinese hamster ovary (CHO) cell culture media.
- Purification of β-Glucocerebrosidase to a near-homogeneous state with specific activity comparable to commercial preparations (40-44 U/mg protein).
- Development of high-capacity, easily regenerable immunoaffinity sorbents.
Conclusions:
- Novel, high-affinity antibodies against β-Glucocerebrosidase were successfully generated.
- Antibody-based affinity chromatography provides an effective method for purifying active β-Glucocerebrosidase.
- The developed immunoaffinity sorbents show promise for bioprocessing and therapeutic enzyme production.
