Related Experiment Videos
Processing of fusion proteins with immobilized factor Xa
Z Assouline1, R Graham, R C Miller
1Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Biotechnology Progress
|January 1, 1995
Summary
Modified factor Xa (FXa) with affinity tags remains active when immobilized. These FXa derivatives can efficiently remove affinity tags from fusion proteins without product contamination.
Area of Science:
- Biochemistry
- Protein Engineering
- Biotechnology
Background:
- Factor Xa (FXa) is a serine protease crucial in coagulation.
- Enzyme immobilization is a key technique for enzyme reusability and purification.
- Affinity tags are widely used for protein purification.
Purpose of the Study:
- To engineer and characterize novel immobilized Factor Xa (FXa) derivatives for efficient affinity tag removal.
- To evaluate the activity and stability of immobilized FXa derivatives.
- To demonstrate the utility of immobilized FXa in protein purification processes.
Main Methods:
- Construction of FXa fused with a cellulose-binding domain (FXa-CBD) and a hexahistidine tail (FXa-H6).
- Immobilization of FXa-CBD onto cellulose and FXa-H6 onto Ni(2+)-NTA agarose.
- Assays to determine the activity and stability of immobilized enzyme derivatives.
- Application of immobilized FXa in cleaving affinity tags from fusion proteins.
Main Results:
- Both FXa-CBD and FXa-H6 derivatives retained enzymatic activity upon immobilization.
- Immobilized FXa derivatives exhibited comparable stability and activity.
- The immobilized FXa systems effectively removed affinity tags from fusion proteins.
- No detectable FXa contamination was observed in the purified products.
Conclusions:
- Immobilized Factor Xa derivatives are effective tools for affinity tag cleavage in protein purification.
- This approach offers a contamination-free method for producing purified proteins.
- Engineered FXa with specific binding domains provides versatile options for enzyme immobilization and application.