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Expression analysis of a modified factor X in stably transformed insect cell lines
T A Pfeifer1, M M Guarna, E M Kwan
1Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. pfeifer@zoology.ubc.ca
Protein Expression and Purification
|October 26, 2001
Summary
Recombinant cellulose-binding domain Factor X (CBDFX) was produced in insect cells, achieving high yields. The secreted CBDFX protein demonstrated proper processing, cellulose binding, and functional Factor X cleavage activity.
Area of Science:
- Biotechnology
- Protein Engineering
- Insect Cell Culture
Background:
- Factor X is a critical enzyme in the coagulation cascade.
- Recombinant protein production in insect cells offers a scalable system.
- Cellulose-binding domains facilitate protein purification and immobilization.
Purpose of the Study:
- To develop a method for producing a modified Factor X protein with a cellulose-binding domain (CBDFX) in insect cell lines.
- To optimize and scale up the production of secreted CBDFX.
- To characterize the functionality of the produced CBDFX protein.
Main Methods:
- Fusion of a cellulose-binding domain (CBD) to Factor X.
- Expression and secretion of CBDFX in Hi5 and Sf9 insect cell lines.
- Scale-up of high-producing cell lines in 2-liter fermentors.
- Analysis of protein processing via amino-terminal sequencing.
- Assessment of cellulose-binding affinity to Avicel.
- Evaluation of activated CBDFX's enzymatic activity on a Factor X cleavage site.
Main Results:
- Stable Hi5 and Sf9 insect cell lines expressing secreted CBDFX were generated.
- Production levels reached 4 mg/liter in Hi5 and 18 mg/liter in Sf9 cell lines.
- Amino-terminal sequencing confirmed proper protein processing.
- CBDFX demonstrated effective binding to the cellulose substrate Avicel.
- Activated recombinant CBDFX efficiently recognized and processed a Factor X cleavage site.
Conclusions:
- Insect cell lines, particularly Sf9, are suitable for high-level production of secreted CBDFX.
- The produced CBDFX is correctly processed, binds to cellulose, and retains enzymatic activity.
- This engineered protein holds potential for applications requiring immobilized and active Factor X.
