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Functional display and expression of chicken cystatin using a phagemid system
A S Tanaka1, C A Sampaio, H Fritz
1Abteilung für Klinische Chemie und Klinische Biochemie, Klinikum Innenstadt, Ludwig-Maximilians Universität, München, Germany.
Biochemical and Biophysical Research Communications
|September 14, 1995
Summary
Functionally active chicken cystatin was successfully displayed on phage surfaces using a recombinant phage antibody system. This method enables the selection of cystatin variants with altered binding affinities to cysteine proteinases.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Cystatins are crucial inhibitors of cysteine proteinases.
- Phage display technology offers a powerful platform for protein engineering and selection.
Purpose of the Study:
- To display functionally active chicken cystatin on filamentous phage.
- To develop a method for selecting cystatin variants with modified enzyme inhibitory properties.
Main Methods:
- A synthetic gene for a modified chicken cystatin was cloned into a phagemid vector.
- The cystatin-gpIII fusion protein was expressed on phage surfaces and as a soluble inhibitor in E. coli.
- Recombinant phages were selected based on binding to carboxymethylated-papain.
Main Results:
- Functionally active chicken cystatin was successfully displayed on phage surfaces.
- The displayed cystatin-gpIII fusion protein showed dose-dependent binding to papain.
- Soluble inhibitor exhibited strong papain inhibitory activity.
Conclusions:
- Phage display is a viable method for presenting functional cystatin.
- This technology can be utilized to evolve cystatin variants with altered papain-binding affinities.