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Expression and purification of chaperone-active recombinant clusterin
Rebecca A Dabbs1, Mark R Wilson1
1Illawarra Health and Medical Research Institute, School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia.
Plos One
|January 28, 2014
Summary
Researchers developed a novel method to produce pure, active recombinant clusterin, a key protein for cellular proteostasis. This breakthrough enables future studies and potential therapeutic applications for this challenging-to-express protein.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Cellular Biology
Background:
- Clusterin is a secreted mammalian chaperone crucial for proteostasis.
- Previous attempts to produce recombinant clusterin have been hampered by poor characterization, inadequate purity, and insufficient methodological detail.
- The structural complexity of clusterin makes its expression and purification challenging.
Purpose of the Study:
- To establish a well-validated and economical method for producing pure, chaperone-active recombinant clusterin.
- To overcome the limitations of previous recombinant clusterin production methods.
- To facilitate future research utilizing recombinant clusterin, including mutagenesis and metabolic labeling studies.
Main Methods:
- Expression of recombinant clusterin was trialed in bacterial, yeast, insect, and mammalian cells.
- The optimal method involved stable transfection of HEK293 cells for expression in protein-free medium.
- Purification utilized a combination of immunoaffinity, cation exchange, and size exclusion chromatography.
Main Results:
- A high yield of recombinant clusterin (7.5–10 µg/ml) was achieved using stably transfected HEK293 cells.
- The purified protein was confirmed to be correctly glycosylated and proteolytically cleaved into its active α- and β-subunits.
- The recombinant clusterin exhibited chaperone activity comparable to human plasma clusterin.
Conclusions:
- A robust and validated method for producing pure, active recombinant clusterin has been established.
- This method provides a theoretically unlimited supply of recombinant clusterin for research and potential therapeutic development.
- The availability of this recombinant protein will enable detailed functional studies and the exploration of clusterin's therapeutic potential.
