Osmolyte mixture for protein stabilization
Inventors
Fisher, Mark T. • Katayama, Hiroo
Assignees
University of Kansas • University of Kansas Medical Center
Publication Number
US-9052323-B2
Publication Date
2015-06-09
Expiration Date
2030-08-27
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Abstract
An osmolyte composition comprising 4 M glycerol and 4M urea for stabilizing previously transient protein folding intermediates as long-lived stable forms. A method to search for other possible stabilizing osmolyte mixtures using a screening array is also provided. These additional osmolyte mixtures may complement or augment the successful 4M glycerol/4 M urea mixture.
Core Innovation
The present invention provides a novel osmolyte composition comprising high concentrations of glycerol and urea (preferably 4 M glycerol and 4 M urea) for the stabilization of partially denatured proteins. This unique composition allows aggregation-prone protein folding intermediates, which are typically short-lived and unstable, to persist in solution as long-lived, stable, non-aggregating forms. The invention further relates to a process for folding completely denatured proteins through the formation of these stabilized protein intermediates.
Conventional protein folding methods face difficulties such as irreversible aggregation of overexpressed or mutant proteins, leading to poor yields in the production and refolding of proteins, especially in biotechnology and pharmaceutical industries. Current approaches like using chaperonins or individual osmolytes may not prevent aggregation or may not be universally effective. The invention addresses this problem by providing a method to prevent large-scale protein misfolding and aggregation during in vitro protein folding, thus enabling the concentration and stabilization of folding intermediates without loss due to aggregation.
Additionally, the invention includes a screening method utilizing an array with different test stabilizing osmolyte compositions. By introducing an unfolded protein into these conditions, adding chaperonin after a delay, and assaying for protein function, the method allows rapid identification of the most effective osmolyte mixtures for stabilizing a given protein. The most efficient combinations (such as the preferred mixture of 4 M glycerol and 4 M urea) can be complemented or augmented by screening for improved stabilization properties, enabling tailored optimization for various proteins.
Claims Coverage
There is one independent claim that defines the principal inventive feature.
Screening method for identifying stabilizing osmolyte compositions for protein folding intermediates
A method comprising: 1. Providing an array with multiple elements, each containing a different test stabilizing osmolyte composition. 2. Introducing a completely unfolded polypeptide capable of binding to a chaperonin into each element to form a composition with partially unfolded intermediates. 3. Introducing a chaperonin into each element after a designated time delay. 4. Replacing the test stabilizing osmolyte composition with a refolding buffer to allow the chaperonin to capture the partially unfolded intermediate. 5. Adding at least one folding osmolyte into each element. 6. Measuring the yield of active polypeptides in each element by assaying for functional activity. 7. Identifying the optimal stabilizing osmolyte composition by comparing yields of active polypeptide across the array.
The key inventive feature lies in the systematic screening and identification method for stabilizing osmolyte compositions, enabling the stabilization and subsequent correct folding of protein intermediates.
Stated Advantages
Prevents large-scale protein misfolding and aggregation during in vitro folding, thereby enhancing yields of active protein.
Enables stable maintenance of aggregation-prone protein folding intermediates, allowing for their concentration and processing without loss due to aggregation.
Provides a rapid screening method for identifying optimal long-term stabilization conditions where protein folding intermediates can be preserved for later folding.
Documented Applications
In vitro protein folding for structural analysis and biotechnological manufacturing of pharmaceutical proteins.
Production and storage of stabilized non-native conformation isomers as vaccine candidates for prevention and treatment of human diseases.
Use of stabilized protein intermediates related to neurodegenerative diseases, for diagnosis and intervention, including proteins implicated in Alzheimer disease, Parkinson disease, prion-associated diseases, and similar disorders.
Rapid screening and identification of optimal stabilizing osmolyte conditions for folding a broad diversity of proteins, benefiting both academic and industrial protein formulation research.
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