Chaperonin and osmolyte protein folding and related screening methods
First Claim
1. A method of folding a denatured polypeptide, comprising the steps of:
- (a) providing a polypeptide in an unfolded state which is capable of binding to a chaperonin;
(b) binding said polypeptide to said chaperonin to form a chaperonin-polypeptide complex for the folding of said polypeptide to its biologically active state; and
(c) adding an osmolyte to said chaperonin-polypeptide complex, thereby promoting the folding of said polypeptide from its unfolded state to its folded state to yield a folded biologically active polypeptide whereby said promoting is greater than that which is achieved using chaperonins and osmolytes alone.
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Abstract
The invention describes an inexpensive in vitro protein folding process for preventing large scale protein misfolding and aggregation, for concentrating aggregation prone chaperonin-protein folding intermediates in a stable non-aggregating form, and for rapidly screening these stable concentrates for the best folding solution conditions. The process comprises: (1) the formation of a chaperone-substrate complex and (2) the release of the substrate using a broad array of folding solutions containing different osmolyte ions, detergents, gradients of ionic strength and pH or other commonly used folding additives. Specifically, when the chaperonin/osmolyte protein process was applied to identify and optimize GSΔ468 bacterial glutamine synthetase mutant refolding conditions that otherwise cannot be folded in vitro by commonly used techniques, 67% of the enzymatic activity was recovered.
4 Citations
29 Claims
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1. A method of folding a denatured polypeptide, comprising the steps of:
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(a) providing a polypeptide in an unfolded state which is capable of binding to a chaperonin;
(b) binding said polypeptide to said chaperonin to form a chaperonin-polypeptide complex for the folding of said polypeptide to its biologically active state; and
(c) adding an osmolyte to said chaperonin-polypeptide complex, thereby promoting the folding of said polypeptide from its unfolded state to its folded state to yield a folded biologically active polypeptide whereby said promoting is greater than that which is achieved using chaperonins and osmolytes alone. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method of folding a denatured polypeptide, comprising the steps of:
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(a) providing a polypeptide in an unfolded state that is capable of binding to an oligomeric chaperonin;
(b) binding said polypeptide to said chaperonin to form a chaperonin-polypeptide complex for the folding of said polypeptide to its biologically active state; and
(c) adding an osmolvte to said chaperonin-polypeptide complex, thereby promoting the folding of said polypeptide from its unfolded state to its folded state to yield a folded biologically active polypeptide whereby said promoting is greater than that which is achieved using chaperonins and osmolytes alone. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification