Osmolyte mixture for protein stabilization
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.
1. A method of screening for a stabilizing osmolyte composition for an partially unfolded intermediate polypeptide of a polypeptide, comprising the steps of:
(a) providing an array having a plurality of elements with each element having a different test stabilizing osmolyte composition therein;
(b) introducing said polypeptide in a completely unfolded state which is capable of binding to a chaperonin to each element to form a polypeptide and test stabilizing osmolyte composition for said partially unfolded intermediate polypeptide;
(c) introducing, after a time delay, said chaperonin into each element;
(d) replacing said test stabilizing osmolyte composition with a refolding buffer so that said chaperonin can capture said partially unfolded intermediate polypeptide;
(e) adding at least one folding osmolyte into each element;
(f) measuring a yield of active polypeptides within each element by assaying for functional activity; and
(g) identifying said stabilizing osmolyte composition for said partially unfolded intermediate polypeptide, said stabilizing osmolyte composition being selected from the different test stabilizing osmolyte compositions in said plurality of elements, by measuring the yield of active polypeptides within each element of said array.
2. The method of screening of claim 1 wherein said partially unfolded intermediate polypeptide is incapable of being folded to its biologically active form by either a chaperonin or a folding osmolyte alone.
3. The method of screening of claim 1 wherein said chaperonin is of the Escherichia coli GroE chaperonin family.
4. The method of screening of claim 3 in which the chaperonin is E. coli GroEL.
5. The method of screening of claim 1 in which one of said different test stabilizing osmolyte composition comprises 2 M to 6 M glycerol and 2 M to 6 M urea.
6. The method of screening of claim 1 further comprising the step of promoting the folding of said polypeptide to its native state by the addition of a co-chaperonin to each of said elements, wherein said co-chaperonin has the ability to bind and dissociate from the chaperonin and aid said chaperonin to achieve correct binding of said polypeptide.
7. The method of screening of claim 1 wherein said identifying step comprises monitoring an enzymatic activity of said polypeptide.
8. The method of screening of claim 1 further comprising the step of adding a nucleotide to each of said elements.
9. The method of screening of claim 8 wherein said nucleotide is selected from the group consisting of ATP or ADP.
10. The method of screening of claim 1 wherein said time delay is 1 to 10 minutes.
11. The method of screening of claim 1 wherein said steps (a) to (g) are repeated.
12. The method of screening of claim 1 wherein said chaperonin is immobilized on a solid support.
13. The method of screening of claim 1 wherein said polypeptide in said introducing step (b) is completely unfolded with urea or guanidine hydrochloride.
14. The method of screening of claim 1 in which one of said different test stabilizing osmolyte compositions comprises 3 to 5 M glycerol and 4 to 5 M urea.
15. The method of screening of claim 1 wherein said partially unfolded intermediate polypeptide retains some native secondary and tertiary structure and is not aggregated.
16. The method of screening of claim 1 wherein said partially unfolded intermediate polypeptide is either malate dehydrogenase or citrate synthase.
17. The method of screening of claim 1 wherein said time delay is up to 24 hours.