Protein folding and methods of using same

Patent metadata

Field	Value
Publication number	US-10457716-B2
Application number	US-201514820290-A
Country	US
Kind code	B2
Filing date	Aug 6, 2015
Priority date	Aug 6, 2014
Publication date	Oct 29, 2019
Grant date	Oct 29, 2019

Abstract

The instant disclosure provides a microscale method for providing correctly folded, and assembled biologically active proteins in an efficient and shorted time frame, compared to conventional protein production techniques. Proteins produced from inclusion bodies and other aggregated protein sources are provided. Microscale production of correctly folded and assembled class I MHC protein and complexes thereof are also provided for, as well as for high throughput production for use in epitope discovery protocols. Microscale production of complex proteins from protein aggregates and preparations containing protein aggregates is provided that requires less than 24 hours of processing time.

First claim

What is claimed is:

1. A method for microscale production of a complex protein comprising at least a first protein of interest and a second protein of interest, wherein at least one protein of interest is a recombinant protein, comprising the steps of:
diluting a denatured solubilized protein preparation comprising a first protein of interest to provide a diluted preparation;
dialyzing the diluted preparation at a temperature of between 4° C. and 32° C. for 1 to 3 hours to provide a first correctly folded first protein of interest preparation; and
adding a volume of a second protein of interest to the first protein of interest preparation to form a complex protein preparation comprising the complex protein;
adding the complex protein preparation to a purification reagent, the purification reagent capable of capturing the complex protein; and
obtaining the complex protein,

wherein the complex protein has a biologically active folded protein structure, said method is completed in less than 24 hours,
at least one protein of interest comprises β 2 -microglobulin (β 2 m), a class I major histocompatibility complex protein heavy chain (MHC-HC), or a class I MHC binding molecule, and
wherein each preparation has a final volume of less than 1 milliliter.

2. The method of claim 1 , wherein the complex protein preparation is purified by a chromatographic method or by a buffer exchange method.

3. The method of claim 1 , wherein said first protein of interest is a recombinant protein and the diluted preparation has a volume of not more than 500 microliters.

4. The method of claim 3 , wherein the recombinant protein is β 2 m or MHC-HC.

5. The method of claim 3 , wherein said first preparation of said protein of interest is incubated with an MHC-binding molecule.

6. The method of claim 1 wherein at least one protein of interest is a class I MHC binding molecule, and said class I MHC binding molecule is a peptide, lipid, glycolipid, metabolite or other molecule having binding affinity for a class I MHC protein.

7. The method of claim 1 wherein the purification reagent is Ab coated beads.

8. The method of claim 1 , wherein said denatured solubilized protein of interest is diluted in a folding buffer in a ratio of between 1:5 to 1:50 by volume.

9. The method of claim 1 , wherein the complex protein preparation is purified by size-exclusion column chromatography, hydrophobic interaction chromatography, ion exchange chromatography, column chromatography, affinity chromatography, or batch purification.

10. The method of claim 1 wherein the first protein of interest is provided from denatured solubilized bacterial inclusion bodies, a precipitated protein aggregate, or a misfolded or inactive protein solution.

11. The method of claim 1 wherein said complex protein comprises a detectable tag having affinity for the purification reagent.

12. A microscale production method for preparing peptide/MHC complexes comprising:
combining a MHC binding molecule of interest with a small volume of folding buffer, and adding a molecular tagged preparation of β 2 m sufficient to yield a final concentration of about 12.5 μM, so as to provide a solubilized first preparation;
incubating said first preparation for 1 hour;
adding MHC-HC to said first preparation so as to provide a final concentration of 5 μM, to provide a second preparation;
incubating said second preparation for 12 hours;
dialyzing said second preparation for 2 hours at 4° C. to about 32° C., to provide a third preparation;
adding said third preparation to a volume of a purification reagent having affinity for the detectable molecular tag to provide a fourth preparation comprising a captured tagged peptide/MHC complex;
eluting the captured tagged peptide/MHC complex with an elution buffer to provide a fifth preparation comprising partially purified protein complex; and
removing contaminating reagents from the fifth preparation to provide a peptide/MHC complex suitable for analysis;

wherein each preparation has a volume of less than 1 milliliter.

13. The microscale production method of claim 12 wherein said purification reagent comprises Ab coated beads.

14. The microscale production method of claim 12 wherein said fourth solution is incubated for 12 hours at a temperature of 4° C.

15. The microscale production method of claim 12 wherein said eluting buffer comprises mixture of 10 mM HEPES (pH 8), 150 mM NaCl, 3 mM EDTA, and 300 mM imidazole.

16. The microscale production method of claim 12 wherein said second preparation is dialyzed against a dialysis membrane having a 7 kDa molecular weight cutoff membrane.

17. The microscale production method of claim 12 , wherein the detectable molecular tag is Histidine.

Assignees

• The Univ Of Notre Dame Du Lac
• Univ Notre Dame Du Lac

Inventors

• Baker Brian M
• Hellman Lance

Classifications

• C07K14/70539 Primary

  MHC-molecules, e.g. HLA-molecules · CPC title

• C07K1/1136

  by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• B01D15/3804

  Affinity chromatography · CPC title

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · CPC title