# US 2016/0355545 A1 · Method for improving protein functionality using vortexing fluid shear foces > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2016/0355545 A1 - Document ID: US20160355545A1 - Status: Published application - Kind: A1 - Application number: 14/913,951 - Filed: 2014-09-03 - Published: 2016-12-08 - Earliest claimed: 2013-09-04 - Assignees: The Regents of the University of California; Flinders University - Applicants: The Regents of the University of California; Flinders University - Inventors: Gregory A. Weiss; Colin L. Raston; Tom Z. Yuan; Callum Ormonde; Stephan Timothy Kudlacek - Web page: https://patentgrep.com/patent/US20160355545A1 ## CPC - C07K 1/113: without change of the primary structure - C07K 1/14: Extraction; Separation; Purification - C07K 14/005: from viruses - C07K 14/47: from mammals - C12N 9/2462: {Lysozyme (3.2.1.17)} - C12Y 302/01017: Lysozyme (3.2.1.17) - C12N 2740/16111: concerning HIV env - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract A method of improving protein functionality includes obtaining a protein such as protein contained within exclusion bodies contained within bacteria or yeast used for the recombinant expression of the protein. The exclusion bodies are solubilized using a chaotropic agent such as guanidine or urea. The protein is then subject to denaturation conditions and optionally purified. A liquid containing the denatured protein is loaded into a vessel that is angled relative to horizontal. The vessel is then rotated in the angled configuration at a rate within that is less than 10,000 RPM for period of time. Refolded protein is formed by the high shear conditions formed in the thin film of fluid formed in the inner surface of the rotating vessel. Refolding can be performed in a batch mode or a continuous mode. The process may be scaled up for industrial applications by using multiple vessels. ## Claims ### Claim 1 A method of improving protein functionality comprising: loading a liquid containing a denatured protein into a vessel that is angled relative to horizontal; and rotating the vessel in the angled configuration at a rate within the range of about 3,000 RPM to about 9,000 RPM for period of time. ### Claim 2 (depends on 1) The method of claim 1, further comprising diluting the denatured protein prior to rotating the vessel. ### Claim 3 (depends on 2) The method of claim 2, wherein the denature protein is diluted with a buffer solution. ### Claim 4 (depends on 1) The method of claim 1, further comprising adjusting the temperature of the liquid contained within the vessel. ### Claim 5 (depends on 1) The method of claim 1, wherein the vessel is rotated for less than sixty (60) minutes. ### Claim 6 (depends on 4) The method of claim 4, wherein the vessel is rotated for less than ten (10) minutes. ### Claim 7 (depends on 1) The method of claim 1, wherein the angle is within the range of about 20° to about 70°. ### Claim 8 (depends on 7) The method of claim 7, wherein the angle is about 45°. ### Claim 9 (depends on 1) The method of claim 1, wherein the vessel is rotated at a rate of about 5,000 RPM. ### Claim 10 (depends on 1) The method of claim 1, further comprising: stopping rotation of the vessel; and removing the fluid from the vessel after rotation. ### Claim 11 (depends on 1) The method of claim 1, wherein the vessel comprises an internal surface having a circular-shaped cross section. ### Claim 12 (depends on 1) The method of claim 1, wherein the vessel comprises an inner surface having a textured surface. ### Claim 13 (depends on 1) The method of claim 1, wherein the protein comprises a recombinant expressed protein. ### Claim 14 (depends on 1) The method of claim 1, wherein the protein comprises a native protein. ### Claim 15 A method of improving protein functionality comprising: continuously loading a fluid containing a denatured protein into a vessel that is angled relative to horizontal; and rotating the vessel in the angled configuration at a rate within the range of about 3,000 RPM to about 9,000 RPM for a period of time; and continuously removing a portion of the fluid from the vessel during rotation of the vessel. ### Claim 16 (depends on 15) The method of claim 15, wherein the continuous loading of the fluid is delivered to the vessel via an inlet conduit terminating within the vessel. ### Claim 17 (depends on 16) The method of claim 16, wherein the portion of the fluid is continuously removed from the vessel via an open end of the vessel. ### Claim 18 (depends on 15) The method of claim 15, further comprising diluting the denatured protein prior to rotating the vessel. ### Claim 19 (depends on 15) The method of claim 15, further comprising adjusting the temperature of the liquid contained within the vessel. ### Claim 20 (depends on 15) The method of claim 15, wherein the vessel is rotated for less than ten (10) minutes. ### Claim 21 (depends on 15) The method of claim 15, wherein the angle is within the range of about 20° to about 70°. ### Claim 22 (depends on 15) The method of claim 15, wherein the method is performed using a plurality of vessels.