# US 2021/0277052 A1 · Recombinant grp94 chaperones and methods of using the same to modulate glycosylation in fusion proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0277052 A1 - Document ID: US20210277052A1 - Status: Published application - Kind: A1 - Application number: 17/191,387 - Filed: 2021-03-03 - Published: 2021-09-09 - Earliest claimed: 2020-03-03 - Assignees: Hauptman-Woodward Medical Research Institute, Inc. - Applicants: Hauptman-Woodward Medical Research Institute - Inventors: Daniel T. Gewirth; John D. Huck - Web page: https://patentgrep.com/patent/US20210277052A1 ## CPC - C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids} - C07K 14/47: from mammals - C12N 9/1051: {Hexosyltransferases (2.4.1)} - C07K 2319/21: containing a His-tag - C07K 2319/43: containing a FLAG-tag - C07K 2319/91: containing a motif for glycosylation - 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) ## Abstract Methods for glycosylation of Grp94 chaperone proteins, including Grp94 fragments, mutants, and fusion proteins, expressed in mammalian cells are presented. The method comprises altering a Pre-N domain of the Grp94 chaperone protein, wherein the alteration of the Pre-N domain of the Grp94 chaperone protein leads to glycosylation at available glycosylation sites downstream of the Pre-N domain of the Grp94 chaperone protein, when expressed in mammalian cells. ## Claims ### Claim 1 A recombinant glucose-regulated protein 94 (Grp94) protein comprising: deletion of seven or more residues from a Pre-N domain of the recombinant Grp94; or substitution of five or more residues of the Pre-N domain with alanine, wherein the recombinant Grp94 is glycosylated at a minor glycosylation site. ### Claim 2 (depends on 1) The recombinant Grp94 protein of claim 1, wherein the recombinant Grp94 comprises SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4. ### Claim 3 (depends on 1) The recombinant Grp94 protein of claim 1, wherein the recombinant Grp94 comprises SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8. ### Claim 4 (depends on 1) The recombinant Grp94 protein of claim 1, wherein the minor glycosylation site comprises a glycosylation sequon, wherein the glycosylation sequon is Asn-X-Ser or Asn-X-Thr, and wherein X is any amino acid except proline. ### Claim 5 (depends on 1) The recombinant Grp94 protein of claim 1 further comprising a histidine tag, a flag tag, or a biotin tag. ### Claim 6 (depends on 1) The recombinant Grp94 protein of claim 1, wherein the recombinant Grp94 protein is a fusion protein and further comprises a glycosylated protein. ### Claim 7 (depends on 6) The recombinant Grp94 protein of claim 6, wherein the glycosylated protein is fused to the C-terminus of the Grp94 via a cleavable linker. ### Claim 8 (depends on 1) A method of producing the recombinant Grp94 protein of claim 1 comprising culturing a host cell including a vector which comprises a nucleic acid encoding the recombinant Grp94 protein under conditions permitting the production of the recombinant fusion protein. ### Claim 9 (depends on 8) The method of claim 8, wherein the vector comprises SEQ ID NO. 35, SEQ ID NO. 36, or SEQ ID NO. 34. ### Claim 10 (depends on 8) The method of claim 8, wherein the vector comprises SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, or SEQ ID NO. 51. ### Claim 11 (depends on 8) The method of claim 8 further comprising a step of recovering the recombinant fusion protein. ### Claim 12 A method of glycosylating a protein comprising: expressing a recombinant fusion protein in a host cell, wherein the recombinant fusion protein comprises (i) a wild type glucose-regulated protein 94 (Grp94) or a recombinant Grp94 and (ii) the protein, wherein the protein fused to the C-terminus of the wild type or recombinant Grp94. ### Claim 13 (depends on 12) The method of claim 12, wherein the recombinant fusion protein comprises the recombinant Grp94, and wherein the recombinant Grp94 comprises deletion of seven or more residues from a Pre-N domain of the recombinant Grp94. ### Claim 14 (depends on 13) The method of claim 13, wherein the recombinant Grp94 comprises SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4. ### Claim 15 (depends on 12) The method of claim 12, wherein the recombinant fusion protein comprises the recombinant Grp94, and wherein the recombinant Grp94 comprises substitution of five or more residues of the Pre-N domain with alanine. ### Claim 16 (depends on 15) The method of claim 15, wherein the recombinant Grp94 comprises SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8. ### Claim 17 (depends on 12) The claim 12, wherein the recombinant fusion protein further comprises a histidine tag, a flag tag, or a biotin tag, and wherein the method further comprises isolating the recombinant fusion protein. ### Claim 18 (depends on 12) The method of claim 12, wherein the protein is fused to the C-terminus of the Grp94 via a cleavable linker, and wherein the method further comprises cleaving the protein from the wild type or recombinant Grp94. ### Claim 19 (depends on 12) The method of claim 12, protein wherein the protein comprises a glycosylation sequon, wherein the glycosylation sequon is Asn-X-Ser or Asn-X-Thr, and wherein X is any amino acid except proline. ### Claim 20 (depends on 19) The method of claim 19, further comprising coexpressing oligosaccharyltransferase (OST) complex.