# US 11,898,187 B2 · Protein glycosylation sites by rapid expression and characterization of N-glycosyltransferases > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,898,187 B2 - Document ID: US11898187B2 - Status: Granted - Kind: B2 - Application number: 16/639,500 - Filed: 2018-08-15 - Published: 2022-07-28 - Granted: 2024-02-13 - Earliest claimed: 2017-08-15 - Assignees: Northwestern University - Applicants: Northwestern University - Inventors: Michael Christopher Jewett; Weston K. Kightlinger; Liang Lin; Milan Mrksich - Web page: https://patentgrep.com/patent/US11898187B2 ## CPC - C12P 21/005: {Glycopeptides, glycoproteins} - C07K 5/1008: {the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala} - C07K 5/101: {the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu} - C07K 5/1013: {the side chain containing O or S as heteroatoms, e.g. Cys, Ser} - C07K 5/1016: {and aromatic or cycloaliphatic} - C07K 5/1021: {with the first amino acid being acidic} - C07K 5/1024: {with the first amino acid being heterocyclic} - C12N 9/1048: {Glycosyltransferases (2.4)} - C12N 9/1051: {Hexosyltransferases (2.4.1)} - C12N 15/1044: {Preparation or screening of libraries displayed on scaffold proteins} - C12Q 1/48: involving transferase - C12Y 204/01: Hexosyltransferases (2.4.1) - G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry} - C07K 2319/91: containing a motif for glycosylation - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - 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) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12Y: ENZYMES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Disclosed are components, systems, and methods for glycoprotein or recombinant glycoprotein protein synthesis in vitro and in vivo. In particular, the present invention relates to components, systems, and methods for identifying amino acid glycosylation tag motifs for N-glycosyltransferases and the use of the identified amino acid glycosylation tag motifs in methods for preparing glycoproteins and recombinant glycoproteins in vitro and in vivo. ## Claims ### Claim 1 A method for synthesizing a glycoprotein or recombinant glycoprotein, the method comprising: (a) expressing in a cell or in a cell-free protein synthesis (CFPS) reaction, a polypeptide comprising the amino acid sequence of a target protein which includes naturally an amino acid motif that includes an asparagine that is glycosylated by N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) or that has been modified to include a heterologous amino acid motif that includes an asparagine that is glycosylated by ApNGT, the amino acid motif or heterologous amino acid motif comprises X−2-X−1-N-X+1-S/T-X+3, wherein X−2 is selected from Gly, Asn, and Tyr; X−1 is selected from Gly and Ala; X+1 is selected from Trp, Val, His, Ala, and Ile; and X+3 is selected from Thr, Met, and Phe; (b) expressing ApNGT in a cell or in a CFPS reaction; and (c) reacting the polypeptide and ApNGT in the presence of a sugar donor, wherein ApNGT glycosylates the amino acid motif or heterologous amino acid motif of the polypeptide with the sugar of the sugar donor to synthesize the glycoprotein or recombinant glycoprotein. ### Claim 2 (depends on 1) The method of claim 1, wherein the target protein is a eukaryotic protein. ### Claim 3 (depends on 1) The method of claim 1, wherein the target protein is a prokaryotic protein. ### Claim 4 (depends on 1) The method of claim 1, wherein step (a) is performed in a prokaryotic cell; step (b) is performed in a prokaryotic cell; or both of step (a) and step (b) are performed in the same prokaryotic cell. ### Claim 5 (depends on 1) The method of claim 1, wherein step (a) is performed in a eukaryotic cell; step (b) is performed in a eukaryotic cell; or both of step (a) and step (b) are performed in the same eukaryotic cell. ### Claim 6 (depends on 1) The method of claim 1, wherein step (a) is performed in a prokaryotic-based CFPS reaction; step (b) is performed in a prokaryotic-based CFPS reaction; or both of step (a) and step (b) are performed in the same prokaryotic-based CFPS reaction. ### Claim 7 (depends on 6) The method of claim 6, wherein step (c) is performed in the same prokaryotic-based CFPS reaction. ### Claim 8 (depends on 1) The method of claim 1, wherein step (b) further comprises expressing in the cell an additional N-glycosyltransferase selected from one of Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp. ### Claim 9 (depends on 1) The method of claim 1, wherein multiple identical, multiple distinct, and/or non-naturally occurring glycans are introduced to a protein by specifically choosing unique sequence:enzyme pairs that allow for orthogonal, and/or parallel and/or independent glycosylation. ### Claim 10 (depends on 1) The method of claim 1, wherein prior to performing step (c), the polypeptide is immobilized on a solid support. ### Claim 11 A method for synthesizing a glycoprotein or recombinant glycoprotein, the method comprising: (a) expressing in a cell or in a cell-free protein synthesis (CFPS) reaction a polypeptide comprising the amino acid sequence of a target protein which includes naturally two or more different amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of the different N-glycosyltransferases comprises N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT), or that has been modified to include two or more different heterologous amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of the different N-glycosyltransferases comprises ApNGT, the amino acid motifs or heterologous amino acid motifs comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs:1-549, wherein at least one of the amino acid motifs or heterologous amino acid motifs comprises X−2-X−1-N-X+1-S/T-X+3, wherein X−2 is selected from Gly, Asn, and Tyr: X−1 is selected from Gly and Ala: X+1 is selected from Trp, Val, His, Ala, and Ile; and X+3 is selected from Thr, Met, and Phe; (b) expressing in one or more cells or in one or more CFPS reactions the two or more different N-glycosyltransferases from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of the different N-glycosyltransferases comprises ApNGT, wherein the two or more different N-glycosyltransferases selected from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of the different N-glycosyltransferases comprises ApNGT, are expressed simultaneously in the same cell or CFPS reaction or sequentially in two or more different cells or two or more different CFPS reactions; and (c) reacting the polypeptide and the two or more different N-glycosyltransferases selected from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of the different N-glycosyltransferases comprises ApNGT, in the presence of two or more sugar donors which are the same or different, wherein the polypeptide is reacted with the two or more different N-glycosyltransferases selected from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of A the different N-glycosyltransferases comprises ApNGT, simultaneously in the same cell or CFPS reaction, or sequentially in two or more different cells or two or more different CFPS reactions, and wherein the two or more different N-glycosyltransferases selected from Actinobacillus spp., Escherichia spp., Haemophilus spp., or Mannheimia spp., wherein at least one of the different N-glycosyltransferases comprises ApNGT, glycosylate the two or more different amino acid motifs or heterologous amino acid motifs of the polypeptide with the sugar of the two or more sugar donors to synthesize the glycoprotein or recombinant glycoprotein. ### Claim 12 (depends on 11) The method of claim 11, wherein prior to performing step (c), the polypeptide is immobilized on a solid support. ### Claim 13 A method for selecting an amino acid motif that is glycosylated by an N-glycosyltransferase, the method comprising: (a) reacting a library of peptides with an N-glycosyltransferase in the presence of a sugar donor, wherein the N-glycosyltransferase glycosylates one or more of the peptides, and wherein the peptides comprise at least 6 amino acids and have a sequence X−2-X−1-N-X+1-S/T-R-C wherein X is any amino acid; (b) detecting glycosylation of the reacted peptides by immobilizing the reacted peptides on a substrate comprising self-assembled monolayers, and performing matrix-assisted laser desorption/ionization mass spectrometry of the immobilized reacted peptides to select the amino acid motif that is glycosylated by the N-glycosyltransferase. ### Claim 14 (depends on 13) The method of claim 13, wherein the library comprises at least 500 peptides. ### Claim 15 (depends on 13) The method of claim 13, wherein the peptides are covalently immobilized on the substrate comprising the self-assembled monolayers. ### Claim 16 (depends on 15) The method of claim 15, wherein the library of peptides comprise a C-terminal Cys, the self-assembled monolayers comprise free maleimides, and the C-terminal Cys of the peptides reacts with the free maleimides to form a bond and covalently immobilize the peptide. ### Claim 17 (depends on 16) The method of claim 16, wherein the self-assembled monolayers comprise alkylthiolates which provide the free maleimides. ### Claim 18 (depends on 13) The method of claim 13, wherein: (i) the library of peptides comprise a C-terminal alkyne, the self-assembled monolayers comprise free azides, and the C-terminal alkyne of the peptides reacts with the free azides to form a bond and covalently immobilize the peptide; or (ii) the library of peptides comprise a C-terminal azide, the self-assembled monolayers comprise free alkynes, and the C-terminal azide of the peptides reacts with the free alkynes to form a bond and covalently immobilize the peptide. ### Claim 19 (depends on 13) The method of claim 13, wherein the bonds formed in (i) and (ii) comprise a 1,2,3-triazole bond. ### Claim 20 (depends on 1) The method of claim 1, wherein the amino acid motif comprises the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 8. ### Claim 21 (depends on 11) The method of claim 11, wherein X−2-X−1-N-X+1-S/T-X+3 comprises the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 8.