Patent ID: 11898187
Assignee: NORTHWESTERN UNIVERSITY
Field: Measurement (Instruments)
Classification: CPC C  G | IPC C  G

Claim 10:
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.