	US 11,898,187 B2
	Protein glycosylation sites by rapid expression and characterization of N-glycosyltransferases
Michael Christopher Jewett, Evanston, IL (US); Weston K. Kightlinger, Evanston, IL (US); Liang Lin, Evanston, IL (US); and Milan Mrksich, Hinsdale, IL (US)
Assigned to Northwestern University, Evanston, IL (US)
Appl. No. 16/639,500
Filed by Northwestern University, Evanston, IL (US)
PCT Filed Aug. 15, 2018, PCT No. PCT/US2018/000185 § 371(c)(1), (2) Date Feb. 14, 2020, PCT Pub. No. WO2019/035916, PCT Pub. Date Feb. 21, 2019.
Claims priority of provisional application 62/545,760, filed on Aug. 15, 2017.
Prior Publication US 2022/0235389 A1, Jul. 28, 2022
Int. Cl. C12P 21/00 (2006.01); C12N 9/10 (2006.01); C12N 15/10 (2006.01); C12Q 1/48 (2006.01); G01N 33/68 (2006.01)

CPC C12P 21/005 (2013.01) [C12N 9/1051 (2013.01); C12N 15/1044 (2013.01); C12Q 1/48 (2013.01); G01N 33/6851 (2013.01)]

21 Claims

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₋₂is selected from Gly, Asn, and Tyr; X₋₁is selected from Gly and Ala; X₊₁is selected from Trp, Val, His, Ala, and Ile; and X₊₃is 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.