US 12,305,211 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, Evanston, IL (US)
Assigned to Northwestern University, Evanston, IL (US)
Filed by Northwestern University, Evanston, IL (US)
Filed on Jan. 4, 2024, as Appl. No. 18/404,392.
Application 18/404,392 is a continuation of application No. 16/639,500, granted, now 11,898,187, previously published as PCT/US2018/000185, filed on Aug. 15, 2018.
Claims priority of provisional application 62/545,760, filed on Aug. 15, 2017.
Prior Publication US 2024/0376510 A1, Nov. 14, 2024
This patent is subject to a terminal disclaimer.
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)] 20 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 an N-glycosyltransferase or that has been modified to include a heterologous amino acid motif that includes an asparagine that is glycosylated by an N-glycosyltransferase, wherein the amino acid motif or heterologous amino acid motif comprises a sequence 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 a cell or in a CFPS reaction the N-glycosyltransferase; and
(c) reacting the polypeptide and the N-glycosyltransferase in the presence of a sugar donor, wherein the N-glycosyltransferase 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.