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US 12,305,211 B2Granted
Protein glycosylation sites by rapid expression and characterization of N-glycosyltransferases
AssigneeNorthwestern University
InventorsMichael Christopher Jewett, Weston K. Kightlinger, Liang Lin, Milan Mrksich
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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.
Claims20
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.
4 more elements
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 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, the amino acid motifs or heterologous amino acid motifs optionally comprising an amino acid sequence selected from SEQ ID NOs:
- 1-549, wherein at least one of the two or more different amino acid motifs or two or more different heterologous amino acid motifs comprise 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 one or more cells or in one or more CFPS reactions the two or more different N-glycosyltransferases, wherein the two or more different N-glycosyltransferases 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 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 simultaneously for example in the same cell or CFPS reaction, or sequentially for example in two or more different cells or two or more different CFPS reactions, and wherein the two or more different N-glycosyltransferases 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.
5 more elements
Dependencies are inferred from the claim text.