Granted Patent
B2
US 12,503,715 · App. 17/771,590 · Granted Dec 23, 2025
Cell-free extract preparation protocol for enrichment of membrane vesicles and increased glycoprotein yields
Inventors:
Michael C. Jewett (Evanston, IL); Jasmine M. Hershewe (Evanston, IL); Katherine F. Warfel (Evanston, IL)
Assignee:
Northwestern University
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Abstract
Disclosed are protocols for preparing cell-free extracts preparation protocols that are enriched in membrane vesicles and use of the disclosed extract in cell-free glycoprotein synthesis methods and platforms for increasing glycoprotein yields.
Claims (7)
1 . A method of synthesizing and glycosylating a target protein in a cell-free glycoprotein synthesis (CFGpS) reaction in vitro, the method comprising:
contacting an expression vector encoding the target protein to an Escherichia coli ( E. coli ) cell S12 homogenized lysate, wherein the E. coli cells are engineered to express heterologous glycosylation machinery:
i) transcribing and translating the target protein in the E. coli S12 homogenized lysate; and
ii) glycosylating the target protein in the E. coli S12 homogenized lysate,
wherein the lysate comprises membrane vesicles enriched in the heterologous glycosylation machinery as compared to an S30 E. coli homogenized lysate, or an S12 or S30 sonicated E. coli lysate.
2 . The method of claim 1 , wherein the glycosylation machinery A comprises one or more proteins selected from PglB, PglO, STT3, NarX, PR and CB1.
3 . The method of claim 1 , wherein the glycosylation machinery comprises a transmembrane oligosaccharyltransferase selected from PglB, PglO, and STT3.
Assignments (2)
CONFIRMATORY LICENSE
Recorded Jan 10, 2023
From: NORTHWESTERN UNIVERSITY
To: DEFENSE THREAT REDUCTION AGENCY, US DOD
Reel/Frame 062337/0356 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Apr 26, 2022
From: JEWETT, MICHAEL C.; HERSHEWE, JASMINE M.; WARFEL, KATHERINE F.
To: NORTHWESTERN UNIVERSITY
Reel/Frame 059738/0472 →
Continuity (2)
Provisional Application
62926166
· Oct 25, 2019
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