US 11,725,224 B2
Methods for co-activating in vitro non-standard amino acid (nsAA) incorporation and glycosylation in crude cell lysates
Michael Christopher Jewett, Evanston, IL (US); Jessica Carol Stark, Evanston, IL (US); and Jasmine Hershewe, Evanston, IL (US)
Assigned to Northwestern University, Evanston, IL (US)
Appl. No. 17/48,331
Filed by NORTHWESTERN UNIVERSITY, Evanston, IL (US)
PCT Filed Apr. 16, 2019, PCT No. PCT/US2019/027733
§ 371(c)(1), (2) Date Oct. 16, 2020,
PCT Pub. No. WO2019/204346, PCT Pub. Date Oct. 24, 2019.
Claims priority of provisional application 62/658,181, filed on Apr. 16, 2018.
Prior Publication US 2021/0147894 A1, May 20, 2021
Int. Cl. C12P 21/00 (2006.01); C12N 1/20 (2006.01); C12N 9/02 (2006.01); C12N 9/10 (2006.01); C12N 9/16 (2006.01); C12N 9/88 (2006.01); C12N 9/00 (2006.01)
CPC C12P 21/005 (2013.01) [C12N 1/20 (2013.01); C12N 9/0051 (2013.01); C12N 9/1051 (2013.01); C12N 9/16 (2013.01); C12N 9/88 (2013.01); C12N 9/93 (2013.01); C12Y 108/01007 (2013.01); C12Y 301/21001 (2013.01); C12Y 402/01047 (2013.01)] 9 Claims
 
1. A cell-free glycoprotein synthesis (CFGpS) reaction mixture in a single reaction vessel comprising:
(1) a cell lysate from a genomically recoded strain of Escherichia coli (E. coli) comprising:
(a) a mutation in an endogenous (prfA) gene resulting in a deficiency the encoded release factor 1 protein;
(b) a mutation in an endogenous O-antigen ligase (waaL) gene encoding an O-antigen ligase protein resulting in a deficiency of the O-antigen ligase protein;
(c) a mutation in an endogenous DNA-specific endonuclease I (endA) gene encoding a DNA-specific endonuclease I protein resulting in a deficiency of the endonuclease I protein;
(d) a mutation in an endogenous GDP-mannose 4,6-dehydratase (gmd) gene encoding a GDP-mannose 4,6-dehydratase protein resulting in reduced expression and/or activity of GDP-mannose 4,6-dehydratase as compared to wild-type E. coli; and
(e) an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO);
(2) a DNA template for expressing a sequence-defined amino acid polymer, a DNA-dependent RNA polymerase for transcribing an mRNA encoding the sequence-defined amino acid polymer, nucleotide triphosphates, amino acids, and an energy source; and
(3) a non-standard amino acid (nsAA), an orthogonal amino-acyl tRNA synthetase (aaRS), or both of an nsAA and an aaRS, wherein the non-standard amino acid (nsAA) comprises a moiety that reacts with a corresponding moiety on a saccharide to conjugate the nsAA to the saccharide; and
(4) one or more components for performing a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.