Cornell University

Northwestern University

Cornell Research Foundation Inc.

Wisconsin Alumni Research Foundation

Research Development Foundation

Matthew DeLisa

Michael Christopher Jewett

Jessica Carol Stark

Thapakorn Jaroentomeechai

David Putnam

Adam Fisher

Thomas Mansell

Chung-Jr Huang

Susana Mendez

Joseph Rosenthal

George Georgiou

Sydnor T Withers, Iii

Cassandra Guarino

Miguel A Dominguez

Charles H Haitjema

Jae-Young Kim

Anne M Doody

Alyse Portnoff

Robert Conrado

Dujduan Waraho

Jeffrey Varner

Dario Mizrachi

Lydia Contreras-Martinez

Amy Karlsson

Hyung-Kwon Lim

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Method for rapid in vitro synthesis of glycoproteins via recombinant production of N-glycosylated proteins in prokaryotic cell lysates

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated carrier proteins. The glycosylated carrier proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated carrier proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated carrier proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Compositions and methods for rapid in vitro synthesis of bioconjugate vaccines in vitro via production and N-glycosylation of protein carriers in detoxified prokaryotic cell lysates

Method for rapid in vitro synthesis of glycoproteins via recombinant production of n-glycosylated proteins in prokaryotic cell lysates

Disclosed are components and systems for cell-free glycoprotein synthesis (CFGpS). In particular, the components and systems include and utilize prokaryotic cell lysates from engineered prokaryotic cell strains that have been engineered to enable cell-free synthesis of glycoproteins.

Cell-free glycoprotein synthesis (CFGpS) in prokaryotic cell lysates enriched with components for glycosylation

The present invention is directed to a cell-free system for producing a glycosylated protein. This system comprises an isolated oligosaccharyltransferase capable of transferring a glycan from a lipid carrier molecule to a glycoprotein target, one or more isolated glycans, where each glycan is linked to a lipid carrier molecule, and a glycoprotein target comprising one or more glycan acceptor amino acid residues or a nucleic acid molecule encoding said glycoprotein target. The present invention further relates to kits and methods for producing a glycosylated protein in this cell-free system.

Prokaryote-based cell-free system for the synthesis of glycoproteins

The present invention relates to an isolated chimeric molecule comprising a degradation domain including a eukaryotic U-box motif and a targeting domain capable of immunospecifically directing the degradation domain to a substrate where the targeting domain is heterologous to the degradation domain. A linker couples the degradation domain to the targeting domain. Also disclosed are compositions as well as methods of treating a disease, substrate silencing, screening agents for therapeutic efficacy against a disease, and methods of screening for disease biomarkers.

Targeted protein silencing using chimeras between antibodies and ubiquitination enzymes

The present invention relates to a nucleic acid construct having a chimeric nucleic acid molecule comprising a first nucleic acid moiety encoding an amphipathic shield domain protein; a second nucleic acid moiety encoding an integral membrane protein; and a third nucleic acid moiety encoding a water soluble expression decoy protein, as well as a chimeric nucleic acid molecule having a first nucleic acid moiety encoding an amphipathic shield domain protein and a second nucleic acid moiety encoding an integral membrane protein. The present invention further relates to an expression vector, a host cell, and a protein encoded by the nucleic acid construct. Also disclosed is a method of recombinantly producing an integral membrane protein in soluble form.

Nucleic acids, vectors, host cells, and methods for recombinantly producing water-soluble membrane proteins

The present invention relates to a probiotic cell transformed with a construct suitable to overexpress and display on the surface of the probiotic cell a fusion protein comprising at least a portion of a transport protein coupled to at least a portion of one or more antigenic proteins or peptides. Probiotic-derived vesicles displaying this fusion protein as well as methods of inducing an immune response using the probiotic cells or vesicles are also disclosed.

Subunit vaccine delivery platform for robust humoral and cellular immune responses

Method for rapid in vitro synthesis of glycoproteins via recombinant production of-glycosylated proteins in prokaryotic cell lysates

The present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality. This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, with the deoxyribonucleic acid molecule being coupled to a stall sequence. A host cell is transformed with the construct and then cultured under conditions effective to form, within the host cell, a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and ribosomes. The protein in the complex is in a properly folded, active form and the complex is recovered from the cell.

Protein discovery using intracellular ribosome display

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutantstrains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

The present invention relates to a prokaryotic host cell comprising eukaryotic glycosyltransferase activity, where the eukaryotic glycosyltransferase activity is eukaryotic dolichyl-linked UDP-GlcNAc transferase activity and eukaryotic mannosyltransferase activity. Also disclosed is a method of producing a glycosylated protein by providing a prokaryotic host cell comprising the eukaryotic glycosyltransferase activity and culturing the prokaryotic host cell under conditions effective to produce a glycosylated protein. Another aspect of the present invention pertains to a method for screening bacteria or bacteriophages by expressing one or more glycans on the surface of a bacteria, attaching a label on the one or more glycans on the surface of the bacteria or on the surface of a bacteriophage derived from the bacteria, and analyzing the label in a high-throughput format. A glycosylated antibody comprising an Fv portion which recognizes and binds to a native antigen and an Fc portion which is glycosylated at a conserved asparagine residue is also disclosed.

Glycosylated protein expression in prokaryotes

Growing community of inventors

Ithaca, NY, United States of America

Matthew DeLisa