Protein glycosylation sites by rapid expression and characterization of N-glycosyltransferases
Inventors
Jewett, Michael Christopher • Kightlinger, Weston K. • Lin, Liang • Mrksich, Milan
Assignees
Publication Number
US-12305211-B2
Publication Date
2025-05-20
Expiration Date
2038-08-15
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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.
Core Innovation
The invention provides components, systems, and methods for synthesizing glycoproteins or recombinant glycoproteins in vitro and in vivo, focusing specifically on identifying and using amino acid glycosylation tag motifs (GlycTags) for N-glycosyltransferases (NGTs). These methods allow the modification of the amino acid sequence of a protein to include a GlycTag, which can then be glycosylated by an expressed N-glycosyltransferase in the presence of a suitable sugar donor.
This approach addresses the problem that existing methods cannot sufficiently explore the experimental landscapes required to accurately predict and design glycosylation sites for specific glycosyltransferases. The patent describes a systematic workflow, including GlycoSCORES, which utilizes cell-free protein synthesis (CFPS) and self-assembled monolayers for mass spectrometry (SAMDI-MS), to analyze and optimize peptide acceptor motifs for NGTs with high throughput and depth. This enables the design of primary amino acid sequences for efficient, site-specific glycosylation.
The invention makes it possible to prepare glycoproteins and recombinant glycoproteins by expressing modified amino acid sequences—including those with identified GlycTags—in vitro (e.g., in CFPS) or in vivo (e.g., recombinant prokaryotic or eukaryotic cells). The system supports both single and multiple distinct glycosylation sites, including site-specific control with unique sequence:enzyme pairs, and provides methods for selecting glycosylation motifs via high-throughput screening using peptide libraries and mass spectrometry-based analysis.
Claims Coverage
The patent claims cover two main inventive features: the synthesis of glycoproteins using defined heterologous glycosylation tag motifs for N-glycosyltransferases, and the parallel or sequential use of two or more different N-glycosyltransferases with different peptide specificities.
Synthesis of glycoproteins or recombinant glycoproteins using heterologous glycosylation tag motifs for N-glycosyltransferases
A method comprising: - Expressing, in a cell or cell-free protein synthesis (CFPS) reaction, a polypeptide that comprises the amino acid sequence of a target protein which either naturally includes, or has been modified to include, an amino acid motif containing asparagine, that is glycosylated by an N-glycosyltransferase. - The amino acid motif or heterologous motif has the sequence X−2-X−1-N-X+1-S/T-X+3, with X−2 selected from Gly, Asn, and Tyr; X−1 from Gly and Ala; X+1 from Trp, Val, His, Ala, and Ile; and X+3 from Thr, Met, and Phe. - Expressing an N-glycosyltransferase in a cell or CFPS reaction. - Reacting the polypeptide with the N-glycosyltransferase in the presence of a sugar donor, resulting in glycosylation at the specified motif by the N-glycosyltransferase. The claim encompasses various types of target proteins (both eukaryotic and prokaryotic), supports in vitro (including prokaryotic-based) and in vivo production, specifies the use of particular N-glycosyltransferases, and includes optional immobilization of the polypeptide on a solid support prior to glycosylation.
Parallel or sequential site-specific control using two or more different N-glycosyltransferases and different glycosylation tag motifs
A method comprising: - Expressing, in a cell or cell-free protein synthesis (CFPS) reaction, a polypeptide comprising the amino acid sequence of a target protein with two or more different amino acid motifs (each containing asparagine) that are glycosylated by two or more different N-glycosyltransferases. At least one motif is of the form X−2-X−1-N-X+1-S/T-X+3, as defined above. - Expressing two or more different N-glycosyltransferases, either simultaneously in the same cell/CFPS reaction or sequentially in different cells/CFPS reactions. - Reacting the polypeptide with the two or more N-glycosyltransferases and two or more sugar donors (the sugars may be the same or different), either simultaneously or sequentially. Each N-glycosyltransferase glycosylates its specific motif(s) on the polypeptide to generate a glycoprotein with multiple, site-specific glycosylation events. This feature claims the ability to introduce multiple identical, distinct, and/or non-naturally occurring glycans into a protein by specifically choosing unique sequence:enzyme pairs to allow for orthogonal, parallel, or independent glycosylation. The claim allows optional immobilization of the polypeptide on a solid support prior to glycosylation.
The claims provide broad protection for the synthesis of glycoproteins or recombinant glycoproteins through rational design and expression of glycosylation motifs recognized by defined N-glycosyltransferases, including multipoint, site-specific control using distinct sequence/enzyme pairs. The inventive features cover both the basic and advanced methods for in vitro and in vivo production using selectable motif/enzyme combinations.
Stated Advantages
N-glycosyltransferase glycosylation systems allow efficient modification of polypeptides without requiring a eukaryotic host or membrane-bound substrates and enzymes.
The technology enables the first glycosylation of human IgG Fc fragment in E. coli cytoplasm by using redesigned sequences for efficient NGT-directed glycosylation.
The system allows production of glycoproteins in the bacterial cytoplasm, eliminating the need for transport to the bacterial periplasm that is required by oligosaccharyltransferase glycosylation methods.
The innovation increases the diversity of glycoproteins producible in bacteria, which is a preferred industrial host.
The SAMDI-MS approach enables rapid, high-throughput study of thousands of peptides and reaction conditions for enzyme characterization, compared to current methods that are limited to much fewer peptides.
GlycoSCORES enables high-throughput and quantitative analysis for GT peptide specificity, allowing systematic and rational design of glycosylation sites for diverse proteins in vitro and in vivo.
The methods support the synthesis and engineering of site-specifically glycosylated proteins, including protein therapeutics and vaccines.
The system can control glycosylation at multiple sites within the same protein using unique glycosylation tags and corresponding transferases, enabling the synthesis of proteins with defined and distinct glycan structures at each site.
Documented Applications
Design of therapeutic polypeptide amino acid sequences for improved glycosylation by an N-linked glycosyltransferase in vitro or in a cell.
High-throughput characterization of glycosyltransferases peptide specificities or engineering of glycosyltransferases for alternative peptide or sugar specificities.
Production of high titers of proteins in industrial bacterial host organisms that are glycosylated site-specifically in the bacterial cytoplasm.
Synthesis and engineering of glycoproteins and recombinant glycoproteins, including protein therapeutics and vaccines, with defined glycosylation patterns.
Site-specific control of glycosylation at multiple sites in a single target protein using unique glycosylation enzyme-motif pairs.
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