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.

Provided herein are enzymatic compositions for protein O-glycosylation and sialylation, methods and systems associated therewith. In particular, the composition for in vivo sialylation of therapeutic proteins. The composition comprises a polypeptide N-acetylgalactosaminyltransferase; a β-1,3-galactosyltransferase; an UDP-Glc/GlcNAc 4-epimerase; a disulfide bond isomerase; and an α-2,3-sialyltransferase or an α-2,6-sialyltransferase. Furthermore, provided herein are compositions for efficient and complete O-glycosylation and di-sialylation of therapeutic proteins.

The present invention, in the field of bioengineering and biotechnology, relates to a method for preparing a recombinant antibody with a unique glycan profile produced by a genome-edited CHO host cell. Specifically, according to a method of the present invention, the TALEN technology is used to edit the FUT8 gene in CHO cells that have been adapted for serum-free suspension growth. The edited CHO host cells can produce recombinant antibodies with a unique glycan profile. The unique glycan profile can be characterized by non-fucosylated N-linked oligosaccharide chains of the antibodies, extremely low N-glycosylation heterogeneity and uniform carbohydrate chains. The antibody prepared by the method of the invention exhibit significantly increased ADCC and greater stability.

The present invention relates to compounds, compositions, and methods are provided for covalently linking a cargo molecule, such as a therapeutic or a diagnostic agent, to a glycan in the Fab region of an antibody. Also provided are methods of modeling and producing antibodies having de novo Fab glycosylation sites. Also provided are antibody carrier conjugates, methods of using the conjugates.

The present invention provides expression vectors for use in an inducible coexpression system, capable of controlled induction of expression of each gene product.

The present application provides methods and uses of O-oligosaccharyltransferase (O-OTases) for generating vaccines. In particular, the present application provides a method of synthesizing a glycoprotein comprising glycosylation of pilin-like protein ComP using a Pg1L.sub.ComP O-OTase. Uses of glycoproteins synthesized by glycosylating ComP using Pg1L.sub.ComP O-OTase, particularly for the preparation of vaccines and the like, including a vaccine to Streptococcus, is also provided.

A genetically modified plant or plant cell with reduced α1,3-fucosyltransferase and β1,2-xylosyltransferase activity compared to a wild type plant or plant cell, wherein less than 10% of the total glycan on a protein produced by the plant or plant cell is α1,3-fucosylated glycan and less than 3% of the total glycan on the protein is β1,2-xylosylated glycan is provided. In one embodiment, the plant or plant cell comprises three T-DNA insertions expressing five copies of RNAi targeting α1,3-fucosyltranserase and three copies of RNAi targeting β1,2xylosyltransferase.

The present invention relates to a process for the enzymatic modification of a glycoprotein. The process comprises the step of contacting a glycoprotein comprising a glycan comprising a terminal GlcNAc-moiety, in the presence of glycosyltransferase that is, or is derived from, a β-(1,4)-N-acetylgalactosaminyltransferase, with a non-natural sugar-derivative nucleotide. The non-natural sugar-derivative nucleotide is according to formula (3):

##STR00001##

wherein A is selected from the group consisting of —N.sub.3, —C(O)R.sup.3, —(CH.sub.2).sub.iC≡C—R.sup.4, —SH, —SC(O)R.sup.8, —SC(O)OR.sup.8, —SC(S)OR.sup.8, —F, —Cl, —Br —I, —OS(O).sub.2R.sup.5, terminal C.sub.2-C.sub.24 alkenyl groups, C.sub.3-C.sub.5 cycloalkenyl groups, C.sub.4-C.sub.8 alkadienyl groups, terminal C.sub.3-C.sub.24 allenyl groups and amino groups. The invention further relates to a glycoprotein obtainable by the process according to the invention, to a bioconjugate that can be obtained by conjugating the glycoprotein with a linker-conjugate, and to β-(1,4)-N-acetylgalactosaminyltransferases that can be used in preparing the glycoprotein according to the invention.

Glycosylated biopharmaceuticals are important in the global pharmaceutical market. Despite the importance of their glycan structures, our limited knowledge of the glycosylation machinery still hinders controllability of this critical quality attribute. To facilitate discovery of glycosyltransferase specificity and predict glycoengineering efforts, here we extend an approach to model biosynthetic pathways for all measured glycans, and the Markov chain modeling is used to learn glycosyltransferase isoform activities and predict glycosylation following glycosyltransferase knock-in/knockout. We apply our methodology to four different glycoengineered therapeutics (i.e., Rituximab, erythropoietin, Enbrel, and alpha-1 antitrypsin) produced in CHO cells, along with o-glycosylation and lipid profiles. Our models accurately predict N-linked glycosylation following glycoengineering and further quantified the impact of glycosyltransferase mutations on reactions catalyzed by other glycosyltransferases. By applying these learned GT-GT interaction rules identified from single glycosyltransferase mutants, our model further predicts the outcome of multi-gene glycosyltransferase mutations on the diverse biotherapeutics. We further apply this to study differential O-glycosylation and lipidomics. Thus, this modeling approach enables rational glycoengineering and the elucidation of relationships between glycosyltransferases and other enzyme classes, thereby facilitating biopharmaceutical research and aiding the broader study of glycosylation to elucidate the genetic basis of complex changes in glycosylation and the lipidome.

Disclosed are new recombinant isoforms of human-like lubricin or PRG4 glycoprotein having outstanding lubrication properties and a novel glycosylation pattern, and methods for their manufacture at high levels enabling commercial production.