The invention discloses a recombinant protein (P-selectin glycoprotein ligand-1 and Neural Retina-specific Leucine Zipper) PSGL-1-NRL chimeric protein comprising a Selectin Binding domain and a non-covalent dimerization domain, which is a leucine zipper and is more preferably the leucine zipper domain of the human or mouse Neural Retina-specific Leucine Zipper. The chimeric protein further comprises a covalent dimerization domain with at least one cysteine suitable to form a disulfide bridge with another chimeric protein to form a homodimer. In the chimeric protein, the PSGL-1 domain corresponds to the extracellular region of Human PSGL-1 and is more preferably the selectin binding region of the mature protein. The chimeric protein is correctly post-translationally modified and is efficiently expressed in a mammalian system. It is sulfated, O-linked glycosylated and sialylated and binds P, E and L selectin, allowing in vivo and in vitro targeting for diagnostic or therapeutic purposes.

The present invention provides a method and compositions utilizing the CRISPR system to disrupt a target gene in eukaryotic cells to produce double allele knock outs. The method finds use in producing afucosylated antibodies with enhanced ADCC activity.

The present disclosure relates to methods of obtaining cell with disrupted fucosylation and obtaining afucosylated protein. The present disclosure employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in a protein producing cell line to produce afucosylated protein. The resulting protein, specifically the resulting monoclonal antibody is completely afucosylated and reveals higher degree of antibody dependent cellular cytotoxicity.

The present invention provides a method and compositions utilizing the CRISPR system to disrupt a target gene in eukaryotic cells to produce double allele knock outs. The method finds use in producing afucosylated antibodies with enhanced ADCC activity.

A quadruple mutant (qm) plant is deficient of functions of core 1,3-fucosyltransferase A (FucTA), core 1,3-fucosyltransferase B (FucTB), 1,2-xylosyltransferase (XylT), and 1,2-N-acetylglucosaminyltransferase II (GnTII), and produces a protein containing hypoallergenic pauci-mannose-type N-glycan that does not include 1,3-fucose and 1,2-xylose residues. A method for producing a transgenic plant for a production of a protein containing hypoallergenic pauci-mannose-type N-glycan that does not include 1,3-fucose and 1,2-xylose residues but includes 1,6-fucose residue includes preparing the quadruple mutant (qm) plant, and transforming the quadruple mutant plant with a recombinant vector containing a gene encoding the human-derived 1,6-fucosyltransferase (FUT8) protein to overexpress FUT8 gene, and selecting a transgenic plant which is deficient of the functions of FucTA, FucTB, XylT and GnTII proteins.

A method of selecting cells having zero fucose level useful as host cells for expressing recombinant proteins is disclosed. The method comprises: (d) introducing genetic mutations into a population of CHO cells by contacting the cells with a methotrexate (MTX), (e) contacting the population of CHO cells comprising mutated cells with a non-toxic fucose binding agent for an amount of time that allows binding of the fucose binding agent to a fucose moiety on a cell membrane of the population of cells, wherein the amount of time does not allow killing of the cells; and (f) depleting from the population of cells comprising mutated cells, a subpopulation of cells which bind the fucose binding agent, thereby selecting cells useful as host cells for expressing recombinant proteins, the selected cells having zero fucose content. There are also disclosed cells and cell lines useful as host cells for expressing recombinant proteins.