主权项 |
1. A method for producing a recombinant sialylated glycoprotein in a Pichia pastoris host cell, the host cell selected or engineered to produce glycoproteins comprising a Gal(1-4)GlcNAc(1-4)Man3GlcNAc2 glycoform, the method comprising the step of transforming into the host cell:
a. a nucleic acid encoding an enzyme having sialyltransferase activity, wherein the nucleic acid encodes a silayltransferase enzyme comprising the amino acid sequence of SEQ ID NO:104 or a catalytic domain thereof; b. a nucleic acid encoding a CMP-sialic acid transporter; and c. one or more nucleic acids encoding a CMP-sialic acid pathway consisting of: a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9-phosphate synthase, and a CMP-sialic acid synthase;wherein, upon passage of the recombinant glycoprotein through the secretory pathway of the host cell, a recombinant glycoprotein comprising a NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNA2 glycoform is produced. |