Methods of producing glycosylated proteins
Described herein are methods of producing glycosylated proteins in vitro and in vivo. The methods include using host cells to produce glycosylated proteins. Also described herein are glycosylated proteins produced using such methods and uses thereof.
1. A host cell comprising (i) a heterologous nucleic acid that encodes a target protein comprising an N-glycosylation consensus sequence of the formula Asn-X-Ser(Thr), wherein X can be any amino acid residue except Pro; (ii) a nucleic acid that encodes a heterologous N-glycosyltransferase (NGT) that adds glucose to an amino acid residue present in said N-glycosylation consensus sequence; (iii) a nucleic acid that encodes a heterologous galactosyltransferase that catalyzes addition of a galactose to said glucose; (iv) a nucleic acid that encodes a heterologous sialyltransferase; (v) a nucleic acid that encodes a heterologous polysialyltransferase (polyST); and (vi) a nucleic acid that encodes a heterologous CMP-Neu5Ac synthetase; wherein the host cell is capable of glycosylating the target protein at the N-glycosylation consensus sequence with glucose-galactose primer and polysialylation.
2. The host cell of claim 1 , wherein said heterologous NGT is the NGT of Actinobacillus pleuropneumonias, the NGT of a species of Haemophilus , the NGT of a species of Mannheimia , the NGT of a species of Bibersteinia , or the NGT of a species of Yersinia.
3. The host cell of claim 1 , wherein said heterologous galactosyltransferase is LgtB of Neisseria meningitidis , the LgtB of Neisseria gonorrhoeae , LgtE of Neisseria meningitidis , CgtB of Campylobacter jejuni , WaaX of Escherichia coli , HP0826 of Helicobacter pylori , or a eukaryotic β4Gal-T1.
4. The host cell of claim 1 , wherein said heterologous sialyltransferase is CstII of Campylobacter jejuni , CstI of Campylobacter jejuni , Lst of Neisseria meningitidis , or Lst of Neisseria gonorrhoeae.
5. The host cell of claim 1 , wherein said heterologous poly ST is a poly ST of Neisseria meningitidis or a homolog thereof, Escherichia coli K1 or a homolog thereof, Mannheimania haemolytica or a homolog thereof, or Moraxella nonliquifacien or a homolog thereof.
6. The host cell of claim 1 , wherein said heterologous CMP-Neu5Ac synthetase is SynB of Neisseria meningitidis.
7. The host cell of claim 1 , wherein said host cell is a prokaryotic host cell or eukaryotic host cell.
8. The host cell of claim 1 , wherein said target protein is a bacterial protein, eukaryotic protein, or therapeutic protein.
9. The host cell of claim 1 , wherein said host cell does not comprise an oligosaccharyltransferase (OST).
10. A method for producing a glycosylated target protein that comprises a glucose assembled at an amino acid residue present in an N-glycosylation consensus sequence; wherein said glucose is linked to a monosaccharide, said method comprising (i) culturing the host cell of claim 1 under conditions suitable for protein production and (ii) isolating said target protein.
11. A method for producing a sialylated target protein, comprising (i) culturing the host cell of claim 1 under conditions suitable for protein production and (ii) isolating said sialylated target protein.
12. A method for producing a polysialylated target protein, comprising (i) culturing the host cell of claim 1 under conditions suitable for protein production and (ii) isolating said polysialylated target protein.
13. A kit comprising the host cell of claim 1 .