	US 7,449,308 B2
	Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
Tillman U. Gerngross, Hanover, N.H. (US); Stefan Wildt, Lebanon, N.H. (US); Byung-Kwon Choi, Norwich, Vt. (US); Juergen Hermann Nett, Grantham, N.H. (US); Piotr Bobrowicz, White River Junction, Vt. (US); Stephen R. Hamilton, Enfield, N.H. (US); and Robert C. Davidson, Enfield, N.H. (US)
Assigned to GlycoFi, Inc., Lebanon, N.H. (US)
Filed on Feb. 20, 2003, as Appl. No. 10/371,877.
Application 10/371877 is a continuation in part of application No. 09/892591, filed on Jun. 27, 2001, granted, now 7,029,872.
Claims priority of provisional application 60/214358, filed on Jun. 28, 2000.
Claims priority of provisional application 60/215638, filed on Jun. 30, 2000.
Claims priority of provisional application 60/279997, filed on Mar. 30, 2001.
Prior Publication US 2004/0018590 A1, Jan. 29, 2004
Int. Cl. C12N 15/00 (2006.01)

U.S. Cl. 435—69.1 [435/254.2; 435/320.1; 435/455]

41 Claims

1. A method for producing a recombinant glycoprotein comprising complex or hybrid N-glycans in a yeast or filamentous fungus host cell that does not display a 1,6 mannosyltransferase activity, the method comprising the step of introducing into the host cell a nucleic acid encoding an alpha-1,2 mannosidase enzyme, the enzyme comprising:

a) an alpha-1,2 mannosidase catalytic domain; fused to

b) a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the enzyme to the ER or Golgi apparatus of the host cell where the enzyme functions optimally;

whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, greater than 30 mole percent of the N-glycan structures attached thereto are converted to a Man₅GlcNAc₂glycoform that can serve as a substrate for additional glycosylation enzymes so that the glycoprotein comprising complex or hybrid N-glycans can be formed.