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US 8,299,228 B2Granted
Expression of Class 2 mannosidase and Class III mannosidase in lower eukaryotic cells
AssigneeGlycoFi, Inc.
InventorStephen Hamilton
Life of this patent
Estimating the term…
Abstract
A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.
Claims15
A nucleic acid molecule encoding a chimeric mannosidase enzyme comprising
- (a) a D. melanogaster mannosidase II catalytic domain fused to a cellular targeting signal peptide selected from the group consisting of Gls1-s, Mns1-s, Mns1-m, S.Sec-s, S.Sec-m, S.Sec-1, P.Sec-s, P.Sec-m, Mnn9-s, Van1-s, Van1-m, Van1-1, Anp1-s, Anp1-m, Anp1-1, Hoc1-s, Hoc1-m, Hoc1-1, Mnn10-m, Mnn11-s, Mnt1-m, Ktr1-s, Ktr2-s, Gnt1-s, Gnt1-m, Gnt1-1, Mnn2-s, Mnn2-1, Mnn5-m, Mnn1-s, Mnn1-m, Mnn1-1, Mnn6-s, and Mnn6-m or
- (b) a C. elegans mannosidase II catalytic domain fused to a cellular targeting signal peptide selected from the group consisting of Gls1-s, Mns1-s, Mns1-m, S.Sec-s, S.Sec-m, S.Sec-1, P.Sec-s, Van1-s, Van1-m, Van1-1, Anp1-s, Hoc1-m, Mnn10-s, Mnn10-m, Mnn10-1, Mnn11-s, Mnn11-m, Mnt1-s, Mnt1-m, Mnt1-1, D2-s, D2-m, D9-m, J3-m, Ktr2-s, Gnt1-s, Gnt1-m, Mnn2-s, Mnn2-m, Mnn2-1, Mnn5-s, Mnn5-m, Mnn1-s, Mnn1-m and Mnn6-m.
An expression system comprising:
- a plasmid comprising:
- (i) a nucleic acid molecule encoding a D. melanogaster mannosidase II catalytic domain fused to a cellular targeting signal peptide selected from the group consisting of Gls1-s, Mns1-s, Mns1-m, S.Sec-s, S.Sec-m, S.Sec-1, P.Sec-s, P.Sec-m, Mnn9-s, Van1-s, Van1-m, Van1-1, Anp1-s, Anp1-m, Anp1-1, Hoc1-s, Hoc1-m, Hoc1-1, Mnn10-m, Mnn11-s, Mnt1-m, J3-m, Ktr1-s, Ktr2-s, Gnt1-s, Gnt1-m, Gnt1-1, Mnn2-s, Mnn2-m, Mnn2-1, Mnn5-m, Mnn1-s, Mnn1-m, Mnn1-1, Mnn6-s, and Mnn6-m or
- (ii) a nucleic acid molecule encoding a C. elegans mannosidase II catalytic domain fused to a cellular targeting signal peptide selected from the group consisting of Gls1-s, Mns1-s, Mns1-m, S.Sec-s, S.Sec-m, S.Sec-1, P.Sec-s, Van1-s, Van1-m, Van1-1, Anp1-s, Hoc1-m, Mnn10-s, Mnn10-m, Mnn10-1, Mnn11-s, Mnn11-m, Mnt1-s, Mnt1-m, Mnt1-1, D2-s, D2-m, D9-m, J3-m, Ktr2-s, Gnt1-s, Gnt1-m, Mnn2-s, Mnn2-m, Mnn2-1, Mnn5-s, Mnn5-m, Mnn1-s, Mnn1-m, and Mnn6-m;
- and
- (iii) a targeting sequence that directs insertion of the plasmid vector into a particular location of the genome of the host cell by homologous recombination.
2 more elements
Dependencies are inferred from the claim text.