Method of producing recombinant proteins with mannose-terminated N-glycans
AssigneeAgency for Science, Technology and Research
InventorZhiwei Song
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Abstract
We describe a method of expressing a recombinant protein comprising mannose-terminated N-glycans from a host cell, the method comprising: (a) introducing a nucleic acid encoding a recombinant protein into a Chinese Hamster Ovary (CHO) cell comprising a mutation in the GnT 1 gene (GenBank Accession Number AF343963) leading to loss of GnT 1 function; and (c) expressing the recombinant protein from the host cell, in which the expressed recombinant protein comprises a mannose-terminated glycan structure, and in which the method does not include a step of introducing functional GnT-1 into the host cell. The method may be used for producing recombinant glucocerebrosidase with a mannose-terminated glycan structure, suitable for treatment or prevention of Gaucher's Disease.
Claims23
A method of expressing a recombinant protein comprising mannose-terminated N-glycans from a host cell, the method comprising:
- (a) introducing a nucleic acid encoding a recombinant protein into a host cell comprising a mutation in the GnT I gene leading to loss of GnT I function, the host cell is a Chinese Hamster Ovary (CHO) cell selected with Ricinus communis agglutinin I (RCA-I) or a descendent thereof;
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- (b) expressing the recombinant protein from the host cell, in which the expressed recombinant protein comprises mannose-terminated N-glycans structure
- in which the method does not include a step of introducing functional GnT I into the host cell.
A method of expressing a recombinant protein comprising mannose-terminated N-glycans from a host cell, the method comprising:
- (a) introducing a nucleic acid encoding a recombinant protein into a host cell comprising a mutation in the GnT I gene leading to loss of GnT I function, wherein the host cell is a Chinese Hamster Ovary (CHO) cell selected with Ricinus communis agglutinin I (RCA-I) or a descendent thereof;
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- (b) expressing the recombinant protein from the host cell, in which the expressed recombinant protein comprises mannose-terminated N-glycans structure;
- wherein the method does not include a step of introducing functional GnT I into the host cell and the CHO cell was selected with RCA-I by culturing CHO cells in the presence of Ricinus communis agglutinin I (RCA-I) and selecting cells which survive the culture.
A JW152 cell (deposited at ATCC under the Budapest Treaty as accession number PTA-9657) comprising a nucleic acid sequence encoding a recombinant protein.
A method of expressing a recombinant protein comprising mannose-terminated N-glycans from a JW152 host cell, the method comprising:
- (a) providing a JW152 host cell (deposited at ATCC under the Budapest Treaty as accession number PTA-9657) comprising a nucleic acid encoding a protein having glucocerebrosidase activity, MUC1, HER2/neu or carcinoembryonic antigen (CEA);
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- (b) allowing the recombinant protein to be expressed from the JW152 host cell in which the method does not include a step of introducing functional GnT I into the host cell.
Dependencies are inferred from the claim text.