Pichia pastoris strains for producing predominantly homogeneous glycan structure
First Claim
1. An engineered strain of Pichia pastoris, comprising:
- a mutant OCH1 allele which is transcribed into a mRNA coding for a mutant OCH1 protein,wherein said mutant OCH1 protein comprises a catalytic domain that (i) comprises residues 45-404 of the wild type OCH1 protein of the amino acid sequence of SEQ ID NO;
2, or (ii) is at least 90% identical to the amino acids corresponding to residues 45-404 of the wild type OCH1 protein of SEQ ID NO;
2 and has α
-1, 6-mannosyltransferase activity;
wherein said mutant OCH1 protein lacks an N-terminal sequence for targeting the mutant OCH1 protein to the Golgi apparatus; and
wherein said engineered strain is deficient in a protease.
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Abstract
Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., α-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.
8 Citations
16 Claims
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1. An engineered strain of Pichia pastoris, comprising:
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a mutant OCH1 allele which is transcribed into a mRNA coding for a mutant OCH1 protein, wherein said mutant OCH1 protein comprises a catalytic domain that (i) comprises residues 45-404 of the wild type OCH1 protein of the amino acid sequence of SEQ ID NO;
2, or (ii) is at least 90% identical to the amino acids corresponding to residues 45-404 of the wild type OCH1 protein of SEQ ID NO;
2 and has α
-1, 6-mannosyltransferase activity;wherein said mutant OCH1 protein lacks an N-terminal sequence for targeting the mutant OCH1 protein to the Golgi apparatus; and wherein said engineered strain is deficient in a protease. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification