# US 8,771,989 B2 · Vectors and yeast strains for protein production: Ca2+ ATPase overexpression

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- Number: US 8,771,989 B2
- Document ID: US8771989B2
- Status: Granted
- Kind: B2
- Application number: 13/938,603
- Filed: 2013-07-10
- Published: 2013-11-28
- Granted: 2014-07-08
- Earliest claimed: 2008-08-12
- Assignees: GlycoFi, Inc.
- Applicants: GlycoFi, Inc.
- Inventors: Byung-Kwon Choi
- Web page: https://patentgrep.com/patent/US8771989B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12P 21/005: {Glycopeptides, glycoproteins}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Lower eukaryote host cells in which an endogenous or heterologous Ca2+ ATPase is overexpressed are described. Also described are lower eukaryote host cells in which a calreticulin and/or ERp57 protein are overexpressed. These host cells are useful for producing recombinant glycoproteins that have reduced O-glycosylation.

## Claims

### Claim 1

A method for producing a recombinant antibody having reduced O-glycosylation comprising:
(a) providing a Pichia pastoris host comprising a deletion or disruption of a β-mannosyltransferase gene and at least one phosphomannosyl transferase gene and a nucleic acid molecule encoding at least one endogenous or exogenous Ca2+ ATPase wherein expression of the Ca2+ ATPase in the host cell is ectopic;
(b) introducing a nucleic acid molecule into the host cell encoding the recombinant antibody: and
(c) growing the host cell under conditions suitable for producing the recombinant antibody.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid molecule comprises an open reading frame encoding the Ca2+ ATPase operably linked to a heterologous promoter.

### Claim 3 (depends on 1)

The method of claim 1, wherein the function of at least one endogenous gene encoding a chaperone protein has been reduced, disrupted, or deleted; and a nucleic acid molecule encoding at least one mammalian homolog of the chaperone protein is expressed in the host cell.

### Claim 4 (depends on 1)

The method of claim 1, wherein the host cell further includes a nucleic acid molecule encoding an Erp57 protein and/or a nucleic acid molecule encoding a calreticulin protein.

### Claim 5 (depends on 1)

The method of claim 1, wherein the host cell is engineered to reduce or eliminate the function of at least one endogenous Pichia pastoris gene encoding a protein O-mannosyltransferase (PMT) protein.

### Claim 6 (depends on 5)

The method of claim 5, wherein the PMT protein is selected from the group consisting of PMT1 and PMT4.

### Claim 7 (depends on 3)

The method of claim 3, wherein the wherein heterologous chaperone protein is human PDI.