Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

Patent metadata

Field	Value
Publication number	US-9873746-B2
Application number	US-201113070583-A
Country	US
Kind code	B2
Filing date	Mar 24, 2011
Priority date	Oct 22, 2003
Publication date	Jan 23, 2018
Grant date	Jan 23, 2018

Abstract

Methods are provided for the synthesis and secretion of recombinant proteins preferably large mammalian proteins or hetero-multimeric proteins at high levels and for prolonged time in polyploid, preferably diploid yeast. These methods use various mating competent yeast, including Pichia . In a preferred embodiment, a first expression vector is transformed into a first haploid cell; and a second expression vector is transformed into a second haploid cell. The transformed haploid cells, each individually synthesizing a non-identical polypeptide, are identified and then genetically crossed or fused. The resulting diploid strains are utilized to produce and secrete fully assembled and biologically functional hetero-multimeric protein.

First claim

What is claimed is:

1. A diploid Pichia pastoris yeast culture which is cultured under conditions whereby the diploid Pichia pastoris yeast culture expresses and secretes into the culture medium a heterologous multichain polypeptide encoded by one or more heterologous DNAs operably linked to a promoter, wherein said one or more heterologous DNAs are expressed by diploid Pichia pastoris yeast cells comprised in said diploid Pichia yeast culture, wherein the heterologous multichain polypeptide is other than an antibody or antibody fragment that specifically binds to a target antigen,
wherein the density of Pichia pastoris in the culture is at least 100 g/L.

2. The diploid Pichia pastoris yeast culture of claim 1 wherein the density of Pichia pastoris in the culture is at least 200 g/L.

3. The diploid Pichia pastoris yeast culture of claim 1 wherein the density of Pichia pastoris in the culture is at least 300 g/L.

4. The diploid Pichia pastoris yeast culture of claim 1 wherein the density of Pichia pastoris in the culture is at least 400 g/L.

5. The diploid Pichia pastoris yeast culture of claim 1 wherein the density of Pichia pastoris in the culture is at least 500 g/L.

6. The diploid Pichia pastoris yeast culture of claim 1 wherein said one or more heterologous DNAs are comprised on a plasmid.

7. The diploid Pichia pastoris yeast culture of claim 1 wherein said one or more heterologous DNAs are integrated into the chromosomal DNA of said yeast.

8. The diploid Pichia pastoris yeast culture of claim 1 , wherein the heterologous polypeptide is selected from the group consisting of a hormone, growth factor, cytokine, lymphokine, enzyme, receptor, cytotoxin, lymphotoxin, immunotoxin, and a tumor polypeptide.

Assignees

• Cregg James M
• Latham John
• Litton Mark
• Schatzman Randall
• Tolstrorukov Ilya
• Keck Graduate Inst
• Alderbio Holdings Llc

Inventors

• Cregg James M
• Latham John
• Litton Mark
• Schatzman Randall
• Tolstrorukov Ilya

Classifications

• C12N15/81

  for yeasts · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title