Multiple Protease Deficient Filamentous Fungal Cells and Methods of Use Thereof
AssigneesGlykos Finland Oy, Novartis AG
InventorsChristopher Landowski, Anne Huuskonen, Ann Westerholm-Parvinen, Markku Saloheimo, Anne Kanerva, Jukka Hiltunen
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Abstract
The present disclosure relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells.
Claims10
1-15. (canceled)
A Myceliophthora thermophila filamentous fungal cell comprising at least one endogenous sep protease having no protease activity and a recombinant polynucleotide encoding a mammalian polypeptide, wherein the gene encoding endogenous sep protease of SEQ ID NO:
- 792, 794 or 797 comprises a mutation that eliminates the activity of the endogenous sep protease.
The Myceliophthora thermophila cell of claim 34 comprising at least three, four, five, six, seven or eight endogenous proteases having eliminated activity.
The Myceliophthora thermophila cell of claim 35, wherein the proteases are selected from the group consisting of aspartic proteases, trypsin-like serine proteases, subtilisin proteases, glutamic proteases, and metalloproteases.
The Myceliophthora thermophila cell of claim 36, wherein the Myceliophthora thermophila cell further has no detectable protease activity of pep4 of SEQ ID NO:
- 499, slp2 of SEQ ID NO:
- 540, and slp3 of SEQ ID NO:
- 546.
The Myceliophthora thermophila cell of claim 34, wherein the mammalian polypeptide is selected from the group consisting of an antibody, a growth factor, an interferon, a cytokine, and an interleukin.
The Myceliophthora thermophila cell of claim 34, wherein the Myceliophthora thermophila cell further comprises ALG3 having eliminated activity.
The Myceliophthora thermophila cell of claim 34, further comprising
- a) an N-acetylglucosaminyltransferase I catalytic domain, and, optionally,
- b) an N-acetylglucosaminyltransferase II catalytic domain.
A method of improving stability of a mammalian polypeptide, comprising
- a) providing the Myceliophthora thermophila cell of claim 34, and
- b) culturing the Myceliophthora thermophila cell such that the mammalian polypeptide is expressed,
- wherein the mammalian polypeptide exhibits increased stability compared to the mammalian polypeptide produced in a corresponding parental Myceliophthora thermophila cell in which the proteases do not have reduced activity.
A method of making a mammalian polypeptide, comprising
- a) providing the Myceliophthora thermophila cell of claim 34;
- b) culturing the Myceliophthora thermophila cell such that the mammalian polypeptide is expressed;
- and,
- c) purifying the mammalian polypeptide.
Dependencies are inferred from the claim text.