Methods for controlling protease production
AssigneeRoal Oy
InventorsMarja Paloheimo, Susanna Mäkinen, Peter Punt, Kari Juntunen, Terhi Puranen, Jari Vehmaanperä
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Abstract
The present description is related to the field of protein production. It introduces novel host cells with low protease activity, a novel protease regulator, its use in expression systems and protein production, and a method of producing host cells for protein production.
Claims16
A host cell comprising at least one inactivated chromosomal gene wherein the at least one inactivated chromosomal gene comprises a nucleic acid sequence encoding a polypeptide comprising a sequence having at least 90% sequence identity with the amino acids 402-533 of SEQ ID NO:
- 13;
- the at least one inactivated chromosomal gene is inactivated by disruption, inhibition of translation or transcription of the chromosomal gene, at least partial deletion, truncation, deletion, insertion, mutation, or silencing, by RNAi, or by CRISPR/Cas9 technology;
- and the host cell has reduced protease activity compared to the host cell without said inactivation.
A method for making a host cell for protein production comprising suppressing endogenous protease gene expression in a host cell by at least partially inhibiting transcription or translation of the polynucleotide encoding a protein comprising an amino acid sequence having at least 90% sequence identity to amino acids 402-533 of SEQ ID NO:
- 13.
A modified polynucleotide encoding a protein comprising an amino acid sequence having at least 90% sequence identity to amino acids 402-533 of SEQ ID NO:
- 13, wherein inactivation of a chromosomal gene comprising the polynucleotide results into suppression of production of endogenous proteases of the host cell compared to a host cell wherein the chromosomal gene comprising the polynucleotide is not inactivated;
- and wherein the modified polynucleotide contains at least one modification resulting into at least partial incapability of a gene product obtainable by transcribing and/or translating a chromosomal gene comprising the modified polynucleotide to induce expression of endogenous proteases in a host cell.
A method of inducing protease expression in a host cell by providing
- a protease regulator inside or in contact with the host cell;
- wherein the protease regulator is selected from the group consisting of:
- a) a polypeptide, or a gene product encoded by the coding sequence of a polynucleotide encoding a protein, comprising an amino acid sequence having at least 90% sequence identity to amino acids 402-533 of SEQ ID NO:
- 13, wherein inactivation of a chromosomal gene comprising the polynucleotide results into suppression of production of endogenous proteases of the host cell compared to a host cell wherein the chromosomal gene comprising the polynucleotide is not inactivated;
- b) a polypeptide, or a gene product encoded by the coding sequence of a polynucleotide encoding a protein, comprising an amino acid sequence having at least 90% sequence identity to amino acids 402-533 of SEQ ID NO:
- 13, wherein inactivation of a chromosomal gene comprising the polynucleotide results into suppression of production of endogenous proteases of the host cell compared to a host cell wherein the chromosomal gene comprising the polynucleotide is not inactivated, the polynucleotide being selected from the group consisting of:
- i) a polynucleotide comprising a sequence having at least 55% sequence identity with the nucleotides 1141-3889 of SEQ ID NO:
- 11;
- ii) the polynucleotide of SEQ ID NO:
- 12 or the coding sequence thereof;
- iii) the polynucleotide of SEQ ID NO:
- 11 or the coding sequence thereof;
- and
- iv) a nucleotide sequence hybridisable with a nucleotide sequence which is complementary to any one of i) to iii) under high stringency conditions;
- c) a polypeptide or a gene product encoded by the polynucleotide of SEQ ID NO:
- 11 or 12;
- and
- d) a polypeptide comprising an amino acid sequence having at least 90% sequence identity to amino acids 402-533 of SEQ ID NO:13.
Dependencies are inferred from the claim text.