Recombinant host cell for expressing proteins of interest
AssigneesBoehringer Ingelheim Rcv GmbH & Co KG, Lonza Ltd., Validogen GmbH
InventorsBrigitte Gasser, Diethard Mattanovich, Markus Buchetics
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Abstract
The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell's capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.
Claims16
A recombinant host cell for manufacturing a heterologous protein of interest, wherein the host cell is a yeast and is engineered to underexpress at least one polynucleotide encoding a KO protein having an amino acid sequence as shown in SEQ ID NO:
- 10, 11, 12 or a functional homologue thereof compared to the host cell prior to genetic manipulation to achieve underexpression, wherein underexpression is achieved by knocking-out the polynucleotide encoding the KO protein or a homologue thereof from the genome of said host cell, disrupting the polynucleotide encoding the KO protein or a homologue thereof in the host cell, disrupting a promoter which is operably linked with said polynucleotide encoding the KO protein or a homologue thereof, replacing the promoter which is operably linked with said polynucleotide encoding the KO protein or a homologue thereof with another promoter which has lower promoter activity, or disrupting expression control sequences of the KO protein or a homologue thereof, wherein the functional homologue has at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 10 or 11 or at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 12, wherein the host cell is engineered to express a heterologous protein of interest.
A yeast host cell obtained by providing a cell which expresses KO protein 1, KO protein 2, and/or KO protein 3, or functional homologue thereof and engineering the cell to underexpress the gene which encodes KO1, KO2 and/or KO3, or functional homologue thereof compared to the yeast host cell prior to genetic manipulation to achieve underexpression,
- wherein underexpression is achieved by knocking-out the polynucleotide encoding the KO protein or a homologue thereof from the genome of said host cell, disrupting the polynucleotide encoding the KO protein or a homologue thereof in the host cell, disrupting a promoter which is operably linked with said polypeptide encoding the KO protein or a homologue thereof, replacing the promoter which is operably linked with said polypeptide encoding the KO protein or a homologue thereof with another promoter which has lower promoter activity, or disrupting expression control sequences of the KO protein or a homologue thereof,
- wherein the KO protein has an amino acid sequence as shown in SEQ ID NO:
- 10, 11 or 12 or the functional homologue has at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 10 or 11 or at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 12, and wherein the host cell is engineered to express a heterologous protein of interest.
A method of increasing the yield of a heterologous protein of interest in a yeast host cell engineered to underexpress at least one polynucleotide encoding a KO protein having an amino acid sequence as shown in SEQ ID NO:
- 10, 11, 12 or a functional homologue thereof compared to the yeast host cell prior to genetic manipulation to achieve underexpression, comprising underexpressing the at least one polynucleotide encoding the KO protein, wherein the functional homologue has at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 10 or 11 or at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 12.
A method of producing a heterologous protein of interest in
- a yeast host cell comprising:
- providing a yeast host cell engineered to underexpress at least one polynucleotide encoding a KO protein having an amino acid sequence as shown in SEQ ID NO:
- 10, 11, 12 or a functional homologue thereof compared to the yeast host cell prior to genetic manipulation to achieve underexpression, wherein the functional homologue has at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 10 or 11 or at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO:
- 12, wherein said yeast host cell comprises a heterologous polynucleotide encoding a protein of interest;
- culturing the yeast host cell under suitable conditions to express the heterologous protein of interest, and optionally
- isolating the protein of interest from the cell culture.
Dependencies are inferred from the claim text.