Recombinant host cell engineered to overexpress helper proteins
Inventors
Gasser, Brigitte • Mattanovich, Diethard • Buchetics, Markus
Assignees
Vaitdogen GmbH • Sandoz GmbH • Boehringer Ingelheim RCV GmbH and Co KG • Universitaet fuer Bodenkultur Wien BOKU • Validogen GmbH • Biomin Holding GmbH • Lonza AG
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Publication Number
US-10865416-B2
Publication Date
2020-12-15
Expiration Date
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.
Core Innovation
The invention concerns improving recombinant protein-expression by manufacturing a heterologous protein of interest in an isolated host cell. The host cell is engineered to overexpress a helper protein that is different from the heterologous protein of interest, and the engineered cell is further configured to express the heterologous protein of interest. The concept defines helper proteins broadly as proteins used to increase the yield of a heterologous protein of interest.
The disclosed helper proteins include HP1–HP10 encoded by specific polynucleotides, and helper protein concepts also include a polynucleotide encoding a helper protein having the amino acid sequence as shown in SEQ ID NO: 1 or a functional homologue thereof. The disclosure also describes optional underexpression of KO proteins to further support increased production of the protein of interest.
The disclosure frames the problem as a need to increase yield and secretion of heterologous proteins produced in host cells, using engineered helper-protein expression. A screening approach is described in which helper protein candidates are identified and functional performance is assessed, including model Fab proteins, with reported yield improvements for selected prime candidates.
Claims Coverage
The document includes one independent claim directed to a manufacturing method, with dependent claims that refine the sequence-identity threshold for the helper functional homologue and narrow the heterologous protein of interest to specific categories.
Overexpressing a helper protein to increase POI yield
Providing an isolated host cell engineered to overexpress a polynucleotide encoding a helper protein having the amino acid sequence as shown in SEQ ID NO: 1 or a functional homologue thereof, wherein the heterologous protein of interest and the helper protein are different proteins, and wherein the yield of the heterologous protein of interest produced by the isolated host cell is increased as compared to a corresponding isolated host cell that does not overexpress the helper protein or the functional homologue.
Culturing and isolating the heterologous protein of interest
Culturing the isolated host cell under suitable conditions to overexpress the helper protein or the functional homologue and express the heterologous protein of interest; and isolating the heterologous protein of interest from the cell culture.
Using a functional homologue with at least 97% identity
The method wherein the functional homologue has an amino acid sequence identity of at least 97% to the amino acid sequence as shown in SEQ ID NO: 1.
Using a functional homologue with at least 99% identity
The method wherein the functional homologue has an amino acid sequence identity of at least 99% to the amino acid sequence as shown in SEQ ID NO: 1.
Heterologous protein of interest is a mammalian polypeptide
The method wherein the heterologous protein of interest consists of a mammalian polypeptide.
Heterologous protein of interest includes a human polypeptide
The method wherein the heterologous protein includes a human polypeptide.
Heterologous protein of interest selected from enumerated categories
The method wherein the heterologous protein of interest is selected from an enzyme, a therapeutic protein, a food additive, a feed additive, an antibody, or an antibody fragment.
Across the independent claim and dependents, the central claim coverage is focused on engineering an isolated host cell to overexpress SEQ ID NO: 1 helper protein or a high-identity functional homologue distinct from the heterologous protein of interest, combined with culturing and isolating the heterologous protein of interest; dependent claims further constrain the helper homologue identity threshold and specify the heterologous protein of interest categories.
Stated Advantages
Increased yield of the heterologous protein of interest compared to a corresponding isolated host cell that does not overexpress the helper protein or the functional homologue.
Increased yield and secretion of heterologous proteins produced in host cells.
Reported yield improvements for selected prime candidates in screening of helper protein candidates.
Documented Applications
Manufacturing a heterologous protein of interest in an isolated host cell.
Protein production contexts for therapeutic proteins and antibodies or antibody fragments.
Food or feed additives, including enzymes and food/feed additive categories, as heterologous protein-of-interest categories in the method.
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