Methods for improving recombinant protein expression
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Publication Number
US-10066231-B2
Publication Date
2018-09-04
Expiration Date
Abstract
Materials and methods are provided which allowed for increased expression of a transfected gene of interest in a recombinant host cell.
Core Innovation
The invention relates to recombinant protein expression methods and expression vectors that increase expression of a gene of interest by reducing translation efficiency of a co-expressed selectable marker. A selectable marker coding sequence is deoptimized while maintaining an identical amino acid sequence to wild-type. The disclosed deoptimization includes modifications that reduce translation efficiency, including least-preferred codons, codon pair bias, tandem codon pair changes, and altered mRNA secondary structure or GC or A+T content, including UTR modifications.
The system uses a CHEF1-based expression vector comprising CHEF1 regulatory DNA, a first polynucleotide encoding a gene of interest, and a second polynucleotide comprising the codon deoptimized selectable marker gene sequence. The first polynucleotide and the second polynucleotide are under transcriptional control of distinct promoters. The deoptimized selectable marker sequences include SEQ ID NO:3 and SEQ ID NO:4.
In documented examples, codon deoptimized DHFR variants are used in CHEF1 expression vectors (pDEF81/pDEF82) co-expressed with FIGI in CHO DG44 cells. The use of deoptimized selectable marker sequences yields increased FIGI titers and altered selection outcomes, including reduced DHFR and increased reporter fluorescence. Productivity improvements are reported as up to about 2–3× over wild type DHFR-selected controls.
Claims Coverage
The independent claim provides coverage for an expression vector architecture with CHEF1 regulatory DNA and a codon deoptimized selectable marker sequence that preserves the selectable marker’s wild-type amino acid identity. It includes multiple inventive features, with dependent claims further restricting host cell context to Chinese hamster, CHO, and CHO DG44 cells.
CHEF1 regulatory DNA with distinct promoters
An expression vector comprising Chinese hamster elongation factor 1 (CHEF1) regulatory DNA, a first polynucleotide encoding a gene of interest, and a second polynucleotide, wherein said first polynucleotide and said second polynucleotide are under transcriptional control of distinct promoters.
Codon deoptimized selectable marker with wild-type identical amino acid sequence
The second polynucleotide comprises a codon deoptimized selectable marker gene sequence encoding a selectable marker protein having an amino acid sequence identical to wild-type.
Specific deoptimized selectable marker sequences (SEQ ID NO:3 or SEQ ID NO:4)
The second polynucleotide comprises SEQ ID NO:3 or SEQ ID NO:4.
The claim set is centered on an expression vector that couples CHEF1 regulatory DNA-driven expression of a gene of interest with distinct-promoter co-expression of a codon deoptimized selectable marker sequence that preserves wild-type amino acid identity, specifically using SEQ ID NO:3 or SEQ ID NO:4. Dependent claims narrow the covered use by specifying the host cell type as Chinese hamster cells, then CHO cells, and further CHO DG44 cells.
Stated Advantages
Increased expression of the gene of interest by reducing translation efficiency of a co-expressed selectable marker.
Increased FIGI titers.
Altered selection outcomes, including reduced DHFR and increased reporter fluorescence.
Productivity improvements up to about 2–3× over wild type DHFR-selected controls.
Documented Applications
Co-expression of FIGI (IgG1 Fc fusion) with codon deoptimized DHFR selectable markers in CHO DG44 cells using CHEF1 expression vectors, with reported changes in FIGI titers and selection outcomes.
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