Methods for improving recombinant protein expression
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9212367-B2
Publication Date
2015-12-15
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 described invention relates to increasing heterologous protein expression in a mammalian host cell. The host cell comprises a first heterologous polynucleotide sequence encoding a heterologous protein and a second polynucleotide sequence encoding a selectable marker protein, and the second polynucleotide includes a sequence modification compared to a wild-type polynucleotide encoding the selectable marker protein.
The sequence modification reduces translation efficiency of mRNA encoded by the second polynucleotide, while the second polynucleotide and the wild-type polynucleotide encode identical amino acid sequences for the selectable marker protein. The heterologous polynucleotide and the modified selectable marker polynucleotide are encoded on a Chinese hamster elongation factor 1 (CHEF1) expression vector.
The document reports an unexpected outcome in which reducing translation of the selectable marker increases selection stringency and can drive higher heterologous protein titers, including FIGI IgG1-Fc expression and altered FACS profiles consistent with reduced DHFR and elevated GOI expression versus wild-type DHFR.
Claims Coverage
The provided partial content includes one independent claim. The claim covers one inventive concept: co-delivery on a CHEF1 expression vector of a heterologous polynucleotide and a sequence-modified selectable-marker polynucleotide that reduces mRNA translation efficiency while preserving identical amino acid sequences, limited to specific selectable-marker polynucleotide sequences.
Co-delivery on a CHEF1 expression vector for heterologous protein expression
A mammalian host cell comprises a first heterologous polynucleotide and a second selectable-marker polynucleotide encoded on a Chinese hamster elongation factor 1 (CHEF1) expression vector.
Selectable marker polynucleotide deoptimized to reduce mRNA translation efficiency
The second polynucleotide sequence for a selectable marker protein has a sequence modification compared to a wild-type polynucleotide, where the sequence modification reduces translation efficiency of mRNA encoded by the second polynucleotide.
Identical selectable-marker amino acid sequences despite mRNA translation reduction
The second polynucleotide and the wild-type polynucleotide encode identical amino acid sequences for the selectable marker protein.
Specific selectable-marker sequences (SEQ ID NO:3 or SEQ ID NO:4)
The second polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
Across the single independent claim, the core inventive coverage is limited to increasing heterologous protein expression by culturing mammalian host cells carrying a CHEF1 expression vector with a target heterologous polynucleotide and a selectable-marker polynucleotide that is sequence-modified to reduce mRNA translation efficiency while preserving identical amino acid sequences, with the selectable-marker sequence constrained to SEQ ID NO: 3 or SEQ ID NO: 4.
Stated Advantages
Reducing translation of the selectable marker increases selection stringency.
Reducing translation of the selectable marker can drive higher heterologous protein titers.
In reported experiments, FIGI productivity is increased with deoptimized selectable marker versus wild-type DHFR.
Selection outcomes are reported as altered FACS profiles consistent with reduced selectable marker and elevated GOI expression versus wild-type DHFR.
Documented Applications
Increasing heterologous protein expression in mammalian host cells, including reported expression of FIGI IgG1-Fc using CHEF1 expression vectors and deoptimized DHFR selectable markers.
Interested in licensing this patent?