Recombinant protein expression using a hybrid CHEF1 promoter
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-9957525-B2
Publication Date
2018-05-01
Expiration Date
Abstract
The invention provides expression vectors and host cells for high-level expression of recombinant proteins. The expression vectors comprise Chinese hamster ovary elongation factor 1-α (CHEF1) transcriptional regulatory DNA elements and a cytomegalovirus (CMV) promoter and/or a human adenovirus tripartite leader (AdTPL) sequence. The invention achieves increased protein expression and better productivity of host cells compared to previously described expression systems.
Core Innovation
The invention relates to hybrid expression vectors that include Chinese Hamster Elongation Factor-1α (CHEF1) transcriptional regulatory DNA together with a cytomegalovirus (CMV) promoter and/or an adenovirus tripartite leader (AdTPL) sequence. The disclosed vectors include polynucleotides set forth in SEQ ID NO: 6 or 7, or polynucleotides at least 90% identical to those sequences, and define CHEF1 regulatory DNA segments and sequence identities.
The background problem addressed is the need to improve recombinant protein expression and productivity in CHO cells when using CHEF1 transcriptional regulatory DNA in the expression cassette. Vectors comprising CHEF1 transcriptional regulatory DNA alone provide a baseline level of protein expression, while the addition of CMV promoter and/or AdTPL provides improved performance compared with vectors lacking a CMV promoter and lacking an AdTPL sequence.
In host cells such as CHO DG44 pools and clonal lines, the disclosed CHEF1-CMV and CHEF1-CMV-AdTPL vector configurations increase protein titer and specific productivity, and shift maximal expression of antibody/glycoprotein expression to later in the culture, including stationary phase. The document further reports increased productivity metrics for CHEF1-CMV and CHEF1-CMV-AdTPL relative to CHEF1 alone.
Claims Coverage
The claims cover two independent aspects: specific CHEF1/CMV/AdTPL expression vectors defined by particular polynucleotides, and methods of increasing protein expression by at least two-fold using those vector features compared to vectors lacking CMV and AdTPL. Across the claim set, the inventive features are refined by defined sequence constraints, selectable marker gene options, and host-cell scope, including CHO DG44.
CHEF1 transcriptional regulatory DNA with CMV and/or AdTPL
An expression vector comprising Chinese Hamster Elongation Factor-1α (CHEF1) transcriptional regulatory DNA and a cytomegalovirus (CMV) promoter and/or an adenovirus tripartite leader (AdTPL) sequence.
SEQ ID NO: 6/7 defined polynucleotide set
The expression vector comprises the polynucleotide set forth in SEQ ID NO: 6 or 7 or a polynucleotide at least 90% identical to the polynucleotide set forth in SEQ ID NO: 6 or 7.
Increasing specific productivity by at least two-fold using CHEF1/CMV/AdTPL
A method of increasing protein expression comprising transfecting a host cell with an expression vector comprising CHEF1 transcriptional regulatory DNA, a CMV promoter and/or an AdTPL sequence, and a polynucleotide encoding a protein of interest, wherein specific productivity is increased by at least two-fold compared to an expression vector comprising a CHEF1 promoter without a CMV promoter and without an AdTPL sequence.
Overall, the claim coverage centers on using CHEF1 transcriptional regulatory DNA together with a CMV promoter and/or AdTPL in vectors defined by SEQ ID NO: 6 or 7, and on the resulting increase in specific productivity of at least two-fold when expressing a protein of interest in a host cell, with further refinements for sequence content, selectable markers, and CHO DG44 host-cell delivery.
Stated Advantages
Improved recombinant protein expression and productivity in CHO cells.
Increased protein titer and specific productivity when CMV and/or AdTPL are added compared to CHEF1 only.
Maximal antibody/glycoprotein expression shifts to later in the culture, including stationary phase.
Documented Applications
Expression and productivity improvement for recombinant proteins in CHO cells, including reporter proteins and antibody/glycoprotein expression.
Use in CHO DG44 pools and clonal lines for increased protein expression and specific productivity.
Interested in licensing this patent?