RECOMBINANT PROTEIN EXPRESSION USING A HYBRID CHEF1 PROMOTER
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.
1 . 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.
2 . The expression vector of claim 1 , wherein the CHEF1 transcriptional regulatory DNA comprises 5′ CHEF transcriptional regulatory DNA.
3 . The expression vector of claim 2 , wherein the 5′ CHEF1 transcriptional regulatory DNA comprises Sequence ID NO: 1 or a polynucleotide at least 95% identical to Sequence ID NO: 1.
4 .- 5 . (canceled)
6 . The expression vector of claim 2 , wherein the 5′ CHEF1 transcriptional regulatory DNA comprises Sequence ID NO: 2 or a polynucleotide at least 95% identical to Sequence ID NO: 2.
7 . (canceled)
8 . The expression vector of claim 2 , further comprising 3′ CHEF1 transcriptional regulatory DNA.
9 . The expression vector of claim 8 , wherein the 3′ CHEF1 transcriptional regulatory DNA comprises Sequence ID NO: 3 or a polynucleotide at least 95% identical to Sequence ID NO: 3.
10 . The expression vector of claim 8 , wherein the 3′ CHEF1 transcriptional regulatory DNA comprises DNA located between position 1 and position 4180 in Sequence ID NO: 3 or a polynucleotide at least 95% identical to DNA located between position 1 and position 4180 in Sequence ID NO: 2.
11 .- 20 . (canceled)
21 . The expression vector of claim 2 comprising a sequence selected from the group consisting of Sequence ID NO: 4 or a polynucleotide at least 95% identical to Sequence ID NO: 4; Sequence ID NO: 5 or a polynucleotide at least 95% identical to Sequence ID NO: 5; Sequence ID NO: 6 or a polynucleotide at least 95% identical to Sequence ID NO: 6; and Sequence ID NO: 7 or a polynucleotide at least 95% identical to Sequence ID NO: 7.
22 .- 24 . (canceled)
25 . The expression vector of claim 2 , further comprising a selectable marker gene.
26 . The expression vector of claim 2 , further comprising a polynucleotide encoding a protein of interest operably linked to the 5′ CHEF1 transcriptional regulatory DNA, the 3′ CHEF1 transcriptional regulatory DNA, the CMV promoter and/or the AdTPL sequence.
27 . A host cell transformed, transduced or transfected with an expression vector according to claim 2 .
28 . The host cell of claim 27 , wherein the host cell is selected from the group consisting of a prokaryotic cell; a eukaryotic cell; a hamster cell; and a Chinese Hamster Ovary (CHO) cell; a non-hamster mammalian cell; and a human cell.
29 .- 33 . (canceled)
34 . A method of increasing protein expression, comprising transfecting a host cell with 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.