Expression from transposon-based vectors and uses
Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence. Some of the vectors are useful for tetracycline-inducible expression. Some of the vectors contain a 5′ PiggyBac ITR and a 3′ PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and a method of producing a protein of interest, in vitro, involving the mammalian host cell.
1. A recombinant expression vector, comprising:
(a) a 5′ PiggyBac ITR comprising the nucleotide sequence of SEQ ID NO:45;
(b) a first expression cassette, comprising:
(i) a control sequence comprising:
(A) a murine cytomegalovirus (mCMV) enhancer sequence, comprising a mCMV enhancer element (mCMV-E) and a CMV promoter (CMV-P) sequence at its 3′ end, operably linked 5′ to a rat EF-1alpha intron sequence;
(B) an intervening first leader sequence operably linked, 3′ to the CMV-P sequence of the mCMV enhancer sequence, and 5′ to the rat EF-1alpha intron sequence; and
(C) a second leader sequence operably linked 3′ to the rat EF-1alpha intron sequence;
(ii) an open reading frame encoding a protein of interest operably linked to the control sequence; and
(iii) a polyadenylation site operably linked 3′ to the open reading frame;
(c) a second expression cassette, comprising:
(i) a weak constitutive promoter, operably linked to an open reading frame encoding a selectable marker; and
(ii) a polyadenylation site operably linked 3′ to the open reading frame; and
(d) a 3′ PiggyBac ITR comprising the nucleotide sequence of SEQ ID NO:47.
2. The vector of claim 1 , further comprising:
(e) a third expression cassette comprising:
(i) a control sequence comprising a promoter;
(ii) an open reading frame encoding a protein of interest operably linked to the control sequence; and
(iii) a polyadenylation site operably linked 3′ to the open reading frame.
3. The vector of claim 2 , wherein the control sequence (i) of the third expression cassette comprises:
(A) a mCMV enhancer sequence, comprising a mCMV enhancer element (mCMV-E) and a CMV promoter (CMV-P) sequence at its 3′ end, operably linked 5′ to a rat EF-1alpha intron sequence;
(B) an intervening first leader sequence operably linked, 3′ to the CMV-P sequence of the mCMV enhancer sequence, and 5′ to the rat EF-1alpha intron sequence; and
(C) a second leader sequence operably linked 3′ to the rat EF-1alpha intron sequence.
4. The vector of claim 1 , wherein the weak constitutive promoter is a deleted SV40 promoter.
5. The vector of claim 4 , wherein the deleted SV40 promoter comprises the nucleotide sequence of SEQ ID NO:53.
6. The vector of claim 4 , wherein the deleted SV40 promoter comprises the nucleotide sequence of SEQ ID NO:46.
7. The vector of claim 1 , wherein the selectable marker is glutamine synthetase, puromycin resistance, neomycin resistance, zeomycin resistance, or dihydrofolate reductase.
8. The vector of claim 1 , wherein the selectable marker is glutamine synthetase.
9. The vector of claim 1 , wherein the selectable marker is encoded by the nucleotide sequence of SEQ ID NO:49, or a degenerate DNA sequence thereof.
10. A mammalian host cell, comprising the recombinant expression vector of claim 1 .
11. The mammalian host cell of claim 10 , wherein the mammalian host cell is a CHO cell.
12. The mammalian host cell of claim 11 , wherein the CHO cell is a CHO-K1 cell, a DXB11 cell, or a DG44 cell.
13. A method of producing a protein of interest, in vitro, comprising culturing the mammalian host cell of claim 10 in an aqueous medium under physiological conditions permitting expression of the protein of interest; and recovering the protein of interest from the medium.