Expression from transposon-based vectors and uses
AssigneeJust-Evotec Biologics, Inc.
InventorsJeffrey T. McGrew, Pauline S. Smidt, E-Ching Ong
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Abstract
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.
Claims13
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.
Dependencies are inferred from the claim text.