Mammalian expression vectors
An expression vector for mammalian cells includes a selection cassette with a nucleotide sequence encoding a glutamine synthetase, operably linked to a PGK promoter and a pA signal. The vector may include the EASE element which is known to promote stable integration of the expression cassettes into the genome. The vector also includes a selection cassette with a nucleotide sequence encoding an enzyme that confers resistance against an antibiotic to a bacterial host as a bacterial selection marker, operably linked to a suitable promoter. The vector further includes an expression cassette for a target polypeptide with an insertion site for a nucleotide sequence encoding the target polypeptide, operably linked to a CMV promoter and a pA signal. The vector also includes a bacterial origin of replication.
1 . An expression vector for mammalian cells, comprising
(a) a first selection cassette comprising a nucleotide sequence encoding a glutamine synthetase as an eukaryotic selection marker, operably linked to a 3-phosphoglycerate kinase (PGK) promoter and a polyadenylation (pA) signal;
(b) a second selection cassette comprising a nucleotide sequence encoding an enzyme that confers resistance against an antibiotic to a bacterial host as a bacterial selection marker, operably linked to a suitable promoter;
(c) an expression cassette for a target polypeptide, comprising an insertion site for inserting a nucleotide sequence encoding the target polypeptide, operably linked to a cytomegalovirus (CMV) promoter and a pA signal;
(d) a bacterial origin of replication; and
(e) an expression augmenting sequence element (EASE).
2 . The expression vector of claim 1 , wherein the antibiotic against which the bacterial selection marker confers resistance is Ampicillin, and wherein the enzyme that confers resistance against Ampicillin to a bacterial host is beta-lactamase.
3 . The expression vector of claim 1 , wherein the bacterial origin of replication is a pUC origin of replication.
4 . The expression vector of claim 1 , wherein the nucleotide sequence encoding the glutamine synthetase is of at least 90% identity to SEQ ID NO: 3, or wherein the glutamine synthetase is a mammalian glutamine synthetase.
5 . The expression vector of claim 1 , wherein the glutamine synthetase has an amino acid sequence of at least 96% identity to SEQ ID NO: 5.
6 . The expression vector of claim 1 , wherein the PGK promoter has a sequence of at least 98% identity to SEQ ID NO: 2, and/or wherein the glutamine synthetase is a CHO glutamine synthetase having at least 97% identity to SEQ ID NO: 5.
7 . The expression vector of claim 1 , wherein the pA signal is a simian virus 40 pA signal.
8 . A recombinant CHO or NS0 host cell, comprising the expression vector of claim 1 .
9 . A method of producing a target polypeptide, the method comprising culturing the recombinant CHO or NS0 host cell of claim 8 under conditions suitable for expressing a heterologous target polypeptide, wherein the expression cassette comprises a nucleotide sequence encoding the target polypeptide inserted at the insertion site.
10 . The method of claim 9 , wherein the target polypeptide comprises an antibody or a fragment thereof.