Methods and Materials for Expression of a Recombinant Protein
Recombinant expression vectors are provided comprising a 3′ UTR of a light chain and an Epstein-Barr virus origin of replication. Also provided are host cells comprising such vectors and methods of producing recombinant protein with such vectors. Additional methods of producing a recombinant protein involve contacting cells with a first and second vector, each of which encode a different polypeptide chain, and wherein the second vector is present in an amount which is about 1.5 to 2.5 times as much as that of the first vector. Cells also can be transfected with a recombinant transient expression vector encoding a protein and are cultured in a medium in a membrane-enhanced culturing vessel to produce recombinant protein.
1 : A recombinant expression vector comprising a promoter, a 3′ UTR of a light chain gene and an Epstein-Barr virus origin of replication (oriP).
2 : The recombinant expression vector of claim 1 , wherein the light chain gene is a murine light chain gene.
3 : The recombinant expression vector of claim 2 , wherein the 3′ UTR comprises a nucleotide sequence of nucleotides 1062-2560 of SEQ ID NO: 1.
4 : The recombinant expression vector of claim 1 , wherein the recombinant expression vector is a recombinant transient expression vector.
5 : The recombinant expression vector of claim 1 further comprising a pUC19 origin of replication or a 5′ UTR intron.
6 : The recombinant expression vector of claim 5 further comprising a pUC19 origin of replication and a 5′ UTR intron.
7 : (canceled)
8 : The recombinant expression vector of claims 5 and 6 , wherein the 5′ UTR intron comprises nucleotides 888-974 of SEQ ID NO: 1.
9 : The recombinant expression vector of claims 5 and 6 , wherein the pUC19 origin of replication comprises the nucleotide sequence of nucleotides 4551-5220 of SEQ ID NO: 1.
10 : The recombinant expression vector of claim 1 further comprising an antibody signal sequence.
11 : The recombinant expression vector of claim 1 , wherein the recombinant expression vector does not comprise an antibody signal sequence.
12 : The recombinant expression vector of claim 1 further comprising a nucleotide sequence encoding a protein or a functional fragment thereof.
13 : The recombinant expression vector of claim 12 , wherein the nucleotide sequence encodes an immunoglobulin chain.
14 : The recombinant expression vector of claim 13 , wherein the immunoglobulin chain is selected from the group consisting of a γ 1 heavy chain, a γ 2 heavy chain, a γ 4 heavy chain, a κ light chain, and a λ light chain.
15 : A method of producing a recombinant protein, comprising contacting a cell with the recombinant expression vector of claim 1 , whereupon a recombinant protein is produced.
16 : A host cell comprising the recombinant expression vector of claim 1 .
17 : The host cell of claim 16 , wherein the host cell is a mammalian cell.
18 : The host cell of claim 17 , wherein the mammalian cell is a human cell.
19 : The host cell of claim 18 , wherein the human cell is a human embryonic kidney cell.
20 : The host cell of claim 19 , wherein the human embryonic kidney cell expresses an Epstein Barr virus nuclear antigen-1 (EBNA-1) protein.
21 : The host cell of claim 20 wherein the host cell is a 293E cell.
22 : A method of producing a recombinant protein, comprising culturing the host cell of claim 16 , whereupon a recombinant protein is produced.
23 - 93 . (canceled)
94 : The recombinant expression vector of claim 1 , wherein the promoter is a viral promoter.
95 : The recombinant expression vector of claim 94 , wherein the viral promoter is a CMV promoter.