Production of proteins using transposon-based vectors
Novel compositions for the in vitro or in vivo production of specific proteins are provided. The compositions comprise components of vectors, such as a vector backbone, a promoter, and a gene of interest that encodes for the protein of interest, and the transposon-based vectors comprising these components. Also provided are methods of making these compositions and methods of using these compositions for the production of desired proteins in vivo or in transfected cells in vitro.
1. A vector comprising:
a modified transposase gene operably-linked to a first promoter, wherein the nucleotide sequence 3′ to the first promoter comprises a Kozak sequence, and wherein a plurality of the first twenty codons of the modified transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;
one or more genes of interest operably-linked to one or more additional promoters, wherein the one or more genes of interest encodes a protein of interest selected from the group consisting of erythropoietin, bone morphogenic protein, high density lipoprotein, platelet derived growth factor, enterokinase, vascular endothelial cell derived growth factor, luteinizing hormone, trastuzumab, etanercept, colony stimulating factor, and human chorionic gonadotropin, and wherein the one or more genes of interest and their one or more operably-linked promoters are flanked by transposase insertion sequences recognized by a transposase encoded by the modified transposase gene; and
one or more insulator elements located between the transposase insertion sequences and the one or more genes of interest, wherein the one or more insulator elements comprise a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4element, or a matrix attachment region element, wherein the start codon of the gene of interest is located about 2502 bp from the one or more insulator elements located 5′ to the start codon of the gene of interest.
2. The vector of claim 1 , wherein the one or more additional promoters are SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a combination thereof.
3. The vector of claim 1 , wherein the one or more genes of interest encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 57 to 72.
4. The vector of claim 1 , wherein the first promoter is a constitutive promoter.
5. The vector of claim 1 , wherein the transposase is a Tn10 transposase.
6. The vector of claim 1 , wherein the Kozak sequence is any one of SEQ ID NOs: 19 to 28.
7. The vector of claim 1 , further comprising a polyA sequence operably-linked to the gene of interest.
8. A vector comprising:
a modified transposase gene operably linked to a first promoter, wherein the nucleotide sequence 3′ to the first promoter comprises a Kozak sequence, and wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;
a multiple cloning site; and
transposon insertion sequences recognized by a transposase encoded by the modified transposase gene, wherein the transposon insertion sequences flank the multiple cloning site, wherein the vector is SEQ ID NO: 18.
9. A method of producing a protein comprising:
transfecting a cell with a vector comprising:
a modified gene encoding for a transposase, wherein the modified transposase gene is operably linked to a first promoter, wherein the nucleotide sequence 3′ to the first promoter comprises a Kozak sequence, and wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;
one or more genes of interest which encodes for the protein or proteins of interest, wherein the one or more genes of interest is operably linked to one or more additional promoters and encodes for the protein or proteins of interest selected from the group consisting of erythropoietin, bone morphogenic protein, high density lipoprotein, platelet derived growth factor, erythropoietin, vascular endothelial cell derived growth factor, luteinizing hormone, trastuzumab, etanercept, colony stimulating factor, and human chorionic gonadotropin; and the one or more genes of interest and their one or more operably-linked promoters are flanked by transposase insertion sequences recognized by a transposase encoded by the modified transposase gene; and,
one or more insulator elements located between the transposase insertion sequences and the one or more genes of interest, wherein the one or more insulator elements comprise a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, or a matrix attachment region element, wherein the start codon of the gene of interest is located about 2502 bp from the one or more insulator elements located 5′ to the start codon of the gene of interest;
culturing the transfected cell in culture medium;
permitting the transfected cell to release the protein or proteins of interest into the culture medium;
collecting the culture medium containing the protein or proteins of interest; and,
isolating the protein or proteins of interest from the culture medium.