Minimal DNA sequence acting as a chromatin insulator and its use in protein expression
View Patent ↗The invention relates to expression vectors comprising a DNA sequence of 146 bp capable of acting as chromatin insulator, to host cells containing such vectors, to a method of producing a desired polypeptide by using vectors containing said sequence and to the use of said DNA sequence.
1. A method of expressing a gene of interest in a Chinese Hamster Ovary (CHO) cell comprising
culturing a CHO cell comprising a vector under conditions that allow for the expression of said gene of interest,
said vector comprising a promoter, a DNA encoding at least one gene of interest and one or more chromatin insulators consisting of SEQ ID NO: 1 and wherein said CHO cell expresses the gene of interest.
2. The method according to claim 1 , wherein the vector further comprises
an enhancer or a functional expressing enhancing fragment thereof.
3. The method according to claim 1 , wherein said vector further comprises one or more DNA sequences coding for regulatory elements selected from 5′UTRs, introns, 3′UTRs, mRNA 3′ end processing sequences, polyadenylation sites, and internal ribosome entry sequences (IRES).
4. The method according to claim 1 , wherein the DNA encodes more than one polypeptide of interest through a polycistronic mRNA.
5. The method according to claim 1 , wherein said vector further comprises one or more DNA elements selected from boundary elements, locus control regions (LCRs), matrix attachment regions (MARs), and elements for recombination and cassette exchange.
6. The method according to claim 1 , wherein the promoter is selected from the group consisting of a viral promoter, a phage promoter, mCMV-IE1, mCMV-IE2, hCMV, SV40, RSV, T7, T3, and a functional expression promoting fragment thereof.
7. The method according to claim 1 , wherein the gene of interest encodes a polypeptide of interest selected from the group consisting of FSH, LH, CG, TSH, a growth hormone, an interferon, TNF binding protein I, TNF binding protein II, IL-18BP, IL-6, IFNAR1, LIF, a mutein, a fragment thereof, a functional derivative thereof, and a fusion protein thereof.
8. The method according to claim 1 , wherein the gene of interest encodes a polypeptide of interest selected from the group consisting of EPO, G-CSF, GM-CSF, a chain of a humanized antibody, a cytokine, a coagulation factor, etanercept, tPA, an integrin, a mutein, a fragment thereof, a functional derivative thereof, and a fusion protein thereof.
9. The method according to claim 1 , wherein the gene of interest encodes a polypeptide of interest selected from the group consisting of adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (tk), xanthine-guanine phosphoribosyltransferase (HPH), multiple drug resistance gene (MDR), ornithine decarboxylase (ODC) N-(phosphonacetyl)-L-aspartate resistance (CAD), puromycin acetyltransferase (PAC), galactokinase, human folate receptor, and a reduced folate carrier.
10. The method according to claim 1 , wherein the gene of interest encodes a polypeptide of interest selected from the group consisting of luciferase, green fluorescent protein, alkaline phosphatase, horseradish peroxidase, and a combination thereof.
11. The method according to claim 1 , wherein one insulator is positioned upstream and one insulator is positioned downstream of the gene of interest.
12. The method according to claim 1 , wherein at least two insulators are positioned upstream and downstream of the gene of interest, respectively.
13. The method according to claim 1 , wherein at least two protein encoding sequences are positioned between the insulators.
14. The method according to claim 13 , wherein the at least two coding sequences code for subunits of a multimeric protein.
15. The method according to claim 14 , wherein the multimeric protein is a hormone comprising a first subunit that is the alpha chain and a second subunit that is the beta chain of a hormone selected from the group consisting of: human FSH, human LH, human TSH, and human CG.
16. The method according to claim 14 , wherein the multimeric protein is an antibody comprising a first subunit that is the heavy chain of an immunoglobulin and a second subunit that is the light chain of an immunoglobulin.
17. The method according to claim 1 , wherein said CHO cell simultaneously expresses two or more genes of interest from the vector.
18. The method according to claim 1 , further comprising the step of isolating the polypeptide of interest from the CHO cells.
19. The method according to claim 7 , wherein said protein of interest is IL-18BP.