Enhanced transfection system
A mammalian cell gene expression vector system comprising (a) an episomal maintenance system (b), a strong promoter/enhancer, (c) a protein transactivation system and (d) DNA coding for a heterologous protein. The episomal maintenance and protein transactivation systems can include sub-elements located on the same or different plasmids within the cell expression system.
1. A composition comprising a plasmid, the plasmid comprising:
a. an oriP element;
b. a first nucleic acid sequence encoding EBNA 1 operably linked to a promoter;
c. a second nucleic acid sequence encoding TAT operably linked to a promoter;
d. a TAR element; and
e. a third nucleic acid sequence encoding a heterologous protein operably linked to a first CMV promoter/enhancer.
2. The composition of claim 1 , further comprising a nucleic acid sequence encoding BMLF1 operably linked to a second CMV promoter/enhancer.
3. The composition of claim 1 , wherein the heterologous protein is selected from the group consisting of IL-4 and IL-2SA.
4. A method for producing a heterologous protein from a mammalian host cell, comprising the steps of:
(a) transforming the mammalian host cell with the composition of claim 1 ;
(b) expanding the host cell of step (a) in culture; and
(c) isolating the heterologous protein from the culture of step (b).
5. A composition comprising a first plasmid and a second plasmid, wherein the first plasmid comprises an oriP element, a first nucleic acid sequence encoding EBNA1 operably linked to a promoter, and a second nucleic acid sequence encoding TAT operably linked to a promoter, and the second plasmid comprises an oriP element, a TAR element, and a nucleic acid sequence encoding a heterologous protein operably linked to a CMV promoter/enhancer.
6. The composition of claim 5 wherein the first plasmid further comprises a nucleic acid sequence encoding BMLF1 operably linked to a promoter.
7. The composition of claim 5 wherein the heterologous protein is selected from the group consisting of IL-4 and IL-2SA.
8. A method for producing a heterologous protein from a mammalian host cell, comprising the steps of:
(a) transforming the mammalian host cell with the composition of claim 5 ;
(b) expanding the host cell of step (a) in culture; and
(c) isolating the heterologous protein from the culture of step (b).
9. A mammalian cell line transformed with a composition comprising a plasmid, the plasmid comprising:
a. an oriP element;
b. a first nucleic acid sequence encoding EBNA 1 operably linked to a promoter;
c. a second nucleic acid sequence encoding TAT operably linked to a promoter;
d. a TAR element; and
e. a third nucleic acid sequence encoding a heterologous protein operably linked to a first CMV promoter/enhancer.
10. A mammalian cell line transformed with a composition, the composition comprising a first plasmid and a second plasmid, wherein the first plasmid comprises an oriP element, a first nucleic acid sequence encoding EBNA1 operably linked to a promoter, and a second nucleic acid sequence encoding TAT operably linked to a promoter, and the second plasmid comprises an oriP element, a TAR element, and a nucleic acid sequence encoding a heterologous protein operably linked to a CMV promoter/enhancer.
11. A mammalian cell line transformed with at least one plasmid, wherein the cell line expresses EBNA1 prior to transformation, the plasmid comprising:
a. an oriP element;
b. a first nucleic acid sequence encoding TAT operably linked to a promoter;
c. a TAR element; and
d. a second nucleic acid sequence encoding a heterologous protein operably linked to a CMV promoter/enhancer.