High level protein expression system
First Claim
1. An HEK293 cell line transformed by a vector system containing a selectable marker operably linked to a weak promoter and a gene of interest operably linked to a strong promoter.
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Abstract
The present application is directed to a system for expressing high levels of a protein of interest in a cell. Preferably the system includes introducing into a mammalian cell line an expression vector that comprises a gene encoding a selectable marker under the control of a weak promoter to facilitate integration of the expression vector into genomic areas that result in high levels of expression, along with a gene encoding the protein of interest. The gene of interest is linked to a strong promoter which results in high levels of expression of the desired protein. In one embodiment, the protein of interest is detrimental or toxic to cells, and the promoter is an inducible promoter. In another embodiment, the protein of interest is a glycoprotein, including membrane proteins, and the cell line has been mutated to inactivate N-acetylglucosamine transferase I to ensure uniform glycosylation. Preferably the cell is a mammalian or an insect cell.
68 Citations
35 Claims
- 1. An HEK293 cell line transformed by a vector system containing a selectable marker operably linked to a weak promoter and a gene of interest operably linked to a strong promoter.
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19. A method of producing high levels of a protein of interest in a host cell line, comprising:
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(a) introducing a vector system into a host cell line, wherein the vector system comprises a selectable marker operably linked to a weak promoter and a gene of interest operably linked to a strong promoter;
(b) selecting stable transformants of said host cell line by isolating those transformants which express the selectable marker at a sufficient level to allow their survival when grown in the presence of a selecting agent; and
(c) culturing said transformants under growth conditions which allow expression of the gene of interest to produce the protein of interest. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A vector system for high level protein expression, wherein the vector system comprises a selectable marker operably linked to a weak promoter and a gene of interest operably linked to a strong promoter.
Specification