Protein expression in rodent cells
AssigneeHoffmann-La Roche Inc.
InventorsUlrich Goepfert, Erhard Kopetzki
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Abstract
A method is described for the expression of heterologous polypeptides in rodent cells. The method comprises the oriP/EBNA-1 episomal replication and maintenance system of the Epstein-Barr-Virus (EBV). With the stable integration of an EBNA-1-protein expression cassette under the control of a promoter into the genome of a rodent cell an EBNA-1-protein expression in the cells was obtained. The heterologous protein is expressed from an episome comprising an EBV origin of replication and a functional expression cassette of said heterologous protein. The invention further comprises transformed rodent cell lines, a method for the production of a heterologous protein in said cell lines and a kit for the construction of said cell lines.
Claims13
A Chinese Hamster Ovary (CHO) cell, wherein said CHO cell
- a) contains a structural gene located in the genome of said CHO cell which encodes and expresses the Epstein Barr Virus Nuclear Antigen 1 (EBNA-1);
- b) contains an episome, wherein said episome comprises
- (i) a prokaryotic origin of replication;
- (ii) a selection marker;
- (iii) a single Epstein Barr Virus (EBV) derived element, wherein the single EBV derived element is an EBV origin of replication (oriP);
- (iv) an expression cassette suitable for the expression of a heterologous polypeptide in said CHO cell, whereby said expression cassette comprises a promoter sequence, a 5′ untranslated region, a nucleic acid sequence encoding said heterologous polypeptide and a 3′ untranslated region comprising a polyadenylation signal;
- wherein said episome does not contain a nucleic acid encoding EBNA-1;
- and
- c) does not contain the large T-antigen of Polyoma virus.
A method for the expression and recovery of a heterologous polypeptide in a CHO cell, characterized in that said method comprises
- a) transfecting a CHO cell with an expression plasmid, wherein the CHO cell comprises a CHO cell stably transfected with the structural gene encoding the EBNA-1 protein and which does not contain the large T-antigen of Polyoma virus and wherein further the expression plasmid comprises
- (i) a single Epstein Barr Virus (EBV) derived element, wherein the single EBV derived element is an EBV origin of replication (oriP),
- (ii) no structural gene encoding the EBNA-1 protein,
- (iii) a prokaryotic origin of replication,
- (iv) a selection marker,
- (v) an expression cassette suitable for the expression of a heterologous polypeptide in said CHO cell, whereby said expression cassette comprises a promoter sequence, a 5′ untranslated region, a nucleic acid sequence encoding said heterologous protein, and a 3′ untranslated region comprising a polyadenylation signal;
- b) culturing said transfected cell under conditions suitable for the expression of said heterologous polypeptide;
- and
- c)recovering said heterologous polypeptide from the culture.
A method for the production of a heterologous immunoglobulin, wherein said method comprises the following steps:
- a) cultivating a CHO cell under conditions suitable for the expression of said heterologous immunoglobulin, wherein the CHO cell
- (i) expresses the Epstein Barr Virus Nuclear Antigen 1 (EBNA-1);
- (ii) contains an episome, wherein said episome comprises
- (i) a prokaryotic origin of replication;
- (ii) a selection marker;
- (iii) a single Epstein Barr Virus (EBV) derived element, wherein the single EBV derived element is an EBV origin of replication (oriP);
- (iv) expression cassettes suitable for the expression of a heterologous immunoglobulin in said CHO cell, whereby said expression cassettes comprises a promoter sequence, a 5′ untranslated region, a nucleic acid sequence encoding said heterologous immunoglobulin and a 3′ untranslated region comprising a polyadenylation signal,
- wherein said episome does not contain a nucleic acid encoding EBNA-1;
- and
- (iii) does not contain the large T-antigen of Polyoma virus, and
- b) recovering said heterologous immunoglobulin from the culture.
Dependencies are inferred from the claim text.