Production of human mutated proteins in human cells by means of homologous recombination
AssigneeRoche Diagnostics GmbH
InventorsAnne Stern, Konrad Honold
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Abstract
The invention concerns a process for the production of muteins of eukaryotic polypeptides in eukaryotic cells by means of homologous recombination. The invention additionally concerns a process for the production of human cells which are suitable for the production of human mutated proteins. Finally the invention concerns the human cells produced by the process and mutated human proteins obtainable therefrom as well as pharmaceutical preparations which contain these muteins.
Claims18
A process for the production of muteins of eukaryotic polypeptides wherein
- (i) a nucleic acid molecule capable of homologous recombination is introduced into eukaryotic cells, which contain more than two chromosomes that comprise an endogenous target nucleic acid sequence coding for an endogenous target polypeptide, the nucleic acid molecule comprising
- (a) at least one nucleotide sequence which is homologous to sequences in the coding region of the target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in the coding region of the target polypeptide and
- (b) a nucleotide sequence coding for a selection marker;
- (ii) the cells are cultured under such conditions that a homologous recombination of the introduced nucleic acid molecule takes place whereby the coding sequence of the endogenous target nucleic acid sequence is mutated by the introduced nucleic acid sequence after the homologous recombination and the mutated endogenous target nucleic acid sequence encodes a mutein of the target polypeptide,
- (iii) the cells in which a homologous recombination has taken place are selected, and
- (iv) the mutein is isolated from the cells, the cell supernatant, or both the cells and cell supernatant, wherein at least part of the mutein is encoded by the endogenous target nucleic acid sequence.
A process for the production of a human cell which expresses a mutein of a human target polypeptide comprising,
- (i) introducing a nucleic acid molecule into human cells, which contain more than two chromosomes that comprise an endogenous target nucleic acid sequence coding for an endogenous target polypeptide, the introduced nucleic molecule comprising
- (a) at least one sequence which is homologous to sequences in the coding sequence locus of the endogenous target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in a coding sequence of a mature form of the endogenous target polypeptide;
- (b) optionally a heterologous expression control sequence for the target nucleic acid sequence, and
- (c) a nucleic acid sequence coding for a selection marker,
- (ii) culturing the cells under such conditions that a homologous recombination of the introduced nucleic acid molecule takes place whereby the coding sequence of the endogenous target nucleic acid sequence is mutated by the introduced nucleic acid molecule after the homologous recombination and the mutated endogenous target nucleic acid sequence encodes a mutein of the target polypeptide,
- (iii) selecting the cells, in which a homologous recombination has taken place, and
- (iv) isolating the selected cells, wherein at least part of the mutein is encoded by the endogenous target nucleic acid sequence.
Dependencies are inferred from the claim text.