Production of human mutated proteins in human cells by means of homologous recombination
OwnerAnne Stern, Konrad Honoldinventors
Life of this patent
Estimating the term…
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.
Claims20
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 a target nucleic acid sequence coding for an endogenous target polypeptide, the said nucleic acid molecule comprising
- (a) at least one sequence section which is homologous to sequences in the gene locus of the target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in the coding region of the mature target polypeptide and
- (b) a nucleic acid section 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 cell contains a mutated target nucleic acid sequence after the homologous recombination which is able to express 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 or/and the cell supernatant.
Process as claimed in one of the previous claims, wherein
- additionally the expression of the target nucleic acid sequence is activated by introducing a heterologous expression control sequence.
Process as claimed in one of the previous claims, wherein
- the nucleic acid section coding for the selection marker is a neomycin phosphotransferase gene.
Process as claimed in one of the previous claims, wherein
- the nucleic acid molecule introduced into the cell additionally contains an amplification gene and the mutated target nucleic acid sequence is amplified after the homologous recombination.
Process as claimed in one of the previous claims, wherein
- the target nucleic acid sequence is a tissue plasminogen activator (t-PA), erythropoietin, insulin, tumour necrosis factor, interleukin or interleukin receptor sequence.
Process as claimed in one of the previous claims, wherein
- the mutein is isolated from the supernatant of cells cultured in suspension.
Process as claimed in one of the previous claims, wherein
- the mutein is isolated from the supernatant of cells cultured in serum-free medium.
Process for the production of a human cell which expresses a mutein of a human target polypeptide, wherein
- (i) a nucleic acid molecule is introduced into human cells which contain a target nucleic acid sequence coding for an endogenous target polypeptide, the said nucleic acid molecule comprising
- (a) at least one sequence section which is homologous to sequences in the gene locus of the target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in the coding region of the mature target polypeptide,
- (b) optionally a heterologous expression control sequence for the target nucleic acid sequence and
- (c) a nucleic acid section 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 cell contains a mutated target nucleic acid sequence after the homologous recombination which is able to express a mutein of the target polypeptide,
- (iii) the cells, in which a homologous recombination has taken place, are selected and
- (iv) the cells selected in this way are isolated.
Dependencies are inferred from the claim text.