Cells and method for producing proteins comprising an...

Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...

Reexamination Certificate

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C435S440000, C435S252800, C435S071200

Reexamination Certificate

active

07736899

ABSTRACT:
The present invention relates to a method for providing bacterial or yeast cells with the capacity to produce a protein, the amino acid sequence of which comprises at least one unconventional amino acid. The method involves (a) introducing at least one missense mutation in a target codon of a gene encoding a protein required for the growth of the bacterial or yeast cells, where the mutated protein synthesized from the mutated gene is not functional in the bacterial or yeast cells. The method also involves (b) selecting the bacterial or yeast cells obtained in (a) in a culture medium which (1) does not contain a nutrient compensating for the loss of functionality of the mutated protein and (2) contains an unconventional amino acid which restores the functionality of the protein required for growth of the bacterial or yeast cells, in which the unconventional amino acid is that encoded by the target codon. The method also involves culturing the bacterial or yeast cells obtained in (b) in a culture medium containing the amino acid encoded by the target codon.

REFERENCES:
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Seale et al. Amino acid replacements resulting from suppression and missense reversion of a chain-terminator mutation in Neurospora. Genetics. vol. 86, No. 2 Pt. 1, pp. 261-274, Jun. 1977.
Chiu et al. Genetic and molecular analysis of a tRNA(Leu) missense suppressor of nudC3, a mutation that blocks nuclear migration inAspergillus nidulans. Genetics. vol. 145, No. 3, pp. 707-714, Mar. 1997.
Drabkin et al. Initiator-elongator discrimination in vertebrate tRNAs for protein synthesis. Mol Cell Biol. vol. 18, No. 3, pp. 1459-1466, Mar. 1998.
Landes et al. A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases. Biochimie vol. 77, pp. 194-203, 1995.
B. Lemeignan, et al., “Phenotypic suppression by incorporation of an alien amino acid”, Journal of Molecular Biology, vol. 231, No. 2, May 1993, pp. 161-166.
V. Döring, et al., “Reassigning Cysteine in the genetic code ofEscherichia coli”, Genetics, vol. 150, No. 2, Oct. 1998, pp. 543-551.
J. D. Bain, et al., “Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code”, Nature, vol. 356, Apr. 9, 1992, pp. 537-539.
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