Mutants of rna polymerases with increased stability

Details Mutants of rna polymerases with increased stability Mutants of rna polymerases with increased stability

C - Chemistry, Metallurgy

12

N

C12N 15/54 (2006.01) C12N 9/12 (2006.01) C12N 15/52 (2006.01) C12P 19/34 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2353792

The present application relates to mutated RNA polymerases from bacteriophages that have increased stability, for example under high temperature conditions. One example of bacteriophage encoded RNA polymerase is the T7 RNA polymerase. 77 is a bacteriophage capable of infecting E. coli cells. Examples of other E. coli infecting T7- like bacteriophages are T3, .slzero.I, .slzero.II, W31, H, Y, A1, croC21, C22 and C23. An example of a Salmonella typhimurium infecting bacteriophage is SP6. The present invention is concerned with the RNA polymerases of T7-like bacteriophages that have been mutated. Due to these mutations the RNAP's have an increased stability. Preferred mutated RNA polymerases according to the invention are mutant RNA polymerases from 77 or SP3 bacteriophages. Due to the high homology between these enzymes, mutations in the T7 gene 1 sequence are likely to have the same effect in the corresponding gene sequence of the T3 bacteriophage. An especially preferred embodiment of the present invention is a T7 RNA polymerase with a serine to proline amino acid change in the protein at position 633 of the amino acid sequence. Since there is 80 % homology between the T7 RNA polymerase and the T3 RNA polymerase the same effects of the 633 serine.fwdarw.proline mutation in the 17 gene may be expected for a 634 serine.fwdarw.proline amino acid mutation in the T3 RNA polymerase.

La présente invention se rapporte à des mutants d'ARN polymérases issus de bactériophages et dotés d'une stabilité accrue, par exemple dans des conditions de température élevée. Un exemple d'ARN polymérase codée par un bactériophage est la T7 ARN polymérase. T7 est un bactériophage pouvant infecter des cellules de E.coli. Des exemples d'autres bactériophages ressemblant au T7 et infectant E.coli sont T3, phi I, phi II, W31, H, Y, A1, croC21, C22 et C23. Un exemple de bactériophage infectant Salmonella typhimurium est SP6. La présente invention se rapporte à des ARN polymérases de bactériophages ressemblant au T7 qui ont subi une mutation. Ces mutations confèrent aux ARN polymérases une stabilité accrue. Les ARN polymérases mutées conformes à la présente invention sont des ARN polymérases mutantes issues de bactériophages T7 ou SP3. En raison de la forte homologie entre ces enzymes, les mutations dans la séquence 1 du gène T7 ont de fortes chances de produire le même effet dans la séquence du gène correspondant du bactériophage T3. Une réalisation particulièrement préférée de la présente invention est une ARN polymérase de T7 comportant un changement d'acide aminé, sérine}proline, dans la protéine, en position 633 de la séquence d'acides aminés. Etant donné qu'il existe une homologie de 80 % entre l'ARN polymérase de T7 et l'ARN polymérase de T3, on peut s'attendre à ce que la mutation 633 sérine}proline sur le gène T7 produise les mêmes effets que la mutation 634 sérine}proline dans l'ARN polymérase T3.

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