Method of controlling cleavage by ompt protease

Details Method of controlling cleavage by ompt protease Method of controlling cleavage by ompt protease

C - Chemistry, Metallurgy

12

P

C12P 21/02 (2006.01) C07K 14/58 (2006.01) C07K 14/585 (2006.01) C07K 14/605 (2006.01) C12N 15/09 (2006.01) C12N 15/62 (2006.01) C12N 15/63 (2006.01)

Patent

CA 2327524

By clarifying the properties of OmpT protease, a novel use thereof as a protease and a novel method of producing a target polypeptide are provided. More particularly, the present invention relates to a method of controlling cleavage of a polypeptide by OmpT protease which comprises converting a sequence site consisting of two arbitrary consecutive amino acids and/or amino acid(s) in the vicinity of said site in said polypeptide into other amino acids, characterized by (1) setting lysine or arginine as the amino acid at the -1-position concerning said site and setting a specific amino acid as the amino acid at the +1-position; and/or (2) setting specific amino acid(s) as the amino acid(s) at the -4-position and/or the -6-position relative to said site; so that desired parts of said polypeptide are cleaved by OmpT protease and/or undesired parts of said polypeptide are not cleaved by ompT protease. For example, the present invention provides a method of treating by OmpT protease a fusion protein having an amino acid sequence wherein the amino acid at the +1- position is X (wherein X is an amino acid other than glutamic acid, aspartic acid or proline); and a method of controlling cleavage by converting the amino acids at the - 6-position and the -4-position concerning the cleavage site into amino acids other than acidic amino acids.

Dans la présente invention les propriétés de la protéase de l'OmpT ont été précisées afin de permettre une application nouvelle en tant que protéase et un nouveau procédé de production d'un polypeptide désiré. Plus particulièrement, la présente invention concerne un procédé de contrôle de clivage d'un polypeptide par la protéase de l'OmpT comportant une conversion du site d'une séquence constituée de deux acides aminés arbitraires consécutifs au sein du polypeptide et/ou d'acide(s) aminé(s) autour dudit site en d'autres acides aminés. Ledit procédé est caractérisé en ce que: (1) l'acide aminé en position -1 lié audit site est la lysine ou l'arginine et l'acide aminé en position +1 est transformé en un acide aminé spécifiques; et/ou (2) l'acide aminé ou les acides aminés en position -4 et/ou en position -6 dudit site sont transformés en des acides aminés spécifiques, empêchant ainsi le clivage des parties indésirables du polypeptide par la protéase de l'OmpT.

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