Polypeptide cleavage method using ompt protease variant

C - Chemistry – Metallurgy – 12 – N

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C12N 15/09 (2006.01) C12P 21/02 (2006.01)

Patent

CA 2540446

A method of cleaving a polypeptide characterized in that, in the case where the amino acid at the P1-position of a desired cleavage site of the polypeptide is arginine or lysine, the amino acid at the P1~-position thereof is one other than aspartic acid, glutamic acid or proline, and one basic amino acid or two or three consecutive basic amino acids are located at an arbitrary part in the amino acid sequence of from P10- to P3-positions or from P3~- to P5~-positions (provided that in the case of having one basic amino acid, it is located at a position other than the P6- or P4-position), the polypeptide is cleaved at the desired site by using OmpT protease or a mutant enzyme thereof having a substitution of the amino acid at the 97th position from the N-end.

L'invention concerne un procédé de clivage de polypeptide. Ce procédé se caractérise en ce que, lorsque l'acide aminé à la position P1 d'un site de clivage désiré du polypeptide est arginine ou lysine, l'acide aminé à la position P1' dudit site de clivage est autre qu'acide aspartique, acide glutamique ou proline, et qu'un acide aminé basique ou deux ou trois acides aminés basiques consécutifs sont situés dans une partie arbitraire de la séquence d'acides aminés allant de la position P10 à P3, ou de la position P3' à P5' (à condition que l'acide aminé basique, s'il est présent, soit situé à une position autre que la position P6 ou P4). Le procédé selon l'invention permet de cliver le polypeptide au niveau d'un site désiré, au moyen d'une protéase OmpT ou d'une enzyme mutante de celle-ci, présentant une substitution de l'acide aminé à la 97ème position à partir de l'extrémité N.

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