Enzymatic method for modification of recombinant polypeptides

C - Chemistry – Metallurgy – 12 – N

Patent

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Details

C12N 15/16 (2006.01) A61K 38/25 (2006.01) A61K 38/26 (2006.01) C07K 14/60 (2006.01) C07K 14/605 (2006.01) C12N 1/21 (2006.01) C12P 21/02 (2006.01) C12P 21/06 (2006.01) A61K 38/00 (2006.01)

Patent

CA 2166870

The method of the invention provides for the formation of a recombinant polypeptide which has been modified at the C-terminal end through the use of a transpeptidation process. The method is suitable for modifying recombinant polypeptides of any source including those which may be commercially available, those derived from recombinant single copy or multicopy polypeptide constructs, or those derived from single or multicopy recombinant fusion protein constructs. The transpeptidation reaction involves contacting an endopeptidase enzyme with a recombinant polypeptide to substitute an addition unit, of one or more amino acids, for a leaving unit, linked to a core polypeptide through a cleavage site recognized by the endopeptidase enzyme. Recombinant polypeptides derived from multicopy polypeptide constructs may be cleaved from the multicopy polypeptide at the N-terminal and C-terminal ends and simultaneously undergo substitution of the leaving unit by the desired addition unit. The invention utilizes known and newly discovered cleavage recognition sites to effectuate the desired modification products.

Procédé de formation d'un polypeptide recombiné dont l'extrémité C-terminale est modifiée par un processus de transpeptidation. Le procédé convient à la modification de polypeptides recombinés de quelque source que ce soit, et notamment de ceux que l'on est susceptible de trouver dans le commerce, de ceux dérivés de structures polypeptidiques recombinées à copie unique ou à copies multiples, ou de ceux dérivés de structures protéiques hybrides recombinées à copie unique ou à copies multiples. La réaction de transpeptidation comporte la mise en contact d'une endopeptidase et d'un polypeptide recombiné afin de remplacer un motif labile lié à un polypeptide de noyau par l'intermédiaire d'un site de clivage reconnu par l'endopeptidase, par un motif d'addition d'un ou plusieurs acides aminés. Les polypeptides recombinés dérivés de structures polypeptidiques à copies multiples peuvent être séparés par clivage du polypeptide à copies multiples au nivau des extrémités N-terminale et C-terminale, et subissent simultanément le remplacement du motif labile par le motif d'addition voulu. Le procédé met en oeuvre des sites de reconnaissance de clivage connus et découverts récemment afin d'obtenir les produits de modification voulus.

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