Commissariat a L'energie Atomique

Frederic Ducancel

Jean-Claude Boulain

Andre Menez

Daniel Gillet

Paul Boquet

Laurence Cattolico

Modified alkaline phosphatases of bacterial origin (BAP or bacterial alkaline phosphatase) which consist of a bacterial alkaline phosphatase sequence in which at least one of the amino acid residues in position 329 or in position 330 is replaced by another amino acid residue, which modified bacterial alkaline phosphatases exhibit both significantly improved enzymic properties and an increased thermal stability, and their applications, in particular in immunoenzymic assays (reagents and diagnostic kits). Method for selecting mutants of alkaline phosphatases which possess significantly improved enzymic properties.

Modified bacterial alkaline phosphatases and their applications

A kit for carrying out an immunoenzymological assay for protein, containing a diagnostic reagent consisting of a hybrid protein comprising, in order, a polypeptide P1 which is an N-terminal portion of mature alkaline phosphatase comprising from 6 to 28 of the N-terminal amino acids of said mature alkaline phosphatase, joined to a polypeptide P2, capable of interacting with an antibody or antigen joined to a polypeptide P3 which is selected from the group consisting of a) the C-terminal remaining amino acids of said mature alkaline phosphatase, b) the mature sequence of alkaline phosphatase, and c) enzymatically active fragments of b).

Hybrid proteins between an extracytoplasmic enzyme and at least another

The disclosed invention involves a DNA encoding a hybrid tripartite protein comprising an amino portion consisting of from 6 to 28 amino acid residues from the N-terminal of a mature E. coli alkaline phosphatase, a central portion corresponding to a heterologous polypeptide and a carboxyl portion which consists of the remainder of the mature E. coli alkaline phosphatase, a different alkaline phosphatase or an enzymatically active fragment of either. The tripartite proteins produced with this DNA can be used as a diagnostic reagent and in the screening of nucleic acid libraries or in the selection of recombinant clones.

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Paris, France

Frederic Ducancel