Cloning and characterization of napsin, an aspartic protease

Details Cloning and characterization of napsin, an aspartic protease Cloning and characterization of napsin, an aspartic protease

C - Chemistry, Metallurgy

12

N

C12N 15/57 (2006.01) C07K 16/40 (2006.01) C12N 9/64 (2006.01) C12N 15/11 (2006.01) C12P 21/00 (2006.01) C12Q 1/68 (2006.01) G01N 33/50 (2006.01)

Patent

CA 2271294

A previously unknown aspartic protease capable of cleavage of proteins by hydrolysis, referred to herein as "napsin", has been cloned from a human liver library. Two cDNA clones have been cloned, sequenced and expressed. These encode isozymes of the protease, referred to as "napsin A" and "napsin B". The gene has also be obtained and partially sequenced. A process for rapid purification of the enzyme using immobilized petpstatin has also been developed, and enzyme isolated from human kidney tissue. Polyclonal antibodies to the enzymes have been made which are also useful for isolation and detection of the enzyme. Similarities to other aspartic proteases, especially cathepsin D, establish the usefulness of the enzyme in diagnostic assays as well as a protease. Either or both the amount or type of napsin expressed in a particular tissue can be determined using labelled antibodies or nucleotide probes to the napsin.

L'invention concerne le clonage, à partir d'une banque de foie humain, d'une protéase aspartique inconnue auparavant, capable d'effectuer un clivage de protéines par hydrolyse et ci-après désignée sous le nom de "napsine". On a cloné, séquencé et exprimé deux clones d'ADN complémentaire qui codent des iso-enzymes de la protéase dénommées "napsine A" et "napsine B". Le gène a également été obtenu et séquencé partiellement. On a également développé un procédé de purification rapide de l'enzyme grâce à une pepstatine immobilisée et on a isolé un enzyme provenant du tissu rénal humain. Des anticorps polyclonaux contre les enzymes ont été produits, ces anticorps permettant aussi d'isoler et de détecter l'enzyme. Des similarités avec d'autres protéases aspartiques, notamment la cathepsine D, démontrent l'utilité de l'enzyme dans des méthodes diagnostiques, ainsi que comme protéase. On peut déterminer la quantité et/ou le type de napsine exprimée dans un tissu particulier en utilisant des sondes de nucléotides ou des anticorps marqués contre la napsine.

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