Premium
A rapid purification procedure for cathepsin L from bovine species and production of specific antibodies
Author(s) -
Sentandreu Miguel A,
Aubry Laurent,
Ouali Ahmed
Publication year - 2004
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.1913
Subject(s) - polyclonal antibodies , chemistry , bovine serum albumin , cathepsin d , ammonium sulfate precipitation , biochemistry , antibody , cathepsin a , cathepsin l , cathepsin , chromatography , affinity chromatography , cathepsin b , enzyme , microbiology and biotechnology , biology , size exclusion chromatography , immunology
Two approaches have been carried out for the production of polyclonal antibodies specific of bovine cathepsin L (EC 3.4.22.15). The first one consisted in the purification of cathepsin L from bovine liver, through the development of an original protocol that can be achieved in 4 days, and further injection in rabbits. The purification scheme included the preparation of an enriched lysosomal extract, selective precipitation with ammonium sulfate and different chromatographic steps including size‐exclusion chromatography, cation‐ and anion‐exchange chromatography and the use of an affinity column for the removal of trace amounts of bovine serum albumin. The final enzyme preparation was purified 8400‐fold and consisted predominantly in the double‐chain form of cathepsin L. This was proved by the presence of a main protein band with M r of 25 kDa, corresponding to the cathepsin L heavy chain, in SDS‐PAGE under reducing and denaturant conditions, and was further confirmed by immunoblotting. The second approach consisted in the generation of polyclonal antibodies against the peptide sequence 99 PECSAANDTGFVDIPQR 115 , specific of the bovine cathepsin L heavy chain. Both types of antibodies were proved to specifically recognize cathepsin L. However, while antibodies raised against the whole protein recognized native cathepsin L, the antipeptide was able to recognize the enzyme in its denatured form. Both of them are adequate for the future development of immunoassays, allowing a rapid and accurate quantification of cathepsin L levels directly in crude extracts of meat and meat‐derived products. Copyright © 2004 Society of Chemical Industry