rapid purification procedure for cathepsin L from bovine species and production of specific antibodies

• Published in: Journal of the science of food and agriculture 2004-12, Vol.84 (15), p.2052-2060
• Main Authors: Sentandreau, M.A, Aubry, L, Ouali, A
• Format: Article
• Language: English
• Subjects: antibodies; beef quality; Biological and medical sciences; cathepsin L; cathepsins; Cattle; enzyme activity; Enzymes; epitopes; Food industries; Fundamental and applied biological sciences. Psychology; Immune system; immunoblotting; liver; Livestock industry; meat; Meat and meat product industries; polyclonal antibodies; proteolysis; purification; rapid methods
• ISSN: 0022-5142; 1097-0010
• DOI: 10.1002/jsfa.1913

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 Mr 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 99PECSAANDTGFVDIPQR115, 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.