Development of affinity purification systems for isolation and proteomic analysis of proteases from Tetrahymena thermophila

Abstract

To better understand the functioning of proteases, we initiated a proteomic study to determine partial primary structures and to identify functional genes for intracellular cysteine proteases from Tetrahymena utilizing affinity chromatography and LCMS/MS (liquid chromatography‐tandem mass spectrometry). A peptidealdehyde tight‐binding protease inhibitor, antipain, was coupled to aminated agarose via carbodiimide condensation, and utilized to isolate proteases from clarified detergent extracts of late exponential phase cells. An immobilized lectin, concanavalin A (Con A), was used to isolate a subset of cellular glycoproteins from the detergent extracts. Protein isolates were subjected to trypsin digestion and the resulting peptides were further purified and sequenced by LCMS/MS. Cell extracts prepared with CHAPS detergent and purified against immobilized antipain, had fewer proteases and fewer contaminating proteins than Triton X‐100 extracts, but yielded higher quality spectra. Con A isolates from CHAPS extracts yielded few proteases. ESI Q‐TOF LC MS/MS confidently identified seven and tentatively identified four proteases from the extracts. Candidate genes for cysteine proteases in the Tetrahymena genome were subjected to phylogenetic analysis and the evolutionary relationships of the identified enzymes were examined. This work was funded by Vassar College and an HHMI‐USEP grant.