Protease purification - plant leaf protease (Apr/02/2006 )
I was wondering about the exact condition that is usually maintained during protease extraction from plant leaf. We do extract proteins from different tissues at about 4 degrees celsius in order to inhibit proteolytic activity. But if our aim is to extract protease then how should I be proceeding? Should I be working at 4 degree celsius all the way, from while making the crude extract, ammonium sulfate precipitation upto activity assay? And I am also about skeptical about the amount of protease present in plant leaf and also the localization of it. Most papers deal with abundant extracellular proteases from bacteria. So there will be no problem of extraction. But with plants, they are either cytoplasmic or vacuolar. So any idea how I might fractionate them?
I understand this protease from particularly plant doesn't interest most people in here. Anyhow I a novice at research level hopefully can get some idea.
you need to work in conditions that minimize the protease's activity, especially if it autolyses. low temperature is a good place to start. if the enzyme requires a cofactor then keep it away (eg- if it is a calpain, maintain edta or egta in the buffers to keep ca away from the enzyme). use of inhibitors is normally not recommended because they may be irreversible or hard to remove.
as for fractionation, if you must you can find procedures for subcellular fractionation. perform one then extract the protease from the fractions.