I want to conduct cellulose hydrolysis experiments using purified cellulase enzymes (endoglucanases and cellobiohydrolases). In literature, researchers have mentioned enzyme purification from commercial cellulase mixtures (such as Celluclast® 1.5 L) using column chromatography, with FPLC (Fast protein liquid chromatography). However, exact protocol of the technique has not been provided. Can somebody provide the detailed protocol?
That depends on what column you want to use - hydrophobic, ionex, affinity, etc.
Thanks Tomas for the reply. In the papers authors (e.g. Jäger et al 2010, Biotechnology for Biofuels 2010, 3:18) have mentioned the use of ionex and hydrophobic interaction columns. However exact protocol is not provided. I am looking for detailed protocol for purification of endoglucanase, CBH I and CBH II from commercial mixture (such as Cellucalst 1.5L).
The exact protocol depends on the column you want to use. As a general rule first you have to equilibrate the column with 3-5 bed volumes equilibration buffer so as to provide optimum binding conditions(in cases where protein will bind to column matrix first and will be eluted later). Then you can load your protein. After that you can elute the protein with elution buffer which is again dependent on the column( e.g. increasing concentration of salt (NaCl) or change of pH in case of Ion exchange, increasing concentration of ethylene glycol in case of Hydrophobic interaction chromatography). Needless to mention every buffer has to used 3-5 bed volumes. Before elution you may include one wash step where unbound or loosely bound proteins may elute. You can chose Column compatible buffers from the column manual. Most of your queries are answered here --> http://chemgroups.northwestern.edu/ohalloran/grouponly/proteinpurification.pdf
If still have some doubt write back.
Best of luck with Purification !
Thanks a lot Virender!!! I will read more from the handbook and will ask you for any doubt.
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