I m purifying it using IEx (DEAE) and GF chrm but not getting success cz of aggregation. In ion exchange the enzyme is getting eluted at low salt concentration of 0.2M while in GF chrom (Sephadex G75) the enzyme is coming out at low elution volume. Any suggestions???
DEAE allows purification according to charge and isoelectric poitn of your protein while GF allows purification based on molecular weight. Are you sure your protein is aggregated? under G75 , protein with molecular mass higher than 75 kDa will be in the void volume. What is the molecular mass of the monomeric form of your lipase? if it is around 35 to 40 kDa under SDS PAGE a dimeric form could be nearby the void volume and eluted quickly without being aggregated.
If the lipase protein is aggregated because of its lipophilicity, you might be able to disaggregate it using a detergent, or some organic solvent.
I am saying the aggregation behavior of protein based on its active band in native PAGE at starting of resolving gel (Not able to move further due to high mol wt), with faint band at around 65 KDa.
I GF I have tried Sephadex G200 also but got same results. In DEAE, the binding is occuring as no active fraction is coming out in initial washing steps with buffer before salt elution, but the protein is eluting at low salt concentration may be because of aggregation.
CHAPS (5 mM) also didnt work in disaggregating the protein.
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