Help to separate fungal proteins - (Mar/15/2006 )
Hello all
I have a small problem.... I am currenting trying to purify an endoxylanase from a crude extract. Using IEX, HIC and gel filtration chromatography I have wittled it down to 3 bands (22, 15 and 7 kDa by SDS PAGE). Using a specific zymogram technique, only the 22 kDa band was enzymatically active after renaturing with 25% IPA. Have had all bands sequenced and the N-termni to 15 amino acid residues are identical!!!
Does anybody have any ideas or maybe some far-fetched explanation???!!!
It looks like you have monomer, homodimer and trimer
I was hoping that was the case but the bands after silver staining were of equal intensity after denaturing with up to 5% DTT and boiling for 10 mins. Under these conditions surely all of the sample would have reverted to its monomeric state
Right, it would be very surprising that trimers survive after such treatment...
True and from the results I got from the zymogram, only the 22 kDa band has xylanase activity. If this were a trimer, then you expect both the 15 kDa "dimer" and 7 kDa "monomer" to retain enzyme activity. In short it has me completely stumped andf there is next to no literature on the subject
Hi,
you obtained 3 bands in SDS-PAGE, that means denature conditions. What about molecular mass of your samples in Gel filtration? gel filtration can help you with native molecular mass.
you obtained 3 bands in SDS-PAGE, that means denature conditions. What about molecular mass of your samples in Gel filtration? gel filtration can help you with native molecular mass.
That is the problem they wont separate by gel filtration. When run on a native PAGE, they are largely represented by one band, whose mass I cannot determine cos it always runs funny. My concern is more towards separation than mass
since only one species appears to exhibit activity, can you try substrate or inhibitor affinity chromatography? how about immunoaffinity?
Have tried using a substrate based chromatography technique but with no joy. To date I have not come across any immunoaffinity methods specific for the enzyme xylanase. I am really stumped!!
you might be able to prepare your own immunoaffinity gel if there are any antibodies available or if you can prepare them yourself (not really that difficult if you have the facilities), but i suspect that the ab will recognize all three peptides. i think that you may be seeing some amazingly well directed cleavage of your enzyme on the c-terminal side. either that or you have three homologous subunits that show a cooperative effect.
you may be able to separate them with inhibitor affinity chromatography. i looked at some of the literature. you may be able to bind taxi-i or taxi-ii to sepharose. of course you would have to first determine that these inhibitors work against your enzyme.