I have cloned and expressed a mammalian protein in E.coli BL21 with GST tag. I use glutathione agarose beads (sigma) for affinity purification (not pre-packed column). I use 0.4% sarkosyl to solubilize my protein from pellet fraction. For 5-10 rounds of purification my protein gets eluted properly. Then it starts running out in flowthrough. Is this problem because of sarkosyl? How can I overcome it?
I would recommend avoiding laurylsarcosine - It is quite a harsh, ionic detergent and may well unfold your protein - if indeed it is folded in the first place.
I notice that you are purifying the protein from the pellet - i.e. the inclusion body fraction - implying that you don't have soluble expression of your target. In that case you really need to purify the inclusion bodies, solubilise them in either urea or guanidinium chloride and then refold the protein using one of several methods - e.g. rapid dilution or dialysis. Do you have an activity/binding assay to confirm that the protein you are purifying is correctly folded?
I would personally avoid the use of a GST tag - it certainly isn't helping make your protein soluble in this case, it is prone to dimer formation and the resin for purification is quite expensive too. Have you tried a His-tag alone at either N- or C-termini? Another tag to try might be a His-SUMO tag.
You may well find that obtaining correctly folded protein in E. coli for your mammalian protein proves very difficult if not impossible. Your best bet may be to simultaneously attempt expression in baculovirus/insect cell and mammalian (e.g. HEK293, CHO cells) systems.
I'm worried by your phrase 5-10 rounds of purification? If the GST tag works and the protein is normally folded then one (or two) passes through a column should be enough. Looks like the protein is aggregating (It is probably very beat up by now). If it were mine I'd move to another method of purification (ion exchange gel?) after the first round. Tagging works as a preliminary method but then you got to move on . I agree with Dodd that less detergent (maybe cholate?) and another tag is prossibly indicated -- I also suggest (His)6 tagging.
Thank you for your suggestions...
@dr. Roger: yes initially i used to get my protein in elut, which after dialysis showed activity. Ok i can go for urea, but is it not harsh too? I had tried his tag before going for gst tag. But i was not getting any expression of the his tagged protein at all, i have no idea why!
@dr. Baxter: i had also tried sodium deoxycholate for solubilizing but to no avail... By 5-10 rounds of purification i mean 5-10 times of passing and eluting and regenerating the column. Glutathione agarose being expensive i cannot discard used beads after 5 odd rounds of affinity chrom.
I will look into sumo tagging... Thank u so much for the suggestions... I will also carry out mammalian transfection...
I think procedure of cleaning and storage of column and most important sample preparation.
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@yamak: thanx a lot... I went through the pdf... But since we recently got his tag column from ge , i am not concentrating on buying glutathione agarose/sepharose column pre packed. M focussing on proper cleaning of my column.
.. Also i have changed my protocol for purification and for now am getting purified protein...
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