I've cloned my GOI into several vectors creating fusion proteins with several tags: MBP, intein, His on N-terminus, His on both termini. Although I have activity in my extract, I'm not able to purify the enzyme using the respective tags, the protein is always in flow-trough.
The only option that seems to be left is to purify it on normal columns with the use of the tags.
Do you have any suggestion, what could I try or something?
I have a silly question. Have you selected the right valve position on the FPLC. Default (at least on ours) is to bypass the column as we always start by preping the lines and pumps with buffer
Actually I usually purify it on low pressure LC, but on FPLC the program is automatic, so I'm pretty sure it goes through the column.
Another silly question: did you mix the different tags so you would have your His-Tagged protein on the chitin beads and vice versa?
Protein in the flow-through usually indicated that either tags are buried in the protein (which in your case is very unlikely) or that the tagged protein does not bind to the resin because of bad buffer conditions (pH, salt, imidazole, DTT, etc.). What are your current buffer conditions?
regarding the his tagged version; did you tried to perform the purification in denaturing condition?
Because in case that the protein will bind to IMAC in this condition, it will confirm the presence of the HIs tag and suggest that you have problem of tag exposition that, in case that also GST and MBP are not able to bind their respectivy affinity coloumn could be due to protein aggregation.
is it your protein completelly soluble on text expressione? Which lysis/binding buffer are you using?
I think your protein of interest is insoluble. After lysis, followed by centrifugation, do check whether your activity is in pellet or in supernatant fraction. If pellet shows activity, then definetly , your protein is insoluble.
No, no, I wrote that I have activity in my extract.
However, as it seems, there is high activity in WT bacteria, so I'm actually trying to purify only small fraction and thus I have most of the activity in the flowthrough.
Are you sure the fusion protein expressed? You say there is high activity in WT bacteria.
I also have a series of stupid questions :-) Is there any chance the activity in your extracts is actually not due to expression of your GOI but due to some bacterial enzymes with similar activities? I assume you sequenced your expression plasmid and checked for the reading frame as suggesested earlier. Did you try to confirm the presence of your tags / and protein in the extract by e.g. western blot?
If you have higher activity in transformed bacteria as compared to WT, it means that your GOI is expressed and active. For the question, I think that you may check the quality of the resine or try using new one, make sure that the pH is well adjusted.
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