Hi, I want to purify my protein by using TEV protein. Can anyone help me?
Hi Dalia,
I would love to help you with your problem, but I am not sure what you meant by your question. Did you mean that you have a recombinant protein with an affinity tag that includes the sequence recognized and cleaved by TEV protease? What is the protein you are trying to purify and what affinity tag did you clone it with? At what stage in your purification do you want to use the TEV protease to remove the tag? I will need a bit more detail about the actual experiment you are trying to do before I can help. Please be as specific and detailed as possible.
Hi Elizabeth,
I have recombinant protein with TEV cleavage site, I try to purify Toxoplasma protein the size about 19.5 kDa after cleavage. I already prepared the TEV protein and I used it to cleave the recombinant one with different dilutions but what happen was Ive got 2 bands the same size of TEV protein 26 kDa and weak band which its my recombinant protein. I dont know the reason for that?
the recombinant tags are GST His LT8.2TEV - fused to protein of interest(SAG). so the final step is to cleave my protein (SAG) from all tags by using the TEV protease site
After the cleavage reaction, the TEV protease should be removed by passing the sample over a nickel column or a glutathione column.
Hi Dalia,
Thank you for your reply. Don't forget that GST is a protein, and it is about 26 kDa in size. The two bands at 26 kDa are the TEV protease, and the cleaved GST-tag. Adam is correct: after the cleavage reaction, you should flow the protein solution over an affinity column to remove the TEV protease, the GST tag and any uncleaved protein. There are several TEV expression constructs in which the TEV protease has it's own polyhistidine tag, and I recommend using one if you aren't already. This is very convenient because it will allow you to flow your protein solution over a nickel affinity column and all the contaminants should bind the resin, and only your pure and cleaved protein will flow through.
The relative density of the band for your protein on SDS PAGE is curious. If the band for the cleaved GST tag is *significantly* stronger, it could indicate that you are producing truncated proteins during expression or you are experiencing significant cleavage from native E coli proteases early on in your protein purification. The best way to determine if this is occurring would be to run some western blots of samples from your expression and purification steps, using an anti-GST antibody (anti-Histag would work but is less specific). You should only see one band at ~45 kDa after protein expression and the GST affinity chromatography step. If you observe multiple bands, then you need to optimize your expression step and/or take measures to inhibit native protease activity.
I hope you found this helpful. Please feel free to respond if you need me to clarify something, or if you have additional issues you'd like help with.
Good Luck!!!
~ Beth from Practichem
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