what's the difference between the purification of GST-fused protein and His- - (Oct/13/2007 )
I am currently busy on protein purification. My major is not biochemstry so quite don't know lots of things. Can anybody tell me what the difference between the purification of GST-fused protein and the purification of His-tagged protein? What's the advantage and disadvantage of them?
I tried both to purify SENP1. But His-tag doesn't work well while GST-fused works. Does anyboday know the reason?
Thanks in advance!
depends on what you mean by 'didn't work' 
basically, the purification tag is not the same. usually one will work better. this depends on many things - pI of the protein, 3-dimensional structure of protein + tag, perhaps one clone produced better than the other, or protein was stuck in inclusion with one and not the other? there are many reasons why one tag could work better than another...it's like comparing apples and oranges
I tried both to purify SENP1. But His-tag doesn't work well while GST-fused works. Does anyboday know the reason?
Thanks in advance!
.Hi
As a general ,people will express the recombinant proteins with tags. As per as GST concerned It s a very big (~25Kd) Protein tag which will make the recombinant protein soluble . Purification is very easy in phophate buffers. Affinity of Gst is more effective and you will get a good resolution(purified protein0. the disadvantage of this system is you have to remove the GST tag from the protein which is again depending on the site specific expression. GST affinity resins are very costly.
As per as the His tag concerned it is 6-8 His Amino acid side chain which will be expressed along with the recombinanat protein. purification was little bit tricky when handling soluble and IB's. There is no need to remove the His tag when it is not effecting the recombinant protein activity.His resins are not much costly. Host proteins are also binds to the resin. So one has to work on column washes and standardize the process.
Gst and His has its own advantages and disadvantages . there may n number of reasons why your protein was not purified with His tag.
how did you confirm that your protein was not purified with his.
Possible reasons:
1. Is your protein expressed with His and have good no of HIs tags?
2.is the His tag accessible to the resin(Ni)?
3.How about the Affinity of the His tag?
4.what about the contaminat load and affinity of that proteins when compare to your protein?
5. have analysed all the laod and flow through and washes?
Gst worked well because the GST it self is a very big molecule and it can easyly accessble to the resin binding site. (it seems you have expressed the protein in soluble form).
Correct me if iam wrong
awaiting for your reply
Regards
Sudhakar m
You may also want to look into other protein tags, such as KT3 and HA epitope.
What Shan wrote is correct. I will only add that 6-His is usually expressed with a linker sequence, so as to minimize the effect of the tag on the characteristics of the protein, so it's actually larger than just the His residues.
I'm sort of in the same situation; learned molecular biology and am now working in a biochemistry lab. I've had constant problems with different tags changing the expression level of my protein, and I would suggest that you try as many tags as you can, time permitting.
Thanks a lot for all you guys reply!
For Shan's questions,
1. Is your protein expressed with His and have good no of HIs tags?
It was the first time we use His tag to SENP1 and we were failed.
2.is the His tag accessible to the resin(Ni)? Yeah, one guy in the lab said he alway uses His tag to the protein which has small MW and it works well. His tag is accesible to Ni.
3.How about the Affinity of the His tag? I am not sure about this.
4.what about the contaminat load and affinity of that proteins when compare to your protein? The bands what I get are just dirty so it is hard to say.
5. have analysed all the laod and flow through and washes? No. I was just busy on follow every step and did not run gel. Next time I should.
A girl in our lab who keep on use GST told me the same advantage of GST. That is GST is soluble and easy to bind. But His may be easy to purify the small protein. not for the protein which has big MW. It may cause misfolding. That's what I get from the girl.
Thanks a lot for all the informations again. Have a good day!