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tag for protein purification


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#1 Angrysh

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Posted 17 April 2013 - 06:05 PM

Hey everyone,

I am now planning an expression of recombinant protein and I want to use HA tag for protein purification. But my supervisor pointed out that for a large scale purification I will need cheaper tag (HA tag requires using of antibodies).

Can anyone suggest a cheaper tag?

Another question, does it make sense to add a restriction sites at the ends of the tag so the expression cassette could be modified (tag removed/changed)?


Thanks for the help!

#2 HOYAJM

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Posted 22 April 2013 - 09:53 AM

You should look into His tags. They are very common and cheap to use. You need a Nickel or Cobalt resin to purify a his-tagged protein. There are cleavable His tags available. Look into pET vectors. They have cloning sites that can be used to add a His tag that is cleavable with enterokinase. Or you could engineer a His6 tag into your forward primer and clone this into the vector of your choice.

#3 Angrysh

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Posted 06 May 2013 - 10:46 PM

Thanks! I'm now choosing between His tag and Strep tag; one more aspect - the tag should not be degraded by T. reesei (expression host) proteases.

#4 mdfenko

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Posted 07 May 2013 - 05:18 AM

you should download the handbook "recombinant protein purification" from ge healthcare.
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#5 labtastic

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Posted 14 May 2013 - 09:42 AM

Strep tags work great but the reagents can be a bit pricey if you're doing lots of purifications Another down side to strep tags is that the binding capacity of strep beads aren't as good as nickle or even cobalt resins. In my opinion, unless you have a specific reason to avoid using a His tag, use it. Reagents are cheap, resin can be reused after being regenerated, and the binding capacity is quite good. Depending on the level of expression, purity can be very good too. If purity isn't perfect, a quick gel-filtration run or some other separation column (ion exchange, hydrophobic interaction, etc) can usually do the trick.

#6 Angrysh

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Posted 21 May 2013 - 09:40 PM

The proteins will be expressed in Trichoderma reesei and according to the previous results (not mine) the His tag doesn't work in Trichoderma.

#7 mdfenko

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Posted 22 May 2013 - 05:07 AM

what about a gst or mbp tag?
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#8 labtastic

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Posted 22 May 2013 - 05:32 AM

Why can't you use a his tag in T. ressei? I would imagine the physical expression of 6 consecutive histidines is perfectly within its capabilities...perhaps some toxicity issue? Or perhaps too many other native T. ressei proteins bind to nickle resin?

Regardless, if that is the case, then a strep tag should work just fine. The other option is a cleavable GST tag, which you can purify using glutathione beads (edit: mdfanko just beat me to it :) ).

Or lastly, you could go "old school" and purify it without a tag. Finding the right purification scheme could take some time but is very much doable, especially if the protein overexpresses reasonably well.

Edited by labtastic, 22 May 2013 - 05:33 AM.





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