Indeed Strep-II (please note there is also a Strep-I which is not as good!) tag is a good tag to try if you can. There are a few caveats that you may want to consider when trying it:
0. if things work out, Strep-II affinity purification can yield essentially pure protein in one step. The question of whether this protein is homogenous or not still needs to be addressed though (via sizing columns, light scattering, etc.) We have had some very mystifying cases where the Strep-II tag just did not work, for no apparent reason - no matter what we tried. (So, in the end we blamed it on the 'hidden' tag scenario). 1. biotin in medium is a potential problem (not as bad for insect cells, but can be bad for yeast) 2. you may want to incubate your lysate with small amounts of avidin, to capture away all the biotinylated eukariotic proteins (Strep-II tag doesn't really bind avidin, but Strep-tactin binds biotin). 3. the resin is pricey and only binds roughly 1 mg/ml of an 'average' (300 aa) protein. It may be reused (very carefully) about 10 times if you handle the column like it's a 2500-year old mummy. Typically 5 or more re-uses result in significant loss of capacity. 4. the tag only works in native conditions as it is structured and binds to a protein that is structured as well. Passing 8M guanidine through a $6000 column only to find out that it's dead afterwards can cause much sadness. 5. the column can degrade very quickly if you have unusually high protease load in your lysate Caveat emptor :) Artem > Hi, > > > > We got some good experiences using the IBA streptag for baculo-expressed > proteins. You'll have to redo the cloning, but it will be worth your while > when you see the first purification step, > > > > cheers > > Joost > > > > -----Original Message----- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Alex > Berndt > Sent: Wednesday, 28 February, 2007 20:21 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Cannot running NTA to purify the protein having > His-tag? > > > > sometimes the insect cell medium intereferes (for whatever reasons) with > nta purifications when they ar employed as a first step in the > purification scheme. i experienced that occasionally. this can easily be > circumvented by doing an ion exchange step beforehand! > > alternatively you might want to introduce a linker between your protein > and the his-tag or create a 8xhis or 10xhis tag to enhance bing to the nta > matrix. make sure you wash your cells from residual medium before you > freeze your pellets. > > > > alex > > > > On 28 Feb 2007, at 19:18, Juergen Bosch wrote: > > > > > > Ngo Duc Tri wrote: > > > > Dear CCP4 users, > > > > I'm purifying a kind of protease having His-tag. The protein is > expressed > in insect cells and broken by sonication. > > I used NTA resin to purify this protein. > > Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B is > 50mM > phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole. > > However, all proteins cannot bind to NTA resin. My protein is eluted in > Flow-through. I also check the NTA resin with the control His-tag. The > western blot also shows that my protein has His-tag. > > > > Do you have any ideas about my problem? I'm really appreciate all of > your > advices how to solve this. Thank you very much! > > > > My best regards, > > TriNgo > > Sungkyunkwan University > > > > You His tag is most likely inaccessible, can you easily change the tag > from e.g the N-terminus to the C-terminus ? Or if you have a structural > homolog you could add the His tag into a loop, which is exposed. > > > > Alternatively you can purify your protein under denaturing conditions > using 8 M urea and refold it if you dare :-) > > > > Juergen > > > > -- > > Jürgen Bosch > > University of Washington > > Dept. of Biochemistry, K-426 > > 1705 NE Pacific Street > > Seattle, WA 98195 > > Box 357742 > > Phone: +1-206-616-4510 > > FAX: +1-206-685-7002 > > > > --- > > Alex Berndt > > MRC Laboratory of Molecular Biology > > Hills Road > > Cambridge CB2 2QH > > UK > > > > mail : [EMAIL PROTECTED] > > phone : +44 (0)1223 402113 > > --- > > > > > > > > > > > This message, including attachments, is confidential and may be > privileged. If you are not an intended recipient, please notify the sender > then delete and destroy the original message and all copies. You should > not copy, forward and/or disclose this message, in whole or in part, > without permission of the sender. >
