Hello Anindito, While Nick and Paul have thrown you some great ideas around the TAP-tag theme (although GST may not be ideal as it dimerises itself), I feel that I've read your initial e-mail differently from them. Maybe you could clarify?
It sounds like you have a specific tag of ~5kDa in mind, presumably for a specific purpose? It's not the case that any old tag will suffice? Are you producing the dimeric protein recombinantly? If so, you have lots of options. If not, you're going to need a chemical modification post-purification (or to establish a recombinant system). Does the insertion of the tag need to be at a specific site within the one protein? Would one of the termini do? Critically, is your dimer a homodimer? If it's a heterodimer, things could be quite easy. All the best, Will. On 27 August 2013 18:50, Paul Paukstelis <shocksofmig...@gmail.com> wrote: > Nicholas is right that it depends a lot on the dimer you are working with. > I worked on a dimeric tRNA synthetase and was able to make heterodimers in > a couple of ways. The most effective was to make a bicistronic version of > the ORFs each with their own S-D. Each had a tag for tandem affinity > purification as Nick suggests. In my case, using two plasmids or even > putting the two ORFs with separate promoters did not result in heterodimers > (probably due to folding/dimerization during translation?). I was able to > express to the two dimeric versions separately, mix, dilute extensively > (which ultimately resulted in a big hit in yield), concentrate, and purify > through the tandem approach. I eventually noticed that even though it was > slow, there was dimer exchange occurring at intermediate concentrations. I > ended up engineering a disulfide at the dimer interface to minimize that. > > --paul > > On 08/27/2013 01:10 PM, Noinaj, Nicholas (NIH/NIDDK) [F] wrote: > >> Anindito, >> >> >> First off, I have never tried this, but here are a few initial thoughts >> on how I might approach this. But first a question, how tight is the dimer >> form? if you mixed dimers with tags and dimers without tags, would you get >> any exchange? If so, you might be able to use this to your advantage. But >> for now, i'll ignore this. >> >> >> Now for how i was thinking of possibly accomplishing the goal you want... >> >> 1- use two vectors or a DUET vector to express two versions of your >> protein, (1) with N-term cleavable (TEV site?) HIS tag and your fusion >> protein, and (2) with N-term cleavable (TEV site?) GST tag only. >> >> 2- express your protein as normal and then purify by (1) running the GSH >> column to capture all GST tagged proteins, followed by a (2) Ni-NTA column >> to capture those with HIS tags. If all works as planned, you would end up >> with one monomer having only HIS-fusion protein tags and one having GST >> tag. You could use SDS-PAGE/Western blot analysis to verify this, since >> the monomers will run at different sizes (you might have to modify your gel >> system to get the best separation....i recommend 5-7% gels for this size in >> MES) AND should be reactive to antiHIS and antiGST antibodies in needed. >> Further, you could get an idea for the ratios of each by comparing the >> overall intensity of the bands under normal staining methods. >> >> 3- to remove the HIS and GST tags, just treat with TEV protease to yield >> your dimer with only one of the monomers containing your fusion protein. >> >> >> I am sure i likely overlooked something in my haste, but you get the >> idea, basically a two step purification with two tags. Again, I haven't >> tried this but seems like it should work to me. hope this helps, good luck! >> >> >> >> >> >> >> Cheers, >> Nick >> >> >> >> >> ------------------------------**-- >> [ Nicholas Noinaj ] >> the Buchanan Lab >> Laboratory of Molecular Biology >> LMB-NIDDK, NIH >> 50 South Drive, Room 4505 >> Bethesda, MD 20892-8030 >> 1-301-594-9230 (lab) >> 1-859-893-4789 (cell) >> noin...@niddk.nih.gov >> [ the Buchanan Lab ] >> http://www-mslmb.niddk.nih.**gov/buchanan/<http://www-mslmb.niddk.nih.gov/buchanan/> >> >> >> >> ______________________________**__________ >> From: Anindito Sen [andysen.to...@gmail.com] >> Sent: Monday, August 26, 2013 11:26 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a >> Dimer protein complex >> >> Dear All, >> >> I need to insert a tag protein of ~5 kDa in one of the molecules of a >> dimer protein (size is ~100kDa). To be more precise - The tag need to get >> itself attached only on one of the dimer molecules. >> >> My expertise are not in Cell biology and therefore any suggestion in this >> regard will be of great help. >> >> Thanks & Best Wishes >> >> >> >> Dr. Anindito Sen (Ph.D) >> Structural Biology >> Graduate School of Medicine >> University of Tokyo >> 7-3-1 Hongo. Bunkyo-ku. Tokyo >> 113-0033. Japan >> Tel & Fax: +81-3-5841-3339 >> >
