Hi,
Can anyone recommend a specific mammalian expression vector that I could use to tag my protein of interest and that would allow me to purify any associated complexes for MS analysis? I am thinking of a vector containing a StrepII tag or similar (ideally in combination with another tag) that allows me to tag my POI and purify complexes by running lysate over a StrepTactin column. I would be grateful for any advice on this topic!
You could use our retroviral vector pOZ.FH that allows one to generate stable cell lines expressing HA-Flag -tagged protein of interest and then perform a tandem affinity purification (Nakatani Y, Ogryzko V. Immunoaffinity purification of mammalian protein complexes.Methods Enzymol. 2003;370:430-44). Sigma now sells a kit for this purification (http://www.sigmaaldrich.com/catalog/product/sigma/tp0010?lang=fr®ion=FR) with agarose-conjugated antibodies and peptides for mild elution. It has been working very well, and not only in our hands.
6xHis tags are pretty popular although you could also use concatemerized versions of Flag or Myc tags as there are good precipitating antibodies bound to beads or columns. There are many commercial vectors of each type.
Like the above response, HIS,FLAG,MYC tags are available to tag POI. There are also commercially available columns from BIO-RAD which you can use to purify. One thing to consider, if you want to do functional assays after purification, there are several reports which indicate that tagging might affect the functions of proteins. Also give a thought about where you want to insert the tag, like C or N-terminus.
As the others suggested, HIS, FLAG, MYC, SBP, Strep-tag II tags should work OK for your purpose and anti-tag bound beads for these are readily available commercially (not cheap, though!). Anyway, I'd suggest to use a system which allows you to elute tagged POI and associated proteins specifically (e.g. elution by competitive peptide, elution with free biotin) to reduce background. Also, each tag is likely to have a different affinity for the resin - check the Kd (or the binding capacity if the Kd is not available). Furthermore, In the case of Flag & Myc, you can add 2-3x Flag or 2-3x Myc in tandem. This will increase the affinity of the tag to the resin i.e. the efficiency of IP. However, be aware that this may also make elution more difficult though you can overcome the problem by increasing amount of competitive peptide as well as repeating the elution step several times. Anyway, HIS, 1x FLAG & SBP tag worked well for me though I never tried a combination of these tags.
Agree with the above - have tried them all, didn't like StrepII. A triple HA tag is now my preferred tag - binding to monoclonal anti-HA agarose resin (Sigma) and mild elution with the HA peptide. You could stick a 3xmyc or 3xFlag tag at the other end if you really want. For down stream MS analysis - what works well is to express your POI (in bulk culture ie >10-15mg total protein). After lysis, split your protein mixture in two - bind half to anti HA and the other to mouse IgG agarose resin and elute both resins the same way. The MS guys should then be able to eliminate most of the non specific peptides binding to the IgG resin - see attached paper about common non-specific contaminants, apart from the amusing title you may find it useful, it mentions tags as well...
You could use our retroviral vector pOZ.FH that allows one to generate stable cell lines expressing HA-Flag -tagged protein of interest and then perform a tandem affinity purification (Nakatani Y, Ogryzko V. Immunoaffinity purification of mammalian protein complexes.Methods Enzymol. 2003;370:430-44). Sigma now sells a kit for this purification (http://www.sigmaaldrich.com/catalog/product/sigma/tp0010?lang=fr®ion=FR) with agarose-conjugated antibodies and peptides for mild elution. It has been working very well, and not only in our hands.
Hi Arshad,
I've only used a GST-tag system for purification of recombinant proteins expressed in E.coli. Also, I have no experience in using the methods described in the literature you referred. I normally use 6x His-tagged TEV for tag removal and do all incubation steps in 50mL falcon tube on a rotator @4C. After washing steps, I wash the POI-bound beads with appropriate digestion buffer twice. Then, I do O/N digestion with TEV @ 4-16C and remove TEV by affinity chromatography with Ni or Cobalt beads or gel filtration if the size of POI and that of TEV are sufficiently different (or you can combine both). As far as I know (from my own experiences and what I heard from my colleagues), the on-column digestion of the tag seems to work for most of proteins. However, it doesn't work well occasionally - we guess that for whatever reason the cleavage site is masked by interaction with a part of POI. Although I never tried on the on-column digest, Precision, Factor Xa and Thrombin worked well for cleavage of a tag after elution & dialysis against digestion buffer,
Anyway, if you want to apply a GST-tag system to mammalian cells to look at interactome or functions of POI, there are at least two major things you need to concern:
1) GST-tag is relatively large (ca. 25kDa) compared with other commonly used tags like His-, Myc, Flag and HA.
2) commonly used GST-tags dimerize.
Hope the above info helps you to design your experiment.
GST, HIS and FLAG tags are the most papular systems in use and you could find many commercially available vectors for these. if you protei of interest is of lower molecular weight, I would personally recommend to use a GST tag .
Strep tag ist working fine, for high yield proteins, the His tag is a nice alternative, since it is cheaper. However, the interaction between the 6x His and the Ni-NTA is not that specific, so your expressed protein must be highly expressed. Otherwise you have to struggle with unspecific binding. FLAG tags are also quite good, but since it is antibody dependant it is more expensive. Especially if your protein is sensitvie to low pH, because in that case you have to elute with the FLAG peptide instead of Glycin pH 3.
you might find the following useful:
pSF-CMV-Puro-COOH-FXa-GST (OG1070) C-terminal GST tag mammalian plasmid from Oxford Genetics.
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