My vote is for His-tag *unless* your protein is of the same general class as Zn-fingers, B-boxes, etc. (weak/multisite/uncertain metal binders) - these proteins often do not fare too well with His-tags because a) the tag residues can participate in artefactual metal-binding sites and b) the use of chelators (imidazole, histidine) can adversely affect the end product. We have had an example fairly recently where his-tag was forming an artefactual half-site with two other residues from a real zinc site, and the resulting protein preparation was brown/red due to the (artefact!) binding of iron. There is also I believe a recent paper highlighting a similar problem with a B-box protein (or a RING finger, don't recall the specifics, sorry).
Artem On Tue, Apr 19, 2011 at 2:28 PM, Harman, Christine < christine.har...@fda.hhs.gov> wrote: > Thanks everyone for your comments. The overall opinion appears to be > that large-scale purification of flag-tag protein is expensive. I 100% > agree and have thought about that plus the dilemma with the flag-tag elution > difficulty (low pH etc) which some of you also pointed out. So I will > probably go with His. Thanks everyone for keeping me frugal. > > Cheers, > Christine > ------------------------------ > *From:* Chun Luo [mailto:c...@accelagen.com] > *Sent:* Tuesday, April 19, 2011 3:18 PM > *To:* Harman, Christine; CCP4BB@JISCMAIL.AC.UK > *Subject:* RE: [ccp4bb] Flag tag vs. his-tag > > His-tag is close to neutral but flag-tag is acidic. > > > > Using flag-tag for affinity purification usually gives cleaner protein but > it costs a fortune due to low binding capacity of commercially available > anti-flag resins. > > > > --Chun > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Harman, > Christine > *Sent:* Tuesday, April 19, 2011 11:11 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Flag tag vs. his-tag > > > > Hi all, > > I am trying to decide between using a N-term his-tag or an N-term flag tag > to be expressed on a protein that I will eventually want to try and > crystallize. I usually don't cleave my tags unless absolutely necessary and > I want to avoid double tagging. I am leaning towards the flag since from my > experience this tag is good for protein interactions (pulldowns), but I > don't know the effects of a flag tag in crystallization trials. Does anyone > have experience with crystallizing protein with flag tags? > > > > Thanks, > > > > Christine > > >
