Hi, I didn't want to get into this because so many good comments and suggestions have already been made - but I guess I will make a wordy and effusive comment of my own :)
In my opinion, the question of how *specific* tags (His, c-Myc) influence crystallization is somewhat misleading. The question infers that there is special significance to His and c-Myc, whereas the data available to us so far strongly suggest that the effect of these tags cannot be readily generalized and is in fact highly dependent on the protein bearing the tag, the details of purification, crystallization, etc. So in this respect I fully expect the crystallization effect of the c-Myc tag to be very similar to that of the His-tag (i.e. context-dependent!). Indeed His-tag sometimes causes proteins to misbehave due to metal crosslinking etc. however this is rather rare. Incidentally, His-tag can also have some very strange post-translational modifications in E. coli (also a very rare phenomenon). The above is not intended to infer that there is no significance to the amino acid composition of the N or C terminal extensions - that is a very different question and I am deeply convinced that such significance in fact does exist. However, such significance is a) much more prominent in the context of individual constructs and b) neither His nor C-myc tags contain 'special' sequences that may be particularly beneficial or particularly disruptive towards crystallization. As a counter-example I would suggest that an Arg-tag (sequence of several arginines) does in fact have an intrinsic detriment with respect to protein crystallization. I am of course suggesting this with relative impunity since the data detailing Arg-tag's influence on crystallization is almost non-existent. I've worked on exactly two c-Myc tagged proteins (for crystallization). In both cases the tags were removed and in both cases the proteins did not crystallize. What helped us to crystallize these molecules was domain hunting and design by which time the c-Myc tag was abandoned due to the costs associated with antibody-affinity resins. This does not in any way imply that the next five proteins we could have tried with this tag on wouldn't have crystallized. The data set is simply too small :) And so my only useful suggestion is the same as that of many other people - do try both. It is often technically easier to leave the (any)-tag on, and it's not very hard to remove it either by enzyme action or via construct engineering. I have personally worked on proteins that only crystallized (in my habds) with a tag and also on proteins that absolutely 'required' the tag to be cut off or deleted for crystallization. The score so far is even as far as I know. In general it seems that 'long and floppy' extensions on the termini are not helping in crystallization so it stands to reason that dual tags (on the same terminus) may be detrimental. Whether dual-tagged proteins with tags on both termini are less likely to crystallize is an open question. Good luck! Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of iulek Sent: Thursday, November 13, 2008 12:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] SUMMARY - crystallization of proteins with His-tag and/or c-myc tags Thanks for all who shared their experiences. I received some personal and public suggestions/comments. So, time for a summary now. My original question was about the influence of his-tag and c-myc-tag on crystallization. Concerning His-tags: some opinions differ, a brief summary of the points raised are - in some cases important for crystallization - choose a method at the start to take out the tag, yet it is more likely to crystallize without it. - cases where crystals came only with his-tag, but also the opposite, either crystals (or better crystals) without his-tag. - a recall that it is not all that difficult to cleave; also, recommendation to check 3C protease and His-SUMO tag ideas, other recommendation, look into the Tagzyme kit. - sometimes His-tags might make the protein prone to aggregation. Also, might bind cations at high pH's (might set up crystal contacts) I see some recommendations/comments: you have the his-tag, go to it first, consider taking out the tag if crystallization trials were unsuccessful. That is a standard in some groups. But..., no one commented about c-myc tags. As I expected, there is no experience with this one yet - ? Jorge
