Anyone who has done som systematic investigations of linker/spacer regions between the typical solubility tags (e.g. TRX, MBP) and protease sites (e.g. 3C, TEV). I am thinking about something like this. For many of the conventional vectors, the MCS could be pretty large, and allow insertion of GOI into any position of choice - making both long and short spacers. Now with the new generation of cloning methods, one could in principle insert genes whereever.

Previously, we have had a discussion on linker between the TF tag and protease site, which might need special consideration since the TF C-terminal "folds back on itself", and could sterically hinder protease treatment. I link to Patrick and Besir's comments here. What about other tags?

Hi Gro,
this is one of the hundreds of question still to be solved in our field, unless a thourough study has been done somewhere. To answer this kind of questions and learn things on a wide spectrum  it would require massive experiments and 2-3 things to really learn something:

- Some careful thinking on the choice of target proteins to choose (which ones, how many different ones...)

- Some careful thinking on the choice of fusion proteins to choose (which ones, how many different ones...)

- A good cloning strategy to be quick and efficient

- A serie of linker length and composition (which ones, how many different ones...)

Probabaly a huge experiment space to cover for a single lab ...

That's where a network like P4EU could be more useful than where we are now (already a nice place to talk and meet and exchange) and make the difference for the advance of our field (let's be optimistic). Among us all we have a huge capacity, we have thousands of targets, lots of cloning vectors, fusions and a fair amount of knowledge and facilities, not mentioning people having prior experience with linkers, fusions...

This could be done in a mode that we haven't really tried among us so far (except maybe on the sequencing project of Sf9 cells... What happened to this BTW?), which would be to see how many people would be interested by this question,  willing to participate and ready to have an input (information, knowledge, cloning vector, cloning capacity, expression screening capacity...). Then we would define the space to cover (say: 4 tags, 10 linkers, 20 proteins...) and fix some items (cloning vector, culture conditions...) choose the items per category (I would go for TRX, SUMO and more for fusion part ) and some of us (the best for the job) would do the work.

Once the plan is done, a bit like SETI one/several labs would do the cloning and send all the clones to several labs in small subgroups of targets (if there are tens of thousands of culture to be done) or to a few HTP labs (SGC, OPPF, else? I'm volunteer for this part) if there are only few thousands culture, then we would gather the results and come to a conclusion. We (SPINE/SPINE 2 labs) have done some similar things in the past. We know that this is a lot of work when it comes to compare the results as even things done in different labs on the same targets  will give different results while we think that we are following the same protocol but that overall we still could come with trends and conclusion. Because of the variability that we observe between 2 labs I would rather use a network where a lot  of people ggive an input at the thinking stage but only very few labs do the jobs to decrease variability issues.

Gro, coming to this point, I realize that to be worth the common effort I think that there might be other points where this way of working all together might be more useful than screening for linker length.

If people are interested to participate to a screening of linker they can answer on this topic. But if people have ideas on more "hot topics" than this one we could open a new topic to discuss collaborative efforts like screening/benchmarking.

My 2 cents.

Renaud

P.S Sorry Gro for the mess in your topic...

Hi Renaud,

wonderful respons! You raise many interesting topics for discussions.

However, I never meant to invite for a collaboration, really (yes, many more interesting questions to collaborate about than linkers). I was just having a question related to linker lengths.

For example, (yes, I could have been more specific in my question), I have recently used - TGTGSS- between MBP and TEV protease. Sometimes we have seen, as you all know, cleaving MBP off by TEV leads to insoluble protein. Other times, cleavage was not complete (adding more rarely helped). This could be explained by soluble aggregates, etc etc. But, I was wondering if the linker residues and/or length could be influencing, aswell. Of course, it is protein dependent, as always.

In the Gateway compatible MBP vector, pHMGWA, the linker region is (at least) - TGTGSYITSLYKKA- (due to the attB1 site), then comes TEV (if you choose to clone it in).

Anyone who has a general or spesific answer?

-Gro