I need that my protein after expression contains C-terminal His-tag and cleavage site before this His-tag (Tev protease / Thrombin ) without any addition signal sequenses and tags on the N-terminal.
Protein is need for NMR experiment. Sequence of protein: FKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKK LGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKG
Another one question; whether there can be a problem with expression such short protein?
Thank you for the all suggestion!
Yes other things being equal, you can expect less mass from a smaller protein. One reason for that is that initiation is a stochiometric mechanism -- whatever fixed amount of protein gets to initiate is going to have less mass, the smaller the protein is.
Except other things are not equal. There isn't a reliable way to predict this short of trying. But there are some rules of thumb -- avoid proteins phylogenetically far away from e. coli; Avoid proteins with advanced structures, like disulfide bonds or subdomains. Avoid small proteins is just one of the rules.
My intuition would be to express this as MBP fusion. It means that you're making a lot of mass (52kDa that you'll throw away later). But it's the best way to get your initiation rate to the highest point it can be.
Place all the tags on the N-terminus. MBP won't help you initiate faster otherwise. There's no point having a c-terminal TEV site. The TEV site is ENLYFQ // G. So with a c-terminal site, all of the ENLYFQ is going to stick to your protein -- which is exactly as big as an uncleavable his-tag would have been. If you put the tags at the N-terminus, you will be stuck with only a foreign G.
Hi Alexander,
your protein will be around 9KDa, so whilst small, it's not really that small and of it's self shouldn't be a problem. As to how well a protein expresses there are many factors and determining the results before hand is problematic.
As for the C-terminal tag I'd not bother with a cleavage site, just have the 6His - as noted by John you will have tail of similar length depending upon the site. For NMR the His will be unstructured and so will not be detected easily.
-Richard
We have many options for excellent protein expression on the Addgene website. Here's one cool kit. https://www.addgene.org/kits/pcrisystem/
You can always call or email us for one of our PhD scientists to give you advice on selecting a plasmid! We do dozens of tech service calls each week.
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