Would you recommend using SumoStar fusion protein in insect cells over other tags as MBP or GST to enhance solubility and production of large (over 150kb) cytosolic proteins?
and if so, where can I get this vector?
Thanks so much!
as usual, it's difficult to say that one tag is superior to another one. Some publications show that SUMOstar can improve expression yield, but improving solubility might not be better (or worse) than GST and MBP. We have seen good results with GST in insect cells with much less degradation or GST-only expression which you can get in E.coli, so things are clearly different in insect cells. Comparing all three with His-tag only would give you (and us eventually) more information.
The sequence of SUMOstar is available from the company's web page (http://www.lifesensors.com/pdf/3101_pI-secSUMOstar_Sequence-1.pdf). The easiest would be to order it or a synthetic construct.
Good luck with your expression!
We've been having a lot of success with MBP-tags in insect cells.
We also had good results with the MBP, but these proteins are large, so I thought I should use the Sumo instead.
When using GST, do you use it codon optimized for insect? I heard GST has a great track record from Dom Esposito, from the NCI.
Thanks so much!
in case of insect cells, size doesn't matter that much.We haven't seen a case where size seemed to be the critical parameter. In fact some of the best expressions are seen with larger target, one of the best was even >300kDa. In most cases, solubility seems to be the main bottleneck.
The GST is the same as in pGEX, because the fragment was just transferred from pGEX-6P-1,-2&-3.