I am trying to express the NSP gene of FMDv. I have cloned in pET-43.1a(+) vector and trying to express it in BL21(DE3)pLysS cells as Nus-tagged fusion protein. However, when I check the induced pellets on the 12.5% SDS-PAGE, it indicates the size of Nus tag at 40kDa, but it should be at about 55-0 kDa.
We have used NusA fusion toexpress plant OMTs in E. Coli (See Guillaumie et al 2013, Plant Physiol vol 162, pp 604–615) as well as other proteins (stilben synthase for exemple) and the NusA fusion protein were always at their expected sizes. However in my experience of protein biochemistry, it does occur that some proteins can have an abnormal mobility in SDS-PAGE, that is migrates at a place that does not reflect their actual size. For exemple it was well know that tonoplastic pyraposphatases migrates at lower apparent molecular size (60 kDa instead of 80+ kDa) because of its high hydrophobicity. I don't know what is NSP gene of FMDv, but you should check the predicted hydrophobicity (dedicated free online sofwares exists for that, check on SwissProt for starter) of the gene product and see if it is very high ?
Also you may rerun your gel including an other Nusa fusion protein of known apparent MW on the SDS-PAGE as a control, just to check that it is not your electrophoresis conditions that shift the apparent MW of your NusA-NSP fusion...
Best regards,
Eric Gomès.
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