I am expressing a ~45 kDa fungal protein in E. coli however with a simple his-tag it proved to be insoluble. I then put it into the pET43 and pET44 vectors to include a solublising Nus tag and it is now soluble and I have purified using the his-tag.
However I get lots of lower molecular weight bands in this purification and no amount of washing or gradient elution improves this. I have had the lower MW bands identified and they have come back with hits to my protein. So I thought maybe they are truncated protein products.
The DNA sequence has been optimised and I have expressed in Rosetta cells with the same banding pattern so it is not a codon issue. I know E. coli struggles to produce proteins > 100 kDa and with the tags my fusion protein is ~110 kDa but I have also just come across the fact that the Nus tag is 491 amino acids of the 495 amino acid NusA protein. I knew NusA was a transcription factor but have just read that one of its purposes is to regulate transcrition termination.
My question is, Is the Nus tag active as NusA? Could the overexpression of NusA by means of the Nus tag be acting on my own fusion protein and causing premature termination? I can't find anything that suggests anyone else has encountered this problem, nor can I find what NusA actually terminates!
Any comments would be greatly appreciated.