Internal RBS for example you can rule out by running denatured Ni-column, since your protein is N-His tagged it will be clear, where the degradation or translational stop comes from;N- or C-terminal.Only if your protein creates a dimer, the second band will copurify in a native Ni-column preparation with your N-terminal tagged version.Stops or degradations can occur if you have eg consecutive rare codons.Then the ribosome can fall off and if the truncated product is stable, it will be possible to copurify.See eg the very beautiful paper by Kapust et al. on : Processive Degradation of Nascent Polypeptides, Triggered by Tandem AGA Codons, Limits the Accumulation of Recombinant Tobacco Etch Virus Protease in Escherichia coli BL21(DE3) in PEP 2002.This is a combination of rare codon effects and degradation.Rare codon effects also seen on Remenyi et al in Acta cryst 2001.Protein has rare codons, coexpresses as short product and also copurifies.

It would be helpful if you provided greater detail. I'm going to assume that you are expressing in E. coli, and are getting a band on SDS-PAGE corresponding to the target protein around 70 kDa, and a second band corresponding to 40 kDa that does not appear in the empty vector control. It also sounds like you have not shown conclusively that this second band is part of the larger 70 kDa, but I will assume it is. It also sounds that no matter what you do regarding protease inhibitors, working fast and at low temperatures, etc. the ratio between these two species remains constant.

In this case, the degradation (if it is indeed degradation) has already taken place before cell lysis. One possibility is proteolytic autoprocessing (is the protein a protease or pro-protease?) or due to a cytoplasmic host protease. In the latter case you could try expressing in a different cell compartment (periplasm), trying different strains or switching hosts altogether. Sometimes it is just easier to get red of it during purification.

Another possibility is the presence of an alternate translational initiation site inside the coding sequence. Check the sequence with something like the RBS Calculator (google for it) and if necessary, do site-specific mutagenesis to eliminate it.

Hope it helps!

hello,

a lot of good remarks have been made by Alejandro and Sebastian. I  assume that you confirmed the relationship between your protein and this 40kDa band by either wesstern blot, N terminal sequencing or mass spec. And of course you sequenced your plasmid before to check if you didn't have any mutations or reading frame shifting.

Also is it heterologous expression? Because I already saw in the past proteins from mycobacterium expressed in E. coli being much degraded whatever the strains or conditions used. It was probably due to some unstable or floppy  domains. Then if your full length protein is still present in good amount it can be worth purifying it and get rid of the truncation this way(as proposed previously)

Also if it is a matter of protein lifetime or stability , it can be worth trying to controle much more the expression in term of time and of level. (using arabinose induced promoter that is fine tuning or inducing with lactose instead of IPTG ) at lower temperature and or for a shorter time. Not sure it will fix the  problem but it may help.

I agree Sebastin's comment, I would first check whether the fragment is part of your protein? by western or Mass spec. Try different expression strains, that will help.

Thank you for all the suggestions provided. As Martin, Sebastian and Anne pointed out, I have confirmed that the 40kDa is part of protein by western blot and so that grey area is cleared. RBS is a good point and I have also heard about iRBS (internal ribosomal binding site). I believe correcting the issue at the nucleotide level will be much better. Expressing at lower temperature is one I have not tried and will try that too.

Thanks..

Try different different temperatures, IPTG levels and strains. Good luck

I can see both bands on gel. I am sure that the second part is also from the protein because western blot confirms it. And also when I purify by Ni-NTA resin, I see it again on the gel. In my gene seq, N-terminal has His tag. That also kind of make sure that after truncation N-terminus of my protein is still intact. My doubt is that the second part is expressed separately by E.coli ? Or even something like this is possible ! I would call it as double expression (expression of target protein and expression of part of protein) rather than a truncation. Confused about this phenomenon.

Internal RBS for example you can rule out by running denatured Ni-column, since your protein is N-His tagged it will be clear, where the degradation or translational stop comes from;N- or C-terminal.Only if your protein creates a dimer, the second band will copurify in a native Ni-column preparation with your N-terminal tagged version.Stops or degradations can occur if you have eg consecutive rare codons.Then the ribosome can fall off and if the truncated product is stable, it will be possible to copurify.See eg the very beautiful paper by Kapust et al. on : Processive Degradation of Nascent Polypeptides, Triggered by Tandem AGA Codons, Limits the Accumulation of Recombinant Tobacco Etch Virus Protease in Escherichia coli BL21(DE3) in PEP 2002.This is a combination of rare codon effects and degradation.Rare codon effects also seen on Remenyi et al in Acta cryst 2001.Protein has rare codons, coexpresses as short product and also copurifies.

Thank you Stier. I will first try doing a denatured Ni-Column and see whats coming up.