Protein expresses wrong size - (Dec/06/2005 )
Hello,
I have a protein which should be 22kDa, but when I analyse the expression in BL21(DE3) I see an over expression band at 13kDa, with nothing at 22kDa. I have tried rosetta and plysS cell strains to no avail. The protein should be N-terminal His tagged, but the 13kDa protein does not bind to a Nickel column. I should also mention that the plasmid was sequenced and checked out. What are the possible explanations for this strange behaviour?
Thanks in advance. 
I have a protein which should be 22kDa, but when I analyse the expression in BL21(DE3) I see an over expression band at 13kDa, with nothing at 22kDa. I have tried rosetta and plysS cell strains to no avail. The protein should be N-terminal His tagged, but the 13kDa protein does not bind to a Nickel column. I should also mention that the plasmid was sequenced and checked out. What are the possible explanations for this strange behaviour?
Thanks in advance.
Well I had a similar trouble with M15 rep4 cells but unfortunately I realize that the overexpress protein (tons of) was in fact a host factor express by the E.Coli strain and not my malaria protein
So I would urge you to check with a specific anti-6His mAb to be sure that it's your stuff and furthermore performed a Maldi tof analysis to be sure that you are dealing with your protein !
Another explanation might be a multiple strat up codons in your sequence and that the initiation do not take place at the forst Met but rather further down the coding sequence !
I also had this trouble with another Falciparum recombiant protein which was in fact the good one but truncated
Hope it helps you
Pesji
It could also be a proteolytic effect - rosetta will help something express that normally doesn't, while pLysS prevents leaky expression until you induce, but they're both based on BL21. Thus, if there is proteolytic activity there, the Rosetta or pLysS modifications aren't going to make a difference.
I'd try subcloning into a new vector, like pTrc99A, and expressing in an alternate strain (JM101, TG1, etc) and see if that makes a difference. If that's not your cup of tea, try a shorter induction.
Pesji's suggestions will help you identify what you're actually dealing with, as well.
Is there an activity associated with this protein you could test for? If it is a truncation, but retains activity, it may not matter.
OK, thanks for all your suggestions.
Andrea. 
May be it is stupid, but are you sure, that you molecular marker of protein weight is in accordance to the SDS gel content.
For example BSA band in the See Blue Invitrogen marker in the Tris-glycine gel runs like a 98 kDa band, and in precast Nu PAGE MES gel runs as a 62 kDa band.
The prestained marker cannot be used for accurate size determination. This is just for estimation only.
You have to use UNSTAINED marker for the exact size determination.
As I can remember, the manual of SeeBlue Plus 2 shows different patterns in different gel types/buffers. I usually align it with my gel type to the manual.