express a big protein in BL21, get an extra band - (May/26/2005 )

Dear fella,
I have try to express a big protein ~100kDa in BL21 strain. It is a big for E coli. and i did 30degree induction for 22 hours and the protein is induced. unfortunately after purified there are two bands on the gel, one is the right size ~100kDa, another one is around 80kDa, I have no idea what is the extra band is. might be truncate one or unfinished translated protein. i do not know. Does anyone has idea what it might be and what can i do to get rid of it.


Thanks lot.

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have your tried seperating the two proteins and seeing if the second has the same function? what method for purification do you use?

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I did not try to separate the two proteins, do you think it is worth to do it? i am using affinity chromatography

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[what type of tag it has -Nterminal or C-terminal and how you purified ?

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passage through gel filtration column will most probabaly resolve your sample into homogeneity.

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that is what i think, but the size of the two protein is quite similar. one is around 100kda, the other is around 80kda.

by the way, do you guys know how to check the Ecoli sequences? my college suggested that might e coli pick up different code for it and it is a stop code so the translation stops and i get a truncated protein which might be a problem

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Hi Cathy
I think you should try to low your expression temp (25 and 20oC) and shorten expression time (20h). I encoumtered the problem and resolved like that.
Good luck!

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Thanks a lot. I will try lower temperature and short time induction

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