More than one protein induced - (Nov/18/2009 )

Hi!

I have a problem with an his tagged protein. I use the T7 expression system in e coli with a pET vector. The problem is that after induction I see 3 fat bands which are induced. This tag is C terminal. When I try purification I purify only the smallest of the 3 proteins. Are this really 3 proteins or is there another possibility? How can I purify the right protein?

Thank you for your help

Hi

there are many possibilities. one is that the other 2 proteins are not the desired ones and they are getting co-induced....cognate chaperones might also get induced.......i often see a 66 kDa and 27 kDa bands, corresponding to e. coli proteins during my purifications.

Don't you know the MW of your desired protein?

Anyhow, u can separate these bands by using FPLC.......depending on the MW of all the three bands, you will have to set up an appropriate gradient. You can also verify the identity of your protein using MALDI-TOF or N-terminal peptide sequencing, or if possible, western blot...

I would think that the protein which comes after binding, will be your desired one.

Hope, this is of some help

all the best!

dora1 on Nov 18 2009, 05:50 AM said:

Just to be clear, you are using a T7 expression system for a c-terminally tagged protein expressed from a pRT vector, afterwhich when staining against the c-terminal tag, you see 3 bands, correct? or are you staining against the protein and seeing 3 bands? I assume also that these bands are not seen if you do a T7 TNT with naked pET vector? Also, how are you purifying your protein, i.e. are you purifying based on the tag?

the protein i am looking for has a MW of 69, 4 kD. the additional bands are present on coomassie gel and on a WB where i stain against the tag. the other bands are about 50 and 35 kD. in the mean time i also found out something new. i induced at 20 °C and was now able to purify (affinity p. for his tag) all tree proteins. at least i see all three bands in the eluates. so you think with FPLC i will be abe to get rid of the other proteins?

Hi

yeah, you should be able to separate them out....

all the best