one pure protein but has two clear bands in SDS-PAGE

EK nobody at elnino.com
Tue Jul 29 13:42:25 EST 2003


<chengzhjun at hotmail.com> wrote in message
news:20030729065240.30227.qmail at ww02.hostica.com...
> We have got several proteins here encountering the same problems! Our
recombinant  protein is on Pet22-b Vector and expressed in Bl21(DE3) E.coli
strain. After purification by NTA-Ni column, we see two clear bands in the
SDS-PAGE. The two bands have a similar concentration and they are very near
to each other. In our protein solutions ,we have already added some protease
inhibitor.
> Anybody can help me to explain this phenomenon? Any suggestion is highly
appreciated.
> By the way,how to attach photos? I want to attach my SDS-PAGE result so
that you can get a direct understanding of my problems.
>
>
>
http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0

No pictures necessary. We discussed this issue before. In our lab, we had
that same problem and went as far as to N-terminal sequencing of proteins in
the 2 bands. Turned out that the lower band (expectedly,because it is less
positive) is the one lacking His-tag. I guess a certain part of your protein
is expressed without the tag, or the tag is cleaved during
extraction/purification. The 2 forms are co-purified probably due to
oligomerization, or due to some other process the protein is involved in.
For example, if the protein binds DNA, the non-tagged protein bound to the
same DNA fragment as the tagged protein would end up in the eluate. We tried
DNAse treatments in our case, but to our surprize the problem went away
after we switched to Ni-resin instead of Co-resin. The only explanation why
Ni could get rid of non-tagged protein I could come up with is that Ni is
usually a stronger binder for His-tagged proteins than Co, and it is
concievable that the stronger binding somehow temporarily affects protein
conformation so that it loses its grip on DNA and consequently non-tagged
proteins "riding" it. Interesting, what metal chelation resin do you use?

Emir





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