Peptide purification

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Fri Mar 1 22:26:03 EST 2002


7 kDa is what, about 65 amino acids ? Would it be possible to obtain this
peptide synthetically ?

As far as digestion with proteases is concerned, I do not feel good about
enterokinase. Do you have an option to re-clone your stuff under a more
specific protease, such as e.g. TEVp ?

An additional option you could try cloning your peptide under a different
protein.

Hope this helps.

A.G.E.

"KH" <harohallik at hotmail.com> wrote in message
news:20020301211735.11841.qmail at ww02.jatek.com...
> Hi
> I have expressed a peptide (7kd) as a fusion with trx, his tag and a
> protease site 5' of the peptide ( enterokinase). I see a thick band around
> 26-17Kd when induced with Iptg and have been able to purify it by hplc.
nOw
> here comes the headache. I have tried to digest the fusion protein (
> separate the tag from the peptide) and purify it by hplc. I have been
unable
> to get sufficient quantity of the product for mass spec. The expression
> system used was PET 32b+ from novagen. I am very tired and fed up with the
> purification.
>
> 2. when i digest the fusion protein with enterokinase i see 3 bands one
> which is approx 7kd(maybe my expected size?) theres is 2 m,ore bands maybe
> 18 and 14 kd. The size of the tag is approx 17-18kd. what is the other
band
> and how to control the digestion of enterokinse? i really am not happy
with
> this method.
> COULD ANY KIND SOUL LOOK THIS UP AND GIVE ME CONCRETE SUGGESTIONS TO
> OVERCOME THIS PROBLEM AND PURIFY MY PEPTIDE??
>
>
> <http://www.biowww.net/forum/read.php?f=1&i=5329&t=5329>
>





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