Peptide purification

KH harohallik at hotmail.com
Fri Mar 1 16:17:35 EST 2002


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??


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