Heparin for purifiying proteins
w.ingram at cmcb.REMOVE-THIS-BIT.uq.edu.au
Tue Jan 26 20:59:43 EST 1999
I am about to embark on some protein production using a the pET expression
system in E. coli. My target protein will be expressed with a 6 histidine
tag and I will be purifying it using Novagen his-bind columns. It has been
suggested that I may also need to further purify with heparin agarose.
This has been done in several publications after the protein was purified
via thrombin cleavage.
What I would like to know is:
1. Will heparin purification be likely to help in my case, or will the
his tag purified protein be very pure anyway after Ni binding and elution?
Does thrombin cleavage give a more ³dirty¹ protein product than his
tagging, thus needing further purification?
2. I would really appreciate some advice on Heparin-Agarose use. Sigma
for example sell various ³types² of Heparin (Type I, Type II, HEP - I-5
etc). I have found lots of info on how heparin is thought to work etc,
but it would be great if someone could point me in the direction of some
good practical references/protocols so I can figure out what is an
appropriate methodology to use with my protein.
Thanks in advance,
Wendy Ingram (PhD Student)
Centre for Molecular and Cellular Biology
University of Queensland
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