His-tag protein-purification

zakaria at ita.fhg.de zakaria at ita.fhg.de
Thu Sep 4 07:22:54 EST 1997


Dear slavica masina

I am haven´t such problems with my proteinpurification. My protein is
about 28,9 kDa big with the 6 His. 
 The Expression  of the protein in E.coli is simple, also the protein is
in Inclusion bodies
First I have to crack the inclusionbodies in a solution of 
      7 M Gdn HCL
      0,5 M NaCl
      0,02 M NaPP  pH 7,5 with sonification 6 times 30 sec under ice
After centrifugation I give the protein on a Ni-Chelat Pro Bond resin from
INVITROGEN. After wasching 3 times with 3 different Buffer I elute my
protein under denaturating cond. wit pH 4,0 
Buffer 1       Washing (Binding Buffer) 6 M Urea 20 mM NaPP pH 7,8
Buffer 2       Washing 6 M Urea 20 mM NaPP pH 6,0
Buffer 3       Washing 6 M Urea 20 mM NaPP pH 5,3
EluteBuffer    Elute   6 M Urea 20 mM NaPP pH 4,0

6 M Urea is enough for denat cond. In the fridge (4C°) 8M Urea isn´t possibble

After eluting I ´am making a Dialyse against 0,1 M Tris/HCl with PMSF and
Aprotenin. The next purification step depends on your protein.

//Does anyone have any sugestions for
> successful purification of His fusion proteins? Or any ideas on ow to
> improve the talon affintiy metod?
       Try Cu instead of Ni



Good luck Marco

-- 
Dr. Hayssam Zakaria
Fraunhofer Institut
Dep. Genetechnology
Nikolai-Fuchs-Str. 1
30625 Hannover
Germany



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