Purification, chromatography, multimeric protein, yeast

[Juan Morales]

Dear researchers:

We are trying to purificate a yeast produced multimeric recombinant 
protein. After the cell disruption the protein of interest is located 
in the pellet. Then it is washed with 1 % Triton X-100. The 
extraction procedure uses 8M Urea in Tris 50 mM pH=9 and afterward 
the sample is renaturalized. In this condition the protein sample is 
stable. At this point the protein of interest is about 20 % of the 
total protein presented here in. The theoretical calculated 
isoelectric point (pI) of this protein is 11, but when we 
precipitated by pH all of the total bulk protein precipitate at pH 
4,5 to 5. Also we have assayed ion-exchange chromatography using 
different matrices. In the following conditions: Tris 50mM Ph = 9 
only in the anionic Q-Sepharose the entire protein sample was 
absorbed while in the cationic ones such as S-Sepharose d 
CM-Sepharose it was not absorbed at all. So the Q- Sepharose was 
studied. At the above mentioned conditions the entire bulk of 
proteins was absorbed to the matrix and the proteins were eluted at 
increasing molarities of NaCl until 2M but the interested protein did 
not elute. We assumed that it precipitated in the column. We decided 
to prove increasing molarities of NaCl in the initial sample. Since 
100 mM to 2,5 mM of NaCl in the initia sample was assayed. In all 
cases the protein of interest together with the bulk of contaminating 
proteins did not interact with the matrix. Only a little part of the 
contaminating proteins were absorbed to the column and this not 
necessary implied a signi cant improvement in the purity of the 
protein of interest. In the same conditions (Tris 50 mM pH=9, NaCl 
2.5 mM) we have tried to prove again cationic interchangers and not 
protein was absorb at all.   We are wondering what are the possible 
causes that in rfere with the absorbing process of the protein sample 
at 2,5 mM of NaCl if at 0 M of NaCl almost all the protein is 
absorbed?

Appreaciate all advice, Nelson
---------------------------------------------------------
Juan Morales Grillo, PhD, Senior Research, Head,
Hepatitis C Department, Vaccines Division, 
Center for Genetic Engineering and Biotechnology.
PO Box 6162, ZP 10600
Cubanacan, Playa, Ciudad Habana, Cuba.
Fax: (53-7) 214764, 218070, 336008
Phone: 53-7-218466/218446/218164; ext 1626, 1194, 1424
E-mail: Juan.Morales at cigb.edu.cu