Call for experiences, Which interfere?

biosci-request at net.bio.net biosci-request at net.bio.net
Tue Sep 14 22:11:50 EST 1999


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 by gel filtration. In this condition the 
protein sample is stable to pH 9. 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, included our protein (?). 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 and 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 interfere 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?, could be some 
presence of Triton X-100?, nucleic acid in the sample?, some yeast 
low product interfere?, quality of water? 

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






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