Methods for preparation of low abundance glycoproteins from mammalian cell supernatants.
Proteins secreted to mammalian cell supernatants are usually in a low concentration and purity, due to the limitation of the expression systems or the presence of a large amount of contaminant proteins from the cell medium. So, initial protein recovery from cell supernatants requires of a highly specific chromatography step. We compared several purification methods based on affinity chromatography for purification of proteins from cell culture supernatants: metal chelate affinity, strep-tag and immunopurification with a monoclonal antibody. Soluble receptor glycoproteins were engineered with the corresponding peptide tag at their C-terminal end. The proteins were expressed in 293T cells and secreted to the cell supernatant, as monitored by sandwich ELISA. Supernatants were run through the different chromatography columns and several purification-related parameters determined. While all column-retained proteins were easily eluted, the chelating and immunopurification chromatography gave the highest yield and the latest method provided a sample with the highest purity. So, in spite of its cost, immunopurification chromatography gave optimal results for purification of a low abundance protein from a cell supernatant. Finally, we applied a protein expression system together with immunopurification chromatography for preparation of a glycoprotein for crystallization.
Ordoño
Desiderio
D
Centro Nacional de Biotecnologia, CSIC, Campus Universidad Autonoma, 28049 Madrid, Spain.
Enjuanes
Luis
L
Casasnovas
José M
JM
D000818:Animals; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D006023:Glycoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C064484:spike protein S, Transmissible gastroenteritis virus