Ion exchange using poorly activated supports, an easy way for purification of large proteins.
Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins. These kinds of proteins have large surfaces to interact by many points with the support. Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp. strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports. A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.
Pessela
Benevides C C
BC
Departamento de Biocatálisis, Instituto de Catálisis CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Munilla
Roberto
R
Betancor
Lorena
L
Fuentes
Manuel
M
Carrascosa
Alfonso V
AV
Vian
Alejandro
A
Fernandez-Lafuente
Roberto
R
Guisán
Jose M
JM
D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000302:isolation & purification*; D013824:Thermus / Q000201:enzymology; D001616:beta-Galactosidase / Q000737:chemistry
D011506:Proteins; D001616:beta-Galactosidase