Selective and mild adsorption of large proteins on lowly activated immobilized metal ion affinity chromatography matrices. Purification of multimeric thermophilic enzymes overexpressed in Escherichia coli.
A strategy to selectively adsorb large proteins on immobilized metal ion affinity chromatography supports is presented. It is based on the fact that large proteins have a large surface that permits the long distance interaction with groups placed quite far apart (very dispersed onto the support surface) in the support, therefore, even using lowly activated supports, these proteins may be able to yield multiple interactions with the support, which is not possible for smaller proteins. This has been shown using a crude extract from Escherichia coli, where only large proteins were adsorbed on supports having 0.25 micromol of metallic groups/g of support. Then, these lowly activated supports have been used for purifying multimeric enzymes from thermophilic organisms (alpha- and beta-galactosidases from Thermus sp. strain T2) cloned and over-expressed in mesophilic ones. A previous heating step of the crude extract destroyed the quaternary structure of all multimeric enzymes from the host (E. coli). Thus, the only large protein remaining in the supernatant of this heated extract are the cloned multimeric thermophilic enzymes, permitting their very simple purification by using only one chromatographic step.
Pessela
Benevides C C
BC
Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus Universidad Autónoma Madrid, Cantoblanco, 28049 Madrid, Spain.
Torres
Rodrigo
R
Fuentes
Manuel
M
Mateo
Cesar
C
Munilla
Roberto
R
Vian
Alejandro
A
Carrascosa
Alfonso V
AV
Garcia
Jose L c
JL
Guisán
Jose M
JM
Fernandez-Lafuente
Roberto
R
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008670:Metals / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013824:Thermus / Q000201:enzymology; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification*
D008670:Metals; D011994:Recombinant Proteins; D000519:alpha-Galactosidase; D001616:beta-Galactosidase