A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli.
The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.
Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Benevides C C
BC
Pessela
Rodrigo
R
Torres
Manuel
M
Fuentes
Cesar
C
Mateo
Miguel
M
Filho
Alfonso V
AV
Carrascosa
Alejandro
A
Vian
Jose L
JL
García
Jose M
JM
Guisán
Roberto
R
Fernandez-Lafuente
D000837:Anion Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects; D013824:Thermus / Q000201:enzymology* / Q000235:genetics; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000528:radiation effects; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000528:radiation effects
D000837:Anion Exchange Resins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D000519:alpha-Galactosidase; D001616:beta-Galactosidase