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

• Published in: Biotechnology progress 2004, Vol.20 (5), p.1507-1511
• Main Authors: Pessela, Benevides C. C., Torres, Rodrigo, Fuentes, Manuel, Mateo, Cesar, Filho, Miguel, Carrascosa, Alfonso V., Vian, Alejandro, García, Jose L., Guisán, Jose M., Fernandez-Lafuente, Roberto
• Format: Article
• Language: English
• Subjects: alpha-Galactosidase - chemistry; alpha-Galactosidase - genetics; alpha-Galactosidase - isolation & purification; alpha-Galactosidase - radiation effects; Anion Exchange Resins; beta-Galactosidase - chemistry; beta-Galactosidase - genetics; beta-Galactosidase - isolation & purification; beta-Galactosidase - radiation effects; Biological and medical sciences; Biotechnology; Chromatography, Ion Exchange - methods; Dimerization; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hot Temperature; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; Multiprotein Complexes - isolation & purification; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - radiation effects; Thermus; Thermus - enzymology; Thermus - genetics
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp049785t

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 α‐galactosidase and a β‐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.