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 Benevides C. C. Pessela, Rodrigo Torres, § Manuel Fuentes, Cesar Mateo, Miguel Filho, Alfonso V. Carrascosa, † Alejandro Vian, † Jose L. Garcı ´a, ‡ Jose M. Guisa ´ n,* and Roberto Fernandez-Lafuente* Departamento de Biocata ´ lisis, Instituto de Cata ´ lisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain, Departamento de Microbiologia, Instituto de Fermentaciones Industriales, C/Juan de la Cierva 3, 28006 CSIC, Madrid, Spain, and Centro de Investigaciones Biolo ´gicas, Departamento de Microbiologı ´a Molecular, C/Ramiro de Maetzu 9, 28040 CSIC, Madrid 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 R-galactosidase and a -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. Introduction Recently, it has been shown that the mechanism of adsorption of proteins on ion exchanger supports (mul- tipoint interactions between the protein and the support) permits the selective adsorption of large proteins com- pared to small ones (1). This was based on the fact that proteins only become adsorbed on the ionic chromato- graphic matrix by multipunctual interactions between several groups placed on the surface of the protein molecule and some groups located on the surface of the support (1). Then, only large enough proteins (that is, covering a large area of the support) could be adsorbed on the matrix when using very lowly activated supports (Scheme 1). Therefore, it may be possible to purify via one simple ionic adsorption any multimeric protein from any contaminant monomeric protein. However, to take full advantage of this, it is necessary to find cases where the only multimeric enzyme present in the enzyme preparation is the target protein. That may be the case of multimeric enzymes from a thermophilic organism expressed in a mesophilic host. These enzymes have a growing interest as a suitable solution for the lack of thermostability of mesophilic enzymes, mainly when the use of high temperatures is convenient (e.g., to prevent microbial contamination in food chemistry) (2-6). More- over, as a result of the problem of the production in the native producers (use of high temperatures, nutrient requirements, productivity) (7, 8), these enzymes are routinely expressed in mesophilic microorganisms (e.g., E. coli)(9-11). The heating of the protein preparation from a meso- philic organism having the multimeric thermophilic enzyme is the conventional way of starting the purifica- tion of thermophilic enzymes expressed in a mesophilic host. This treatment precipitates many of the proteins of the host microorganisms, depending on the pH, ionic strength, etc. (12). It is possible that this treatment may destroy the quaternary structure of most of the meso- philic multimeric proteins, considering their fairly low stability under drastic conditions (13). If this was the * To whom correspondence should be addressed. Tel: 34 91 585 48 09. Fax: 34 91 585 47 60. E-mail: rfl@icp.csic.es/jmguisan@ icp.csic.es. † Instituto de Fermentaciones Industriales. ‡ Centro de Investigaciones Biolo ´gicas. § Permanent address: Escuela de Quı ´mica, Facultad de Cien- cias, Edificio Camilo Torres, Universidad Industrial de Santander, Bucaramanga, Colombia. Scheme 1. Adsorption of Large and Small Protein on Ionic Exchangers 1507 Biotechnol. Prog. 2004, 20, 1507-1511 10.1021/bp049785t CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 08/17/2004