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Applied and Environmental Microbiology, April 2003, p. 1967-1972, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.1967-1972.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Overproduction of Thermus sp. Strain T2 ß-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

Benevides C. C. Pessela,1 Alejandro Vian,1 César Mateo,2 Roberto Fernández-Lafuente,2 José L. García,3 José M. Guisán,2 and Alfonso V. Carrascosa1*

Department of Microbiology, Instituto de Fermentaciones Industriales,1 Department of Biocatalysis, Instituto de Catálisis,2 Department of Molecular Microbiology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain3

Received 22 October 2002/ Accepted 7 January 2003

A novel thermostable chimeric ß-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the ß-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the ß-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.

* Corresponding author. Mailing address: Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. Phone: 34-915622900. Fax: 34-915644853. E-mail: avcarrascosa{at}ifi.csic.es.

Applied and Environmental Microbiology, April 2003, p. 1967-1972, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.1967-1972.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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