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Applied and Environmental Microbiology, August 2004, p. 4642-4647, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4642-4647.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag

Cristina Moldes,{dagger} José L. García, and Pedro García*

Department of Molecular Microbiology, Centro de Investigaciones Biológicas, 28040 Madrid, Spain

Received 26 January 2004/ Accepted 21 April 2004

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.

* Corresponding author. Mailing address: Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain. Phone: 34 91 8373112. Fax: 34 91 5360432. E-mail: pgarcia{at}cib.csic.es.

{dagger} Present address: UMR 163, CEA Cadarache, 13108 St. Paul lez Durance, France.

Applied and Environmental Microbiology, August 2004, p. 4642-4647, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4642-4647.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hernandez-Rocamora, V. M., Maestro, B., Molla-Morales, A., Sanz, J. M. (2008). Rational stabilization of the C-LytA affinity tag by protein engineering. Protein Eng Des Sel 21: 709-720 [Abstract] [Full Text]  


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