This article is part of the supplement: The 4th Recombinant Protein Production Meeting: a comparative view on host physiology .

Poster Presentation

Efficient, antibody-mediated allosteric activation of an immobilized, E. coli beta-galactosidase recombinant biosensor

Rosa M Ferraz^1,3, Anna Arís¹, Gregorio Álvaro² and Antonio Villaverde¹

¹  Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain

²  Departament d' Enginyeria Química, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain

³  Departament de Matemàtica Aplicada IV, Universitat Politècnica de Catalunya. Campus Nord, Jordi Girona 1-3, 08034, Barcelona, Spain

corresponding author email

from The 4th Recombinant Protein Production Meeting: a comparative view on host physiology
Barcelona, Spain. 21–23 September 2006

Microbial Cell Factories 2006, 5(Suppl 1):P43doi:10.1186/1475-2859-5-S1-P43

Published:

10 October 2006

© 2006 Ferraz et al; licensee BioMed Central Ltd.

Background

Allosteric biosensors are based on engineered reporter enzymes responsive to analyte binding through detectable changes in the specific activity [1,2]. Since antibodies are efficient allosteric effectors, such devices are especially useful for the diagnosis of infectious diseases. In previous studies, we have introduced an antigenic peptide from the HIV structural protein gp41, spanning amino acids 579 to 613 of the Env precursor [3], into a permissive site of E. coli beta-galactosidase, resulting in the chimeric protein NF795gpC. In the presence of immune sera or anti-peptide antibodies, the soluble enzyme is efficiently activated in a fast and homogeneous immunoassay [3,4]. To further develop biosensor devices in solid phases, with wider applicability in field conditions, we have here explored the allosteric properties of NF795gpC when immobilized in an agarose substrate.

Results

The immobilization process was optimised by using a commercial beta-galactosidase and different crosslinked types of agarose, from which we finally considered 4BCL as the optimum for the assay because it permitted the total accommodation of the protein in the agarose porus. We monitored the binding process through activity assays (at pH 9.5) along time in supernatant and suspension, always controlling the stability of the protein [5]. The decrease of activity in the supernatant and not in the suspension indicates the efficient joining of the soluble protein to the support.

After that, we checked the activation capacity of immobilized NF795gpC upon the exposure to an anti-peptide antibody. In figure 1 it is compared the allosteric activation of the immobilized NF795gpC beta-galactosidase and its soluble version. Clearly, the protein maintains its allosteric activation properties even in its immobilized form, although with a differential, and slightly delayed profile regarding the soluble form.

Conclusion

NF795gpC is responsive to the allosteric modification mediated by anti-peptide antibodies even when immobilized in an agarose support, proving that the conformational modifications induced by the adaptive binding and supporting activation do not require the protein in solution. The different activation kinetics observed in soluble and immobilized enzyme versions could be due to either structural constraints to the active site conformational modulation or to a differential accessibility of the antigenic peptide (the allosteric receptor) to activating antibodies. The obtained results are promising regarding the possible use of allosteric biosensors in solid-phase platforms.

Acknowledgements

This work has been funded by BIO2004-00700 from MEC, Spain and 2005SGR-00956 (AGAUR). Rosa Maria Ferraz is recipient of a doctoral fellowship from Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya i del Fons Social Europeu.

References

1. Villaverde A: Allosteric enzymes as biosensors for molecular diagnosis.

   FEBS Lett 2003, 554:169-172. PubMed Abstract | Publisher Full Text

2. Ferraz RM, Vera A, Arís A, Villaverde A: Insertional protein engineering for analytical molecular sensing.

   Microb cell Fact 2006, 5:15. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text

3. Ferrer-Miralles N, Feliu JX, Vandevuer S, Müller A, Cabrera-Crespo J, Ortmans I, Hoffmann F, Cazorla D, Rinas U, Prévost M, Villaverde A: Engineering regulable E. coli beta-galactosidases as biosensors for anti-HIV antibody detection in human sera.

   J Biol Chem 2001, 276:40087-40095. PubMed Abstract | Publisher Full Text

4. Ferraz RM, Arís A, Villaverde A: Profiling the allosteric response of an engineered beta-galactosidase to its effector, anti-HIV antibody.

   Biochem Biophys Res Commun 2004, 314:854-860. PubMed Abstract | Publisher Full Text

5. Álvaro G, Fernández-La Fuente R, Blanco RM, Guisán JM: Immobilization-Stabilization of Penicillin G Acylase from Escherichia coli .

   Appl Biochem Biotechnol 1990, 26:181-195. PubMed Abstract

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