A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme

Villaverde, Antonio; Feliu, Jordi X.; Arı́s, Anna; Harbottle, Richard P.; Benito, Antoni; Coutelle, Charles

doi:10.1002/(sici)1097-0290(19980805)59:3<294::aid-bit5>3.0.co;2-6

Cited by 7 publications

(2 citation statements)

References 37 publications

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“…Protein engineering allows a modular design of non‐natural polypeptides, in which different functions associated with independent domains from different origins can be combined in a desirable way to serve specific purposes. Examples of such multifunctional hybrid proteins include vehicles for delivery of targeted DNA [1] or functional enzymes [2] and molecular sensors [3]. The Escherichia coli β‐galactosidase is a homotetrameric enzyme [4] widely employed as a convenient tool in molecular and cell biology.…”

Section: Introductionmentioning

confidence: 99%

Engineering of Escherichia coli β‐galactosidase for solvent display of a functional scFv antibody fragment

2002

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Protein engineering allows the generation of hybrid polypeptides with functional domains from di¡erent origins and therefore exhibiting new biological properties. We have explored several permissive sites in Escherichia coli L L-galactosidase to generate functional hybrid enzymes displaying a mouse scFv antibody fragment. When this segment was placed at the amino-terminus of the enzyme, the whole fusion protein was stable, maintained its speci¢c activity and interacted speci¢cally with the target antigen, a main antigenic determinant of foot-andmouth disease virus. In addition, the antigen-targeted enzyme was enzymatically active when bound to the antigen and therefore useful as a reagent in single-step immunoassays. These results prove the £exibility of E. coli L L-galactosidase as a carrier for large-sized functional domains with binding properties and prompt the further exploration of the biotechnological applicability of the scFv enzyme targeting principle for diagnosis or other biomedical applications involving antigen tagging.

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Section: Introductionmentioning

confidence: 99%

Engineering of Escherichia coli β‐galactosidase for solvent display of a functional scFv antibody fragment

2002

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“…Despite the widespread use of Escherichia coli β‐galactosidase as a molecular marker ([5], and references therein), its 3D structure was not solved until a few years ago [6], this fact having restricted its rational engineering. However, insertional approaches have led to the identification of tolerant sites for the accommodation of foreign, functional peptides, and their presentation on solvent‐exposed surfaces of the active enzyme [7–14]. Furthermore, the positioning of an antigenic viral peptide in the activating loop of β‐galactosidase (amino acids 272–287) resulted in a promising molecular sensor (protein M278VP1) for the quantitative detection of specific antibodies [15].…”

Section: Introductionmentioning

confidence: 99%

Distinct mechanisms of antibody‐mediated enzymatic reactivation in β‐galactosidase molecular sensors

Feliu¹,

Ramírez²,

Villaverde³

1998

FEBS Letters

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The antibody-mediated reactivation of engineered Escherichia coli L L-galactosidases J. Biol. Chem. 271, 21251^21256] has been thoughtfully investigated in three recombinant molecular sensors. Proteins M278VP1, JX772A and JX795A display the highly antigenic G-H loop peptide segment of foot-and-mouth disease virus VP1 protein, accommodated in different solvent-exposed loops of the assembled tetramer. These chimaeric enzymes exhibit a significant increase in enzymatic activity upon binding of either monoclonal antibodies or sera directed against the inserted viral peptide. In JX772A but not in M278VP1, the Fab 3E5 antibody fragment promotes reactivation to the same extent as the complete antibody. On the other hand, M278VP1 K m is reduced by more than 50% in the presence of activating serum, this parameter remains invariable in JX772A and it is only slightly modified in JX795A. In these last two proteins, significant k cat variations can account for the increased enzymatic activity. Alternative reactivation mechanisms in the different L L-galactosidase probes are discussed in the context of the bacterial enzyme structure and its tolerance to antibody-induced conformational modifications.z 1998 Federation of European Biochemical Societies.

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Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression

Arı́s

Feliu

Knight

et al. 2000

Biotechnol. Bioeng.

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A recombinant, multifunctional protein has been designed for optimized, cell‐targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin αVβ3 binding, a DNA‐condensing poly‐L‐lysine domain, and a complete, functional β‐galactosidase protein that serves simultaneously as purification tag and DNA‐shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase‐reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell‐targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid‐based DNA‐condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non‐viral modular DNA transfer vectors that conserve the cell‐target‐ ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 689–696, 2000.

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A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme

Cited by 7 publications

References 37 publications

Engineering of Escherichia coli β‐galactosidase for solvent display of a functional scFv antibody fragment

Engineering of Escherichia coli β‐galactosidase for solvent display of a functional scFv antibody fragment

Distinct mechanisms of antibody‐mediated enzymatic reactivation in β‐galactosidase molecular sensors

Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression

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