Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein - Analytical Methods & Tools for Lyophilization

Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein
Engineering Village 2
? 2006 Elsevier Inc
Accession number: 06169823388

Title: Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein

Authors: Morera, Yanelys; Lamdan, Humberto; Bequet, Monica; Ayala, Marta; Rojas, Gertrudis; Munoz, Yasmiana; Gavilondo, Jorge V.

Author affiliation: Cancer Research Department, Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Havana 10600, Cuba

Serial title: Biotechnology and Applied Biochemistry

Abbreviated serial title: Biotechnol. Appl. Biochem.

Volume: v 44

Issue: n 1

Issue date: April 2006

Publication year: 2006

Pages: p 45-53

Language: English

ISSN: 0885-4513

CODEN: BABIEC

Document type: Journal article (JA)

Publisher: Portland Press Ltd, London, W1N 3AJ, United Kingdom

Abstract: Human VEGF121 (vascular endothelial growth factor isoform 121) was produced as a recombinant fusion protein with GST (glutathione S-transferase) in Escherichia coli. After affinity purification with glutathione, the GST-VEGF121 fusion protein preparation was used to obtain antibodies in mice against commercial hrVEGF (human recombinant VEGF) through immunization. It was also employed successfully to select specific anti-human VEGF antibody fragments of human origin employing phage-display technology. The fusion protein preparation was separated in monomeric, dimeric and oligomeric forms using size-exclusion chromatography. The dimers were recognized by a soluble VEGF receptor 2-Fc chimaera, and stimulated the growth of human umbilical-vein endothelial cells in vitro in a similar fashion to a commercial hrVEGF. The presence of GST in the fusion protein apparently did not affect the correct assembly of dimers and display of residues critical for receptor recognition. The two-step purification method reported in the present paper involves no laborious renaturalization methods, yields 10 mg/l of the mixture of different aggregation states after affinity chromatography, and 5 mg/l of the biologically active dimer after gel filtration, thus providing a source of material for the development of new anti-angiogenic therapeutic molecules. ? 2006 Portland Press Ltd.

Number of references: 42

Ei main heading: Proteins

Ei controlled terms: Bacteria - Growth kinetics - Escherichia coli - Purification - Antibodies - Dimers

Uncontrolled terms: Angiogenesis - Fusion protein - Glutathione S-transferase - Phage display - Vascular endothelial growth factors

Ei classification codes: 804.1 Organic Compounds - 461.9 Biology - 461.2 Biological Materials - 802.3 Chemical Operations - 461.9.1 Immunology - 815.1.1 Organic Polymers

Treatment: Experimental (EXP)

DOI: 10.1042/BA20050169

Database: Compendex

Compilation and indexing terms, ? 2006 Elsevier Inc. All rights reserved
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