Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG.

Abstract

Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model. We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1g of protein per 1 kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50 g to 1 kg of processed leaf biomass.

• Authors: J F Buyel, J A Bautista, R Fischer, V M Yusibov
• Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chromatogr B Analyt Technol Biomed Life Sci) Vol. 880 Issue 1 Pg. 19-26 (Jan 01 2012) ISSN: 1873-376X [Electronic] Netherlands
• PMID: 22134037 (Publication Type: Journal Article)
• Copyright: Copyright © 2011 Elsevier B.V. All rights reserved.

Chemical References

• Buffers
• Papillomavirus E7 Proteins
• Papillomavirus Vaccines
• Recombinant Fusion Proteins
• Vaccines, Subunit
• oncogene protein E7, Human papillomavirus type 16
• Glycoside Hydrolases
• licheninase

Topics

• Blotting, Western
• Buffers
• Chromatography, Gel
• Chromatography, Ion Exchange
• Electrophoresis, Polyacrylamide Gel
• Glycoside Hydrolases (metabolism)
• Human papillomavirus 16 (chemistry, immunology)
• Molecular Weight
• Papillomavirus E7 Proteins (chemistry, immunology, isolation & purification)
• Papillomavirus Vaccines (chemistry, metabolism)
• Recombinant Fusion Proteins (chemistry, immunology, isolation & purification)
• Tobacco (chemistry, virology)
• Vaccines, Subunit (chemistry, immunology)