title: 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: Fraunhofer USA Center for Molecular Biotechnology, Newark, DE 19711, USA. johannes.buyel@rwth-aachen.de
authors: J F
authors: JF
authors: Buyel
authors: J A
authors: JA
authors: Bautista
authors: R
authors: R
authors: Fischer
authors: V M
authors: VM
authors: Yusibov
mesh_terms: D015153:Blotting, Western; D002021:Buffers; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D052162:Human papillomavirus 16 / Q000737:chemistry* / Q000276:immunology; D008970:Molecular Weight; D050725:Papillomavirus E7 Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D053918:Papillomavirus Vaccines / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D014026:Nicotiana / Q000737:chemistry / Q000821:virology; D022223:Vaccines, Subunit / Q000737:chemistry / Q000276:immunology
chemical_list: D002021:Buffers; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines; D011993:Recombinant Fusion Proteins; D022223:Vaccines, Subunit; C059731:oncogene protein E7, Human papillomavirus type 16; D006026:Glycoside Hydrolases; C028020:licheninase