Purification of human papillomavirus-like particles expressed in silkworm using a Bombyx mori nucleopolyhedrovirus bacmid expression system
| Autor: | Enoch Y. Park, Shintaro Kobayashi, Robert Minkner, Rina Baba, Yae Kurosawa, Tatsuya Kato, Shinichiro Suzuki |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Human papillomavirus Virus Cultivation Clinical Biochemistry Ion chromatography 01 natural sciences Biochemistry Analytical Chemistry 03 medical and health sciences Column chromatography Affinity chromatography 010608 biotechnology Silkworm Animals Papillomaviridae Purification Detection limit Chromatography Ion-exchange Ion exchange Chemistry Elution Virion Cell Biology General Medicine BmNPV bacmid Bombyx Chromatography Ion Exchange Nucleopolyhedroviruses 030104 developmental biology chromatography Target protein Ultracentrifuge Virus-like particle |
| Zdroj: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 1096 |
| ISSN: | 1873-376X |
| Popis: | A three-stage chromatography protocol for the purification of human papillomavirus-like particles (HPV-LPs) from the silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system was developed. For host cell DNA separation, anion exchange chromatography was used after screening for a suitable stationary phase. Using the two separation principles of cation exchange chromatography and metal affinity of ceramic hydroxyapatite (CHT) as a second stage, the amount of baculovirus in the sample was reduced to less than the detection limit of qPCR. The CHT separation was optimized with respect to the elution buffer used; 150–600 mM sodium phosphate, pH 7.2, resulted in the highest recovery of HPV-LPs. Using heparin chromatography, it was possible to reduce the sample volume and to thus highly concentrate the target protein during the separation of contaminating proteins. During the second purification stage, over 99.3% of the DNA was removed, and no infectious baculoviruses remained. After concentration by heparin column chromatography, over 99.9% of the DNA and protein had been removed. The purity achieved by this method exceeds that obtained by DDDDK-tag-based affinity chromatography and sucrose gradient ultracentrifugation, which were used as comparative purification methods. The 3-stage purification of HPV-LPs from silkworm fat bodies described here was a proof of concept and is a scalable method, but the overall yield remains to be improved. |
| Databáze: | OpenAIRE |
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