Improving the large scale purification of the HIV microbicide, griffithsin

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• 作者：Joshua L Fuqua ; Valentine Wanga ; Kenneth E Palmer
• 关键词：Griffithsin ; Tobacco mosaic virus ; Protein purification ; N. benthamiana ; Bentonite ; Microbicide ; HIV
• 刊名：BMC Biotechnology
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：15
• 期：1
• 全文大小：2,044 KB
• 参考文献：1. Mori, T, O’Keefe, BR, Sowder, RC, Bringans, S, Gardella, R, Berg, S (2005) Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp. J Biol Chem 280: pp. 9345-53 CrossRef
  2. Nixon, B, Stefanidou, M, Mesquita, PM, Fakioglu, E, Segarra, T, Rohan, L (2013) Griffithsin Protects Mice from Genital Herpes by Preventing Cell-to-Cell Spread. J Virol 87: pp. 6257-69 CrossRef
  3. Emau, P, Tian, B, O’Keefe, BR, Mori, T, McMahon, JB, Palmer, KE (2007) Griffithsin, a potent HIV entry inhibitor, is an excellent candidate for anti-HIV microbicide. J Med Primatol 36: pp. 244-53 CrossRef
  4. O’Keefe, BR, Vojdani, F, Buffa, V, Shattock, RJ, Montefiori, DC, Bakke, J (2009) Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component. Proc Natl Acad Sci U S A 106: pp. 6099-104 CrossRef
  5. Giomarelli, B, Schumacher, KM, Taylor, TE, Sowder, RC, Hartley, JL, McMahon, JB (2006) Recombinant production of anti-HIV protein, griffithsin, by auto-induction in a fermentor culture. Protein Expr Purif 47: pp. 194-202 CrossRef
  6. Kouokam, JC, Huskens, D, Schols, D, Johannemann, A, Riedell, SK, Walter, W (2011) Investigation of griffithsin’s interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate. PLoS One 6: pp. e22635 CrossRef
  7. Moulaei, T, Shenoy, SR, Giomarelli, B, Thomas, C, McMahon, JB, Dauter, Z (2010) Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity. Structure 18: pp. 1104-15 CrossRef
  8. Ziolkowska, NE, Shenoy, SR, O’Keefe, BR, McMahon, JB, Palmer, KE, Dwek, RA (2007) Crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin. Proteins 67: pp. 661-70 CrossRef
  9. Brakke, MK, Pelt, N (1969) Influence of bentonite, magnesium, and polyamines on degradation and aggregation of tobacco mosaic virus. Virology 39: pp. 516-33 CrossRef
  10. O’Keefe, BR, Giomarelli, B, Barnard, DL, Shenoy, SR, Chan, PK, McMahon, JB (2010) Broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family Coronaviridae. J Virol 84: pp. 2511-21 CrossRef
  11. Takebe, Y, Saucedo, CJ, Lund, G, Uenishi, R, Hase, S, Tsuchiura, T (2013) Antiviral Lectins from Red and Blue-Green Algae Show Potent In Vitro and In Vivo Activity against Hepatitis C Virus. PLoS One 8: pp. e64449 CrossRef
  12. Ishag, HZ, Li, C, Huang, L, Sun, MX, Wang, F, Ni, B (2013) Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo. Arch Virol 158: pp. 349-58 CrossRef
  13. Hoorelbeke, B, Xue, J, Liwang, PJ, Balzarini, J (2013) Role of the Carbohydrate-Binding Sites of Griffithsin in the Prevention of DC-SIGN-Mediated Capture and Transmission of HIV-1. PLoS One 8: pp. e64132 CrossRef
  14. Meuleman, P, Albecka, A, Belouzard, S, Vercauteren, K, Verhoye, L, Wychowski, C (2011) Griffithsin has antiviral activity against hepatitis C virus. Antimicrob Agents Chemother 55: pp. 5159-67 CrossRef
  15. Xue, J, Gao, Y, Hoorelbek
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Biotechnology
  Life Sciences
  Plant Breeding/Biotechnology
  Stem Cells
  Transgenics
  
• 出版者：BioMed Central
• ISSN：1472-6750

文摘

Background Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery. Results We achieved >99% pure griffithsin API by generating the initial green juice extract in pH?4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2sub> mixture, and final purification with Capto?multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88?±-% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained. Conclusions The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.