title: Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.
abstract: Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus.
authors.affiliation: Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
authors.forename: Lik Chern Melvin
authors.initials: LC
authors.lastname: Tan
authors.forename: Anthony Jin Shun
authors.initials: AJ
authors.lastname: Chua
authors.forename: Li Shan Liza
authors.initials: LS
authors.lastname: Goh
authors.forename: Shu Min
authors.initials: SM
authors.lastname: Pua
authors.forename: Yuen Kuen
authors.initials: YK
authors.lastname: Cheong
authors.forename: Mah Lee
authors.initials: ML
authors.lastname: Ng
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D002460:Cell Line; D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods*; D009033:Culicidae / Q000235:genetics; D003715:Dengue / Q000517:prevention & control*; D003716:Dengue Virus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D014901:West Nile Fever / Q000517:prevention & control*; D014902:West Nile virus / Q000235:genetics*
chemical_list: D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins