title: Alternative purification method for recombinant measles viral nucleoprotein expressed in insect cells by ion-exchange chromatography.
abstract: Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections.
authors.affiliation: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
authors.forename: Han Saem
authors.initials: HS
authors.lastname: Lee
authors.affiliation: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
authors.forename: You-Jin
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
authors.forename: Jeongsun
authors.initials: J
authors.lastname: Yang
authors.affiliation: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
authors.forename: Hee Sook
authors.initials: HS
authors.lastname: Yoon
authors.affiliation: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
authors.forename: Seung Tae
authors.initials: ST
authors.lastname: Kim
authors.affiliation: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea. Electronic address: tigerkis@gmail.com.
authors.forename: Kisoon
authors.initials: K
authors.lastname: Kim
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis / Q000097:blood; D001769:Blood / Q000276:immunology; D002555:Cerebrospinal Fluid / Q000276:immunology; D002852:Chromatography, Ion Exchange / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008457:Measles / Q000175:diagnosis*; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D061987:Sf9 Cells; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus
keywords: Antigenicity; CSF; ELISA; F; Ion-exchange chromatography; MeV; Measles virus; N; Nucleoprotein; Purification; cerebrospinal fluid; enzyme-linked immunosorbent assay; fusion protein; measles virus; nucleoprotein