Alternative purification method for recombinant measles viral nucleoprotein expressed in insect cells by ion-exchange chromatography

• Published in: Journal of virological methods 2014-03, Vol.197, p.55-62
• Main Authors: Lee, Han Saem, Kim, You-Jin, Yang, Jeongsun, Yoon, Hee Sook, Kim, Seung Tae, Kim, Kisoon
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Viral - analysis; Antibodies, Viral - blood; Antigenicity; Blood - immunology; Cerebrospinal Fluid - immunology; Chromatography, Ion Exchange - methods; Enzyme-Linked Immunosorbent Assay - methods; Humans; Ion-exchange chromatography; Measles - diagnosis; Measles virus; Nucleoprotein; Nucleoproteins - genetics; Nucleoproteins - isolation & purification; Purification; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Sf9 Cells; Spodoptera; Viral Proteins - genetics; Viral Proteins - isolation & purification
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.11.015

•Ion-exchange chromatography was used to purify recombinant measles virus nucleoprotein (rMeV N).•The rMeV N had antigenicity similar with native viral protein.•The method could be applicable for high-throughput production of the rMeV N. 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.