Purification of the Sendai virus nonstructural C protein expressed in E. coli, and preparation of antiserum against C protein

Abstract

An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter of E. coli expression plasmid ptac12-Bam. A new protein produced in E. coli after induction was purified to near homogeneity. The purified protein was found to be identical with the C protein predicted from the C gene cDNA in molecular weight, isoelectric point, amino acid composition, and the amino acid sequence at the N-terminal of the protein as well as those of several fragments obtained on V8 protease digestion. Antiserum raised against the purified protein specifically reacted with the C protein in infected cells. Using this antiserum, the localization of the C protein in infected cells was examined by immunofluorescence, which revealed that it appeared in the cytoplasm but not in nuclei.

Association of the Sendai virus C protein with nucleocapsids

“…When infected L t k -cells were treated with the anti-C antiserum, the C protein was clearly observed but only in the cytoplasm where there were many strong fluorescent granules ( Fig. 1 A), thus confirming our previous observations [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. A similar pattern of fluorescence was observed when infected cells were probed with the anti-NP antiserum (Fig.…”

“…At the confluent stage, cells were infected with the Z strain of Sendai virus (moi 10) as described previously [19]. Sendai virus particles were prepared from the medium of an infected LLC-MK2 cell culture 48 h after infection [28] or the allantoic fluid of embryonated eggs 3 days after infection [23], and were purified by discontinuous sucrose gradient centrifugation [24].…”

“…To prepare anti-P and anti-NP antisera, the eluted proteins were individually injected into rabbits intramuscularly with complete Freund's adjuvant. Preparation of the anti-C antiserum was described previously [19].…”

“…Aiming to elucidate its function, we prepared a high titered antiserum against the C protein, injecting purified C protein synthesized in E. coli into guinea pigs. This antiserum specifically recognized the C protein produced either in E. coli or infected vertebrate cells [19]. Using the antiserum, we investigated the possible presence of C protein in Sendai virus virions and demonstrated that it is tightly associated with Sendai virus nucleocapsids.…”

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

“…The labelled cells were disrupted in 25 mM Tris-HCl buffer (pH 8n0) containing 0n5 % Triton X-100, 0n5% sodium deoxycholate and 5 mM NaCl. Nuclei were removed by centrifugation at 12 000 g for 5 min and the resulting cell extract was subjected to immunoprecipitation analysis using antiserum against the SeV Fushimi strain or antiserum against the C protein (Omata- Yamada et al, 1988) and Protein A-Sepharose 4B (Pharmacia). The precipitated proteins were electrophoresed in 8 % or 14 % (for the detection of the C protein) SDS-polyacrylamide gel.…”