Summary
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 ofE. coli expression plasmid ptac12-Bam. A new protein produced inE. 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Amann E, Broisus J, Ptashne M (1983) Vectors bearing a hybridtrp-lac promotor useful for regulated expression of cloned genes inEscherichia coli. Gene 25: 167–178
Barrett T, Shrimpton SB, Russell SEH (1985) Nucleotide sequence of the entire protein coding region of canine distemper virus polymerase-associated (P) protein mRNA. Virus Res 3: 367–372
Bellini WJ, Englund G, Rozenblatt S, Arnheiter H, Richardson CD (1985) Measles virusP gene codes for two proteins. J Virol 53: 908–919
Burnette WN (1981) „Western blotting“: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195–203
Curran J, Kolakofsky D (1988) Ribosomal initiation from an AUG codon in the Sendai virus P/C mRNA. EMBO J 7: 245–251
Curran JA, Richardson C, Kolakofsky D (1986) Ribosomal initiation at alternate AUGs on the Sendai virus P/C mRNA. J Virol 57: 684–687
Dethlefsen L, Kolakofsky D (1983) In vitro synthesis of the nonstructural C protein of Sendai virus. J Virol 46: 321–324
Etkind PR, Cross RK, Lamb RA, Merz DC, Choppin PW (1980) In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses. Virology 100: 22–33
Galinski MS, Mink MA, Lambert DM, Wechsler SL, Pons MW (1986) Molecular cloning and sequence analysis of the human parainfluenza 3 virus mRNA encoding the P and C proteins. Virology 155: 46–60
Giorgi C, Blumberg BM, Kolakofsky D (1983) Sendai virus contains overlapping genes expressed from a single mRNA. Cell 35: 829–836
Gupta KC, Kingsbury DW (1985) Translational modulationin vitro of a eukaryotic viral mRNA encoding overlapping genes: ribosome scanning and potential roles of conformational changes in the P/C mRNA of Sendai virus. Biochem Biophys Res Commun 131: 91–97
Hidaka Y, Kanda T, Iwasaki K, Nomoto A, Shioda T, Shibuta H (1984) Nucleotide sequence of a Sendai virus genome region covering the entirc M gene and the 3′ proximal 1013 nucleotides of the F gene. Nucleic Acids Res 12: 7965–7973
Ishida Y, Fujita T, Asai K (1981) New detection and separation method for amino acids by high-performance liquid chromatography. J Chromatogr 204: 143–148
Kingsbury DW (1974) The molecular biology of paramyxoviruses. Med Microbiol Immunol 160: 73–83
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Lamb RA, Choppin PW (1977) The synthesis of Sendai virus polypeptides in infected cells. II. Intracellular distribution of polypeptides. Virology 81: 371–381
Lamb RA, Choppin PW (1978) Determination by peptide mapping of the unique polypeptides in Sendai virions and infected cells. Virology 84: 469–478
Lamb RA, Mahy BW, Choppin PW (1976) The synthesis of Sendai virus polypeptides in infected cells. Virology 69: 116–131
Luk D, Sanchez A, Banerjee AK (1986) Messenger RNA encoding the phosphoprotein (P) gene of human parainfluenza virus 3 is bicistronic. Virology 153: 318–325
McConahey PJ, Dixon FJ (1980) Radioiodination of proteins by the use of the chloramine-T method. In: Vunakis HV, Langone JJ (eds) Methods in enzymology, vol 70. Academic Press, New York, pp 210–213
O'Farrell PZ, Goodman HM, O'Farrell PH (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12: 1133–1142
Portner A, Murti KG, Morgan EM, Kingsbury DW (1988) Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy. Virology 163: 236–239
Portner A, Gupta KC, Seyer JM, Beachey EH, Kingsbury DW (1986/87) Localization and characterization of Sendai virus nonstructural C and C' proteins by antibodies against synthetic peptides. Virus Res 6: 109–121
Sakai Y, Suzu S, Shioda T, Shibuta H (1987) Nucleotide sequence of the bovine parainfluenza 3 virus genome: its 3′ end and the genes of NP, P, C, and M proteins. Nucleic Acids Res 15: 2927–2944
Shioda T, Iwasaki K, Shibuta H (1986) Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN, and L proteins. Nucleic Acids Res 14: 1545–1563
Shioda T, Hidaka Y, Kanda T, Shibuta H, Nomoto A, Iwasaki K (1983) Sequence of 3,687 nucleotides from the 3′ end of Sendai virus genome RNA and the predicted amino acid sequences of viral NP, P, and C proteins. Nucleic Acids Res 11: 7317–7330
Taira H, Kanda T, Omata T, Shibuta H, Kawakita M, Iwasaki K (1987) Interferon induction by transfection of Sendai virus C gene cDNA. J Virol 61: 625–628
Tanford C (1962) The interpretation of hydrogen ion titration curves of proteins. In: Anfinsen CB Jr, Anson ML, Bailey K, Edsall JT (eds) Advances in protein chemistry, vol 17. Academic Press, New York, pp 69–165
Tarr GE (1977) Improved manual sequencing methods. In: Hirs CHW, Timasheff SN (eds) Methods in enzymology, vol 97. Academic Press, New York, pp 335–357
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Omata-Yamada, T., Hagiwara, K., Katoh, K. et al. Purification of the Sendai virus nonstructural C protein expressed in E. coli, and preparation of antiserum against C protein. Archives of Virology 103, 61–72 (1988). https://doi.org/10.1007/BF01319809
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01319809