Sendai virus protein

Nonionic detergents are probably the most suitable additives when further purification by lEC is required. Winkler et al. [26] used SDS in the elution buffer for SEC and were able to purify the eluted proteins further by RPC without prior removal of the SDS. We were less successful using a similar procedure for the purification of Sendai virus proteins. All Sendai virus membrane proteins containing SDS were eluted at the same position in RPC with low yields. In some cases [27,28], a volatile eluent has been used for chromatography, which facilitates further purification by one of the other modes of HPLC. 

Figure 1. Calibration curves for three Polyol columns. Calibration curves of three SE-HPLC columns (100 mm X 4.6 mm i.d.) (Serva, Heidelberg, FRG). Particle size and pore size respectively were (a) 5 jim, 50 nm (b) 5 >tm, 30 nm (c) 10 /rni, 30 nm. The flow-rate was 0.1 ml/min and 10 /rl samples of the tetramer of Sendai virus protein HN (272 kd), BSA (68 kd), ovalbumin (43 kd) and trypsin-inhibitor (20 kd) were applied to the column.

In 1957 Isaacs and Lindemann noted that virally infected cells produced a protein that conferred viral resistance to naive cells [Ij.They called the substance interferon. The properties of interferons immediately captured the imagination of virologists. However, clinical research was limited by the inability to produce interferon in sufficient quantity and to adequately purify the protein. The first major step in overcoming these problems was the discovery by Grosser that human leukocytes could produce interferons when stimulated in culture by Sendai virus [2]. 

Frye and Edidin provided a striking visual demonstration of the dynamic nature of membrane structure. They labeled proteins on the plasma membranes of two samples of cells with fluorescent dyes, human cells with a dye that emitted red light, and mouse cells with a dye that emitted green light (fig. 17.15). The two populations of cells then were mixed and treated with Sendai virus, which causes individual cells to fuse. Immediately after the fusion, red fluorescence from the human proteins could be seen on one half of the hybrid membrane, and green fluorescence from the mouse proteins on the other half. But within a few min, the two types of proteins were intermingled over the entire surface. 

Figure 1 Procedure of gene transfer by Sendai virus (HVJ)-liposomes. DNA and nuclear proteins are incorporated into liposomes by vortex-ultrasonication-annealing method, and the liposomes are fused with UV-inactivated Sendai virus. The resulting fusigenic viral liposomes can fuse with cell membrane to introduce DNA and nuclear protein complex directly into the cytoplasm. Nuclear protein such as HMG-1 can enhance the expression of foreign DNA in the nucleus.

Tomita, N., Higaki, J., Ogihara, T., Kondo, T and Kaneda, Y. (1994) A novel gene-transfer technique mediated by HVJ (Sendai virus), nuclear protein, and liposomes. Ca. Detec. Preven. 18(6), 485 491. 

Flavovirus membrane and capsid proteins Oestrogen synthetase Sendai virus detergent extract Nucleoproteins Nucleoproteins Calmodulin... 

Interferon and synthetic fragments Sendai virus membrane protein Interleukin I fragment Heparin-binding growth factors fi-Cisein tryptic peptides Bacteriophage T4 gene 43 protein tryptic peptides V-C4 RBC membrane protmns H-C4... 

The presence of informosomelike RNPs has been demonstrated also in the case of Ehrlich ascites carcinoma cells infected with Sendai virus (Volkova et al., 1969). The cytoplasmic extracts of the cells labeled for 30 minutes with uridine contain virus-specific RNA in the form of particles with a sedimentation coefficient 45S (the sedimentation coefficient of the complete virus equals 57S). The buoyant density of the RNP peak in CsCI equals 1.43 to 1.44 g/cm. Although these properties are compatible with the idea that they are informosome, the particles, and in particular their protein component, should be characterized in more detail before reaching a definite conclusion. Recently SOS virus RNA-containing particles with p = 1.40 g/cm have been found in HeLa cells infected with poliovirus (Huang and Baltimore, 1970), although the authors have some doubts about the reality of these complexes. 

FIGURE 1 Procedure for gene transfer using HVJ-liposomes. DNA and nuclear protein (HMG-1) are enclosed in liposomes by vortexing and sonication, and the liposomes are treated with HVJ (Sendai virus). The resulting HVJ-liposomes deliver the DNA-HMG-1 complex into the cytoplasm, and the complex migrates rapidly into the nucleus. 

At a low concentration of detergent, mainly hydrophilic proteins were eluted during the salt gradient [29]. A second step with a high concentration of detergent in the elution buffer resulted in elution of the membrane proteins. Integral membrane proteins of Sendai virus [29], herpes simplex virus [30,30a], and Plasmodium falciparum [31 ] were purified by this approach. 

Triton X-100 extracts of Sendai virus and several strains of the closely related Newcastle disease virus (NDV) were subjected to SEC. The elution patterns are shown in Fig. 3. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the tetramer and the dimer of the Sendai virus hemagglutinin-neuraminidase (HN) protein (272 and 136 kDa, respectively) and the monomeric fusion (F) protein (65 kDa) were present in peaks 1, 2, and 3, respectively. These peaks are followed by an extremely large peak that contains Triton X-100 micelles. The elution patterns of the other viruses show that there are notable differences when they are compared with that of Sendai virus. In some cases, for example, the strains La Sota and Texas, the most prominent protein eluting from the column is an HN monomer. In other cases, multimeric forms of the HN protein that differ from Sendai HN protein were eluted (strains Mukteswar, Florida, and Herts). 

Figure 3 HPSEC of a Triton X-100 extract ot purified Sendai and Newcastle disease virions. Virus was grown in 10-day-old embryonated chicken eggs and purified and extracted as described [5]. A TSK 4000SW (600 x 7.5 mm ID) column was eiuted with 50 mM sodium phosphate, pH 6.5, containing 0.1% SDS. Samples were boiled for 2 min in 4% SDS prior to chromatography. The flow rate was 1 mL/min and the absorbance was monitored at 280 nm. 1, Tetramer of the hemagglutinin neuraminidase (HN) protein of Sendai virus 2, dimer of HN 3, Sendai virus fusion protein F , Triton X-100.

The elution patterns obtained after RPC of a detergent extract of Sendai virus are shown in Fig. 5. A Sendai virus extract contains the tetramer and dimer of the HN protein, the F protein, and the matrix protein M (38kDa). Disulfide bridges in these proteins were reduced with dithiothreitol, producing two components of the F protein, FI and F2 (13-15 and 50kDa, respectively), M protein, and the monomeric form of HN (67 kDa). This mixture was subjected to RPC on an (a) C )( column (100 A), (b) C column (250 A), and (c) phenyl column (1000 A). The elution patterns in Fig. 5 show that there is no problem in eluting the two... 

RPC is limited to relatively small membrane proteins, although the availability of large-pore-size column materials allows successful chromatography of proteins such as a 67-kDa Sendai virus membrane protein [51]. 

Structure determination of a Galectin-3-carbohydrate complex. Conformational analysis of partially folded proteins illustrated by application to the molecular recognition element of Sendai virus nucleoprotein. ... 

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