Purification and characterization of the reovirus cell attachment protein σ1

Yeung, Michael C.; Gill, M. John; Alibhai, Suleiman S.; Shahrabadi, M Shamsi; Lee, Patrick W.K.

doi:10.1016/0042-6822(87)90417-x

Cited by 30 publications

(16 citation statements)

References 21 publications

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“…The addition of urea did not noticeably affect the antigenicity of the recombinant products. This is consistent with previous reports showing that urea concentrations as high as 10 M, while increasing protein solubility, did not negatively effect the binding and haemagglutination activities of bovine rotavirus VP8 * (Lee et al, 1995) or reovirus l proteins (Yeung et al, 1987).…”

Section: Discussionsupporting

confidence: 93%

Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector

Pérez-Filgueira

Brayfield

Phiri

et al. 2004

Journal of Virological Methods

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Pérez-Filgueira, D. M.; Brayfield, B. P.; Phiri, S.; Borca, M. V.; Wood, Charles; and Morris, Thomas Jack, "Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector" (2004 AbstractProduction of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10-15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world.

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Section: Discussionsupporting

confidence: 93%

Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector

Pérez-Filgueira

Brayfield

Phiri

et al. 2004

Journal of Virological Methods

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“…The oligomerization status of the reovirus cell attachment protein at has been controversial . It was initially thought that protein al, which is located at the 12 vertices of the viral icosahedron, exists as a dieter (Lee et al ., 1981 ;Bassel-Duby et al, 1985 ;Yeung et al, 1987) . Subsequent studies based on migration rate in FIG .…”

Section: Discussionmentioning

confidence: 99%

“…Protein al is a minor outer capsid protein situated at each vertex of the outer capsid of the viral icosahedron (Lee et al, 1981 ;Furlong et at, 1988) . This protein functions as the reovirus cell attachment protein (Lee et at, 1981 ;Armstrong et al, 1984) and the viral hemagglutinin (Weiner et al, 1978 ;Yeung et al ., 1987) .…”

Section: Introductionmentioning

confidence: 99%

Biochemical and biophysical characterization of the reovirus cell attachment protein σ1: Evidence that it is a homotrimer

et al. 1991

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The oligomerization state of the reovirus cell attachment protein sigma 1 (49K monomeric molecular weight) was determined by biochemical and biophysical means. Full-length (protein product designated A) and C-terminal truncated (protein product designated B) serotype 3 reovirus S1 mRNA transcripts synthesized in vitro were cotranslated in a rabbit reticulocyte lysate, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under conditions which allowed for the identification of oligomeric forms of sigma 1. A total of four oligomeric protein bands (corresponding to A3, A2B1, A1B2, and B3, respectively) was consistently observed, which suggests that the protein is made up of three monomeric subunits. Biophysical characterization of purified sigma 1 using column filtration and sucrose gradient sedimentation analysis confirmed the highly asymmetric shape of sigma 1 and allowed us to determine the molecular weight of the native protein to be approximately 132K (a trimer). Similar biophysical analysis on the two tryptic fragments of the sigma 1 [N-terminal fibrous tail (26K monomeric molecular weight) and the C-terminal globular head (23K monomeric molecular weight)] yielded molecular weights of 77K and 64K, respectively, both again corresponding to trimers. We therefore conclude that protein sigma 1 is a homotrimer and provide, with supportive experimental evidence, a rationale for the anomalous behavior of the oligomeric protein in SDS-polyacrylamide gels, which, coupled with chemical cross-linking studies, has in part led to the previous suggestion that sigma 1 might be a higher order oligomer.

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“…Alpha-helical coiled-coil structure has also been suggested for reovirus fibers by the presence of heptad repeats in the sequence (Bassel-Duby et al, 1985) and circular dichroism spectroscopy (Yeung et al, 1987). However, from circular dichroism spectroscopy it has become clear that the adenovirus fiber (Green et al, 1983) and bacteriophage T4 short (Burda et al, 2000) and long fibers (Selivanov et al, 1987) consist almost exclusively of beta-structure.…”

Section: Introductionmentioning

confidence: 99%

Review: Conformation and Folding of Novel Beta-Structural Elements in Viral Fiber Proteins: The Triple Beta-Spiral and Triple Beta-Helix

Mitraki

Miller

Raaij

2002

Journal of Structural Biology

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Purification and characterization of the reovirus cell attachment protein σ1

Cited by 30 publications

References 21 publications

Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector

Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector

Biochemical and biophysical characterization of the reovirus cell attachment protein σ1: Evidence that it is a homotrimer

Review: Conformation and Folding of Novel Beta-Structural Elements in Viral Fiber Proteins: The Triple Beta-Spiral and Triple Beta-Helix

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