Immunogold labeling using an anti-MIMI_L725 monoclonal antibody further revealed gold particles surrounding the capsid core structure of mimivirus, confirming the exposure of the corresponding epitope to the surface (Fig

Immunogold labeling using an anti-MIMI_L725 monoclonal antibody further revealed gold particles surrounding the capsid core structure of mimivirus, confirming the exposure of the corresponding epitope to the surface (Fig. encode bona fide proteins that are likely to participate in well-integrated processes. (mimivirus) is the largest virus isolated so far (23). Based on its highly specific characteristics, this double-stranded-DNA icosahedral virus (47) is the first member of the new family (33, 43). Computational annotation of its 1.2-Mb genome (33) revealed many atypical features, including the presence of key translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (of types IA, IB, and II) (2, 33). Another unique characteristic of mimivirus is the presence of nearly identical promoter sequence motifs upstream of half of its 911 protein-encoding genes (42), which are presumably associated with proteins expressed during the early or late-early phase. Only 23% of the predicted coding genes exhibit convincing homology to proteins Sophoradin of known function, and 39% of them do not exhibit Sophoradin a clear (E values, 10?5) sequence database match (33). Such coding regions without sequence similarity to other genes in databases are considered orphan open reading frames (ORFs) and termed ORFans (12). The origin and function of ORFan genes are still a matter of controversy, with opinions ranging from considering them pieces of junk DNA (1, 8, 40, 44) to seeing them as quickly evolving sequences encoding normally expressed functional proteins (38, 39). Recent clinical evidence raised the possibility that mimivirus might be a human pathogen causing pneumonia (4, 24, 34), as suspected when it was first isolated from a cooling tower following an outbreak of pneumonia (23). Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral Rabbit polyclonal to Piwi like1 proteins associated with viral particles (19, 29, 49). We now present the application of this technique to the largest known, and presumably most complex, viral particle, (23) was purified through a sucrose gradient (25%) and washed twice with phosphate-buffered saline (PBS) in the presence of protease inhibitors (Total; Roche, Mannheim, Germany). The producing pellet was solubilized in 40 mM Tris-HCl, pH 7.5, supplemented with 2% (wt/vol) sodium dodecyl sulfate (SDS; Sigma-Aldrich) and 60 mM dithiothreitol (DTT), followed by 5 min of heating at 95C. The insoluble portion was eliminated by centrifugation (12,000 proteins resolved by 2D gel electrophoresis were transferred onto nitrocellulose membranes (Semi-Phor unit; Hoefer Scientific, San Francisco, CA). Membranes were then clogged in PBS supplemented with 0.2% Tween 20 and 5% nonfat dry milk (PBS-Tween-milk) for 1.5 h before incubation with anti-sera. The sera were from BALB/c mice immunized by three intraperitoneal injections (with 15-day time intervals between injections) of 5 g of purified viral particles resuspended in CpG as an adjuvant. After 1 h of incubation (1:6,400 dilution in PBS-Tween-milk), membranes were washed three times with PBS-Tween and probed with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (1:1,000; Amersham). Detection was achieved by chemiluminescence (ECL; Amersham). SDS-PAGE coupled with electrospray ionization-ion-trap MS/MS analysis. An sample (175 g) prepared as explained above was separated by SDS-PAGE on an 11% acrylamide gel (22), and the proteins were exposed later on by Coomassie blue R-250 staining. Bands of 1-mm thickness were systematically slice from your gel, resulting in 49 pieces to be analyzed. These bands were reduced and alkylated before trypsin digestion, and the producing peptides were analyzed through liquid chromatography (LC) (Finnigan Surveyor HPLC system; Thermo Electron, San Jose, CA) coupled to an electrospray ionization-ion-trap mass spectrometer (LCQ-Deca XP; Thermofinnigan). Tryptic peptides were resuspended in 20 l formic acid at 5% in water, and 1/20 of the whole extract from each piece of gel was desalted on a C18 trapping column (Zorbax 300SB-C18 [5-m inner diameter, 5 by 0.3 mm]; Agilent) by a 20-min washing step with 0.1% formic acid. Peptide separation was achieved by a 60-min linear gradient of acetonitrile (0 Sophoradin to 65%) in 0.2% formic acid on a C18 reverse-phase column (PicoFrit column [5-m BioBasic C18, 300-? pore size, 75-m inner diameter, 100-mm long, 15-m tip]). The peptides were then ionized at a capillary heat of 160C having a 2-kV aerosol voltage. A Sophoradin collision energy of 35% was applied for the MS/MS check out events. The MS/MS spectrum of the three most intense peaks was acquired after each full MS scan. The dynamic exclusion features were set.

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