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Preparative SDS-PAGE Electrophoresis of a Recombinant Epstein-Barr Virus Encoded Protein and Its Application in Serodiagnostic Test Systems, Rev C

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Cloning and Expression of the Viral Protein in E. coli
Since we were interested in both cloning and expressing the entire coding region of the protein, and no appropriate restriction sites were present at the genomic level, PCR was used to modify the ends of the target gene. With this approach, it was possible to construct a recombinant pQE-40 expression vector (QIAGEN GmbH, Hilden, Germany) that harbors the coding region in a proper reading frame downstream of the murine dihydrofolate-reductase (DHFR) gene. The plasmid was introduced into a suitable E. coli strain and protein production was induced with IPTG (isopropyl-β-D-thiogalactopyranoside). Bacterial cells were harvested by centrifugation, resuspended in three volumes of sonication buffer (50 mM Na-phosphate, pH 7.8, 300 mM NaCl, 0.1 mM phenylmethylsulfonylfluoride), containing 5 g/ml DNase I and 1 mg/ml lysozyme, and incubated for 10 min at room temperature. Subsequently, the suspension was sonicated on ice (150 W, 5 x 30 sec) and centrifuged at 4,000 x g for 10 min, and aliquots were analyzed by SDS-PAGE to monitor expression of the recombinant protein (Mini-PROTEAN II electrophoresis system).

The recombinant p23-DHFR fusion protein was expressed at a high level (constituting up to 50% of the total cellular protein), and a preferential localization in inclusion bodies was observed. As demonstrated in Figure 1, the supernatant contained little p23-DHFR (lane 1), and most of the recombinant protein was located in the insoluble fraction (lane 2). Since this insoluble fraction consists of some E. coli proteins, bacterial cell wall debris, and p23-DHFR, inclusion body-directed expression represents a co
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