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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

nvenient purification step for this recombinant antigen.

Fractionated Solubilization of the Inclusion Body Preparation
To convert p23-DHFR into a soluble form, the inclusion body preparation was successively incubated with suspension buffer (0.1 M Na-phosphate, pH 8.0, 10 mM Tris-HCl, pH 8.0) containing 2 M, 4 M, 6 M, and 8 M urea, respectively. After centrifugation, each step of this solubilization procedure was monitored by analyzing the soluble and insoluble fractions on a Coomassie Blue-stained SDS-PAGE gel. Most of the contaminating E. coli proteins were solubilized at 26 M urea (Figure 1, lanes 35) and were discarded with the supernatants, whereas most of the 49 kD recombinant antigen (23 kD p23 + 26 kD DHFR) was solubilized at 8 M urea and thus separated from bacterial cell debris, which remains insoluble. The corresponding immunoblot, developed with a pool of EBVpositive sera, demonstrates the immunological reactivity of the expressed protein (Figure 1B). Nevertheless, additional purification steps were necessary to obtain the recombinant protein in such a purity that it could be used as an antigen in immunological test systems.

Usually, metal chelate affinity chromatography (MCAC) is recommended for the purification of His-tagged proteins. In the presence of 8 M urea, which was essential to keep the protein in solution, no satisfactory purification could be observed during MCAC procedures (data not shown). Therefore, continuous elution electrophoresis was applied for a further purification of the recombinant protein solubilized in 8 M urea.


Preparative SDS-PAGE
To reduce the sample volume, the E. coli inclusion body preparation (suspension
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