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

Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens. In the past few years the specificity and sensitivity of serodiagnostic assay systems have been considerably improved by the use of purified recombinant EBV antigens. After cloning and high-level expression of a particular viral protein as DHFR fusion protein in Escherichia coli, we purified the recombinant antigen to near homogeneity with the help of continuous elution electrophoresis. Sera from both EBVpositive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23. The recombinant antigen seems to be a useful diagnostic marker for EBV infection, since antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera contained either IgM or IgG anti-p23 antibodies (or both). Introduction In the field of clinical diagnosis the determination of specific antibodies against distinct structural or functional antigenic proteins of a given pathogen is the most commonly used diagnostic tool for the detection of viral infections. Although most of the established test systems still use natural antigen from different sources, the advent of genetic engineering opens up the possibility of producing proteins of limited natural availability. Recombinant technology has already proven to be an excellent alternative for the production of specific antigens, which are able to improve sensitivity as well as specificity of derivative test systems. In general, the production of recombinant antigens for diagnostic purposes is inexpensive compared to the use of purified natural antigens. The major problems associated with such test systems are identification of viral antigens that guarantee a certain serological diagnosis, their expression, and, most importantly, the subsequent purification of the recombinant proteins to near homogeneity. 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 convenient 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 2–6 M urea (Figure 1, lanes 3–5) 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 buffer, 8 M urea) was dialyzed extensively against Tris-HCl (50 mM, pH 8.0). The white precipitate was collected and boiled in 1 ml sample buffer for 5 min. The sample was loaded onto a 12% cylindrical SDS-PAGE gel and electrophoresed as indicated in Table 1. Collection of 2 ml fractions was started when the Bromophenol Blue band passed the gel matrix. Table 1. Model 491 Prep Cell Running Conditions Resolving gel 12% acrylamide/2.6% C (cross-linker) Resolving gel length 5.5 cm in a 28 mm gel tube Resolving gel buffer 0.375 M Tris-HCl, pH 8.8 Stacking gel 4% acrylamide/2.6% C Stacking gel buffer 125 mM Tris-HCl, pH 6.8 Running buffer 25 mM Tris-HCl, 0.192 mM glycine, 0.1% SDS Elution buffer 25 mM Tris-HCl, 0.192 mM glycine, 0.1% SDS Sample buffer 62 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 1 mM EDTA, 0.005% Bromophenol Blue Sample The inclusion body preparation (solubilized in 6 M urea) was precipitated and resuspended in 1 ml sample buffer Elution rate 0.2 ml/min