Conjugation of DnaK, OmpA and Tul4 Proteins of <i>F</i>. <i>tularensis</i> SchuS4 to TMV.

<p>Purified OmpA, DnaK and Tul4 proteins were combined with purified TMV and incubated with EDC and NHS for 0, 30 min, 1, or 2 hours as described in Methods section. Two μg of TMV or recombinant proteins DnaK, OmpA, Tul4 or 4 μg of the TMV-protein mixtures were resolved on an 8–16% SDS-PAGE gel to observe conjugation products indicated by changes in the molecular masses of the starting materials. <b>(A)</b> Conjugation of DnaK, OmpA and Tul4 to a single TMV virion to generate TMV-monoconjugate vaccine. The progress of conjugation process was observed over a period of time: Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV-protein mix, 0 min; Lane 2 = TMV-protein mix, 30 min; Lane 3 = TMV-protein mix,1 hour; Lane 4 = TMV-protein mix, 2 hours. <b>(B, C, D)</b> Kinetics of DnaK, OmpA and Tul4 TMV-protein conjugations over a two hour incubation period to generate TMV-protein conjugates. The individual TMV-protein conjugates were then admixed to generate TMV-multiconjugate vaccine. Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV; Lane 2 = Recombinant protein; Lane 3 = TMV-protein mix, 0 hour; Lane 4 = TMV-protein mix, 1 hour; Lane 5 = TMV-protein mix, 2 hours. In all cases, 2 hour time points were used for scale-up and vaccine preparation. Solid arrows indicate TMV-protein conjugate(s), dashed arrows indicate free TMV or free proteins.</p>