Test Expressions for Signal Sequence Variants

For each signal sequence variant protein expression was carried out as described above. After the overnight induction, the final culture OD600 was noted from a 1/20 dilution of an 2 mL aliquot for each signal sequence variant. Each aliquot was pelleted, washed once with PBS, pelleted again and resuspended in sufficient 2% OTG (w/v, octyl-thioglucoside, Gold Biotechnologies) in PBS to normalize each sample’s cell density based upon the measured OD600. Cells were lysed by gentle rocking in this solution at room temperature (20 °C) for 10 minutes. Clarified lysate was prepared with non-reducing SDS-PAGE sample buffer and split into boiled and un-boiled samples for SDS-PAGE analysis. LTIB is resistant to SDS and only denatures upon heating yielding a simple analysis for determining whether expressed B subunits are assembled into pentamers or are monomeric. Finally, it was noted that OD600 measurements were inversely correlated with apparent over-expression as determined by SDS-PAGE analysis as is anticipated if B subunit expression was resource limiting.