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Figure 1
SDS–PAGE, native PAGE and SEC-MALLS demonstrating that the PD1 domains cause irreversible aggregation. (a) SEC-MALLS chromatogram of IMAC-purified PD1-GCN4. The degree of aggregation, as observed by the LS peak at the void volume (∼5 min), the multiple dRI and UV peaks, and an incorrect molecular weight, demonstrated that the purified protein was not amenable to crystallization. (b) Native PAGE of PD1-GCN4 in the presence of increasing concentrations of urea. Increasing the urea concentration had no effect on migration and hence no effect on aggregation. Lane 1, carbonic anhydrase; lanes 3–7, PD1-GCN4 in urea at varying (0, 0.5, 1, 2 and 4 M) concentrations. (c) SDS–PAGE of HsfPD1 purified by IMAC (lanes 1–8) and SEC (lanes 9–13). High levels of expression were evident (lanes 4, 5 and 6) after the proteins were separated on a gradient gel (4–20%) and visualized with Coomassie Blue. Lane M, molecular-weight marker (labelled in kDa); lane 1, unbound; lanes 2–3 and 8, wash; lanes 4–7, IMAC elution fractions; lanes 9–13, SEC fractions. (d) SEC-MALLS chromatogram of IMAC- and SEC-purified HsfPD1 [the peak corresponds to one SEC fraction, lane 12 in the SDS–PAGE gel in (c)]. Alignment of the LS, UV and dRI peaks, and a correct molecular weight, confirmed the presence of trimeric, non-aggregating protein. |
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