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. 2018 Jul 26;1(1):12–20. doi: 10.1021/acsptsci.8b00017

Figure 3.

Figure 3

Formation and purification of WT-Gs and DN-Gs containing heterotrimers by oxyntomodulin with GLP-1R. (a,b) Size exclusion chromatography of complexes initiated with 1 μM oxyntomodulin revealed higher yield of complexes with the DN-Gs (b) relative to that seen with WT-Gs (a), although both had relatively low yields for complex formation (the elution position of the complexes is illustrated by the red arrow). (b) Increasing the concentration of oxyntomodulin to 50 μM in the Oxyn:GLP-1R:DN-Gs:Nb35 preparation during the initiation step markedly improved the yield of complexes, which could be purified to homogeneity by an additional size exclusion chromatography step (d). (e) Coomassie stained gel from the peak in panel d, illustrating high purity and recovery of each of the component proteins. (f) 2D class averages from negative stain EM demonstrate the presence of complexes suitable for single-particle cryo-EM structure determination.