Biophysical Characterization and Stability of Gcg-XTEN.

Gcg-XTEN was produced recombinantly in E. coli and purified to homogeneity using three column steps (see methods). (A) SDS-PAGE analysis of the purified protein product (lane 2). Molecular weight markers are shown in lane 1 with relevant size markers labeled at the left. Note that the true molecular weight of the molecule is 16305 daltons (confirmed by mass spectrometry; not shown). Slow migration in SDS-PAGE relative to globular protein standards is typical of XTEN fusion proteins due to differences in primary amino acid composition. (B) Glucagon receptor (GcgR) Ca²⁺-flux assay comparing the efficacy of Gcg-XTEN to unmodified glucagon. Calculated EC50 values for each curve fit are shown. (C) Reverse phase C18 HPLC analysis and (D) Size exclusion chromatography HPLC analysis of the purified Gcg-XTEN construct at the time of production. (E) Reverse phase C18 HPLC analysis and (F) Size exclusion chromatography HPLC analysis of Gcg-XTEN after 6 months storage at either −80°C (black), 2–8°C (blue), or 25°C (red). Note the scale is expanded in panel E to better illustrate the appearance of minor peaks at 25°C.