FIGURE 8.

Purification of GOAT as a monomer with at least two stable bands. A, colloidal Coomassie Brilliant Blue (CBB) staining of purified mouse GOAT after tag cleavage and ion exchange chromatography reveals three bands. Four identical lanes of mouse GOAT were used as input for electroelution. B, bands in A are stable species, not interconverting gel artifacts. Stained bands were excised, electroeluted, and concentrated and then rerun and silver-stained as compared with the original purified GOAT. C, size exclusion chromatography of cleaved GOAT and standard proteins. Sizes of proteins in the standard are shown in parentheses. The calculated molecular mass for monomeric GOAT in FC-16 micelles is 140.7 kDa. TEV-cleaved GOAT alone has a calculated molecular mass of 50.6 kDa. Empty FC-16 detergent micelles alone are ∼72.5 kDa and do not result in UV absorbance signal at the concentration used. D, interference optical monitoring data from analytical ultracentrifugation of GOAT were fit in SEDFIT to a C(s) model. Fringes over time are shown in progression from red to black; absorbance data (not shown) was similar. Approximately 90% of the signal in the sample fit to a single peak at 1.9 S, corresponding to an S (20 °C, water) of 5.4 and an approximate molecular mass of 110 kDa. E, anti-FLAG immunoblot of GOAT-3xFLAG in SF9 cells with the indicated mutations shows that multiple bands seen are not due to post-translational modification on asparagine or lysine or at residue Cys-368 or Ser-309. Asn-free and Lys-free GOATs include all of the Asn and Lys mutations listed in other constructs. F, treatment with hydrazine or hydroxylamine does not alter the banding pattern of purified GOAT. Mouse GOAT was incubated overnight at the indicated temperatures and concentrations with hydrazine and hydroxylamine (pH 8.0). A Coomassie Brilliant Blue-stained gel is shown; mouse GOAT used in this experiment was eluted from FLAG resin using 100 mm glycine, pH 3.5, and retains the C terminus of the 3xFLAG tag.