| Proinsulin expressed in pET 21b was purified as outlined in the pET system manual (Novagen, Madison, WI) using the BugBuster™/benzonase inclusion body purification protocol. The resulting pellet was resuspended in 5 ml/g (original weight of bacteria) 70% formic acid and incubated for 10–15 min. The sample was centrifuged at 10,000 × g for 10 min at 20 °C. The supernatant was applied to a Bio-Gel P-2 equilibrated with 200 mM Tris/HCl, 8 M urea/5 mM EDTA pH 8.7. The elution of protein was monitored at 280 nm at a flow rate of 1.0 ml/min and all peaks were collected. Protein concentration of the peaks was determined using the Bradford protein assay [7]. The protein peak is the first peak in the elution profile. Fig. 2 shows the analysis of the proinsulin expression with SDS-PAGE. The molecular weight of the induced and expressed feline proinsulin (lanes 3 and 8) of approximately 9500 was consistent with the deduced molecular weight.The protein was further processed according to the method described by Mackin and Choquette [8]. The purification involves reduction of the protein with dithiothreitol (60 mM final concentration). Argon was bubbled into the solution, the tube was filled with argon and the cap tightened immediately. The sample was incubated at 50 °C for 30 min, then cooled to room temperature and applied to the same Bio-Gel P-2 column which was equilibrated with 50 mM glycine/NaOH, pH 10.5, 1 mM EDTA, flow rate of 1 ml/min. The protein peak was collected.The amount of folded proinsulin was determined using reverse-phase high-performance liquid chromatography (RP-HPLC; Vydac Polymer 5 μm, 4.6 mm × 250 mm column; Grace Vydac, Hesperia, CA). The column was equilibrated with 75% H2O/25% acetonitrile. The sample was prepared for injection to contain a final solution of 0.1% TFA, 4% acetonitrile and 100 mM HCl. The filtered sample was injected and eluted using a two-slope gradient of 0.1% trifluoroacetic acid (TFA) (A) and 0.1% TFA in 20% H2O/80% acetonitrile (B) increasing B from 25 to 35% within 6–10 min and then from 35 to 65% in 10–23 min. The flow rate was 0.7 ml/min. Folded proinsulin was purified using a reverse phase column (Vydac Polymer 5 μm, 10 mm × 250 mm; Grace Vydac, Hesperia, CA) and the same conditions as described above, however, the flow rate was increased to 3.0 ml/min. Human recombinant proinsulin (Eli Lilly, Indianapolis, IN) was used as a standard. Molecular weight analysis of recombinant feline proinsulin was determined by mass spectroscopy (Proteomics Facility, University of Georgia, Athens, GA). |