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. Author manuscript; available in PMC: 2022 Apr 14.
Published in final edited form as: Methods Enzymol. 2021 Jun 18;656:545–571. doi: 10.1016/bs.mie.2021.05.008

Figure 4.

Figure 4.

Representative analysis of proline analog incorporation after POI expression. a. SDS-PAGE analysis of proinsulin expression and purification. After proline analog incorporation into proinsulin (10.7 kDa), the inclusion body (IB) fraction was solubilized in 8 M urea. After incubation with Ni-NTA resin, the flow-through (FT), wash (W; 20 mM imidazole), and rinse (R; no imidazole) fractions were collected. Proinsulin was eluted under low pH (3.0) conditions, and fractions were collected, then pooled. b, c. MALDI-TOF spectrum (b) and analysis (c) of proline analog incorporation after GluC digestion. Relevant proinsulin fragments: 33RGFFYTPKTRRE (expected: 1557.8 Da) and 33RGFFYT–4R-OH–KTRRE (1573.8 Da).