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Figure 1
The expression of insoluble D-HEWL in E. coli followed by refolding increases the yield of protein production by more than threefold. (a) Chromatogram from the denaturing SEC, yielding pure unfolded D-HEWLEC, which eluted at 0.6–0.7 CV. (b) Fractions from the denaturing SEC on a 12% SDS–PAGE gel. Lane S, Precision Plus Protein Dual Xtra Standards (Bio-Rad); lane I, injected sample of unpurified D-HEWLEC from the inclusion-body washing steps; lanes F1–F9, collected fractions from the denaturing SEC as indicated at the top of (a). Fractions F5–F7 were used in subsequent refolding experiments. (c) Refolding SEC chromatogram, where monomeric D-HEWL elutes at 0.9 CV. The fractions eluting before and after the monomeric refolded D-HEWLEC are likely to be misfolded or oligomeric and partially unfolded forms of D-HEWLEC, respectively. This is followed by the elution of the guanidine–HCl and the DTT from the denaturing buffer, as shown by the increase in conductivity. (d) Comparison of the D-HEWL expression yields between the two systems, E. coli and P. pastoris. *Considering an average refolding yield of 20%, the final yield of D-­HEWLEC production is 37 mg l−1 without further denaturing and refolding of the misfolded, oligomeric and partially unfolded fractions.

IUCrJ
Volume 8| Part 3| May 2021| Pages 372-386
ISSN: 2052-2525
https://doi.org/10.1107/S2052252521001299