SPINK1 variants
and human trypsin isoforms were expressed and purified as previously
described.18 (link) In brief, SPINK1 was expressed
in SHuffle T7 Express E. coli (New
England Biolabs, Frankfurt am Main, Germany). After induction and
overnight expression at 16 °C, cells were lysed by sonication
and purified by immobilized metal ion affinity chromatography (IMAC).
His-tags were removed by cleavage with the HRV3C protease, and proteins
were purified again by IMAC. Human trypsin isoforms were expressed
in E. coli BL21 (DE3) (New England
Biolabs) overnight at 30 °C. Inclusion bodies were washed before
solubilization and refolded in 0.9 M Gdn-HCl, 0.1 M Tris pH 8, 2 mM
EDTA, 1 mM l-cysteine, and 1 mM l-cystine at 4 °C
overnight. Refolded trypsin isoforms were purified by IMAC and activated
with enterokinase before use. Bovine and porcine trypsin were commercially
available (Sigma-Aldrich, Taufkirchen, Germany) and were thus not
expressed recombinantly. Protein purity and homogeneity were verified
by tricine SDS PAGE and analytical size exclusion chromatography.
Calibration of the analytical size exclusion can be found in the work
of Susemihl et al.19 (link)