Pmxb10 vector

All chemicals and reagents were purchased from Sigma unless stated otherwise. The PCR reagents, restriction enzymes and B-PER reagent were obtained from Thermo Scientific. The cysteine-alkyne bifunctional linker (Figure S1A, Supplementary Materials) was purchased from Eurogentec, the azido-propylamine linker from Jena bioscience (Figure S1B, Supplementary Materials) and the N-hydroxysuccinimide (NHS) derived ester linker 2,5-dioxopyrrolidin-1-ylhex-5-ynoate (Figure S1C, Supplementary Materials) was self-synthesized according to Jagadish et al. [55 (link)]. The pMXB10 vector, E. coli SHuffle^® T7 competent cells and chitin resin were purchased from New England Biolabs. The two recombinant human VCAM1 antigens (hVCAM1) were bought from R & D Systems (MW of 270 kDa) and Peprotech (MW of 180 kDa) while the recombinant mouse VCAM1 (mVCAM1) antigen was purchased from Bioconnect (Huissen, The Netherlands, MW of 95 kDa). The ellipsometer and silicon wafers were bought from Synapse B.V. (Maastricht, The Netherlands) and the Biacore™ (Diegem, Belgium) C1 sensor chips from GEHealthcare. The SPR experiments were performed with a Biacore™ T200 model (GE Healthcare).

The coding regions of cystatin family genes lacking the stop codon and signal peptide sequences were cloned (Supplementary Table S2 at JXB online) and inserted into the pMXB-10 vector (NEB). The resulting plasmids were transformed into Escherichia coli BL21 (DE3) (Novagen). The recombinant cystatins were expressed and purified according to the manufacturer’s instructions. The purified cystatins were re-purified by ion exchange chromatography with a Bio-Scale™ Mini UNOsphere™ Cartridge Q/S or a Bio-Scale™ Mini CHT Type I Cartridge (Bio-Rad) on BioLogic DuoFlow™ system (Bio-Rad). The final protein concentrations were quantified using a Coomassie Plus kit (Thermo) with bovine serum albumin as the standard.