Biological activities and secondary structures of variant forms of human salivary cystatin SN produced in Escherichia coli

Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have produced several recombinant human salivary cystatin SN (reCsnSN) variants. These include a N-terminal-truncated form (aa 17-121), a C-terminal-truncated form (aa 1-102) and two deletion mutants (A12-16 and A56-60). A large amount of the insoluble fusion protein (approx. 15 mg/l) was produced in each case. These were solubilized with urea and refolded by dialysis. The GST carrier was then cleaved with thrombin and the reCsn variants (except A56-60) were purified by anion-exchange chromatography. The CysP inhibitory activities against papain, and bovine and human cathepsin B, and secondary structures of the reCsnSN variants were determined and compared to natural salivary CsnSN. The full-length reCsnSN, the N-truncated and the Δ12-16 variants inhibited the CysP activity of papain and displayed circular dichroism (CD) spectra similar to that of natural CsnSN. On the other hand, the Δ56-60 mutant and the C-truncated variant exhibited very little inhibitory activity towards papain. The CD spectrum of the C-truncated variant indicated a change in the secondary structure (e.g., a decrease in β-sheet and an increase of an α-helical content). Neither the natural nor the full-length reCsnSN or the A12-16 mutant exhibited any inhibitory activity towards bovine and human cathepsin B.