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 f oreign sequences as fusion proteins with a glutathione S-transferase ( GST) carrier, we have produced several recombinant human salivary cyst atin SN (reCsnSN) variants. These include a N-terminal-truncated form (aa 17-121), a C-terminal-truncated form (aa 1-102) and two deletion m utants (Delta 12-16 and Delta 56-60). 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 t hen cleaved with thrombin and the reCsn variants (except Delta 56-60) were purified by anion-exchange chromatography. The CysP inhibitory ac tivities against papain, and bovine and human cathepsin B, and seconda ry structures of the reCsnSN variants were determined and compared to natural salivary CsnSN. The full-length reCsnSN, the N-truncated and t he Delta 12-16 variants inhibited the CysP activity of papain and disp layed circular dichroism (CD) spectra similar to that of natural CsnSN . On the other hand, the Delta 56-60 mutant and the C-truncated varian t exhibited very little inhibitory activity towards papain. The CD spe ctrum of the C-truncated variant indicated a change in the secondary s tructure (e.g., a decrease in beta-sheet and an increase of an alpha-h elical content). Neither the natural nor the full-length reCsnSN or th e Delta 12-16 mutant exhibited any inhibitory activity towards bovine and human cathepsin B.