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 (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 then cleaved with thrombin and the reCsn variants (except delta 56-60) 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 delta 12-16 variants inhibited the CysP activity of papain and displayed circular dichroism (CD) spectra similar to that of natural CsnSN. On the other hand, the delta 56-60 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 beta-sheet and an increase of an alpha-helical content). Neither, the natural nor the full-length reCsnSN or the delta 12-16 mutant exhibited any inhibitory activity towards bovine and human cathepsin B.
Bobek
L A
LA
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo 14214.
Ramasubbu
N
N
Wang
X
X
Weaver
T R
TR
Levine
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015891:Cystatins / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D004926:Escherichia coli; D014644:Genetic Variation; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D010957:Plasmids; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012463:Saliva / Q000378:metabolism*; D055333:Salivary Cystatins; D017384:Sequence Deletion
C525171:CST1 protein, human; D015891:Cystatins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D055333:Salivary Cystatins; D005982:Glutathione Transferase; D002401:Cathepsin B; D010206:Papain