| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
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| Research Abstract |
1. To study the structural and functional relationship and the therapeutic potentials of salivary cystatins, we have established an E.coli system enabling a high level of expression and secretion of cystatins. The cDNAs encoding the precursors (141 residues) of cystatin S,cystatin SA and cystatin SN were expressed in E.coli JM109 with isopropyl-beta-thiogalactoside induction. The cystatins expressed in E.coli cells were detected by anti-cystatin S antibody and purified from the periplasmic fractions prepared by cold osmotic-shock treatment. The amino acid sequences of recombinant cystatins were determined by automated gas-phase Edman degradation. The precursor proteins were post-translationally secreted and assembled in the space between cytoplasmic membrane and outer membrane of E.coli cells as the mature cystatins (121 residues). A mutation (-18R - W) in the signal of cystatin S reduced its accumulation in the periplasmic space remarkably. The replacement of the amino (N-) terminal t
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hird residue of the signal of cystatin SA (-18W) and of cystatin SN (-18Q) by arginine facilitated the translocation of the cystatins across the cytoplasmic membrane. Cystatin SN lacking the N-terminal 17 residues and two molecular forms of cystatin SA lacking, respectively, the N-terminal 4 residues (WSPQ) and 6 residues (WSPQEE) were occasionally purified, suggesting that cystatins SA and SN have been under-gone further proteolysis by proteinases other than signal peptidase I of E.coli. Recombinant cystatins produced by the system showed virtually the same inhibitory properties for papain, ficin, and cathepsins (B,C,and H). Recombinant cystatins S and S (117R-W) did not inhibit Arg-gingipain or Lysgingipain from Porphyromonas gingivalis, however, the recombinant proteins inhibited the growth of P.gingivalis.
2. The chimeric genes consisting of three exonic units from the genes of cystatin S and cystatin C,S1C2C3 (exon 1 of cystatin S - exon 2 of cystatin C - exon 3 of cystatin C), S1C2S3 (exon 1 of cystatin S - exon 2 of cystatin C - exon 3 of cystatin S) and S1S2C3 (exon 1 of cystatin S - exon 2 of cystatin S - exon 3 of cystatin C) were made by the polymerase chain reaction (PCR). The genes were expressed in E.coli JM109 cells with isopropyl-beta-thiogalactoside induction. Three chimeric proteins (S1C2C3, S1C2S3 and S1S2C3) were purified by column chromatography from periplasmic fractions of E.coli. Two chimeric proteins, S1C2C3 and S1C2S3, were found to be strong inhibitors for papain, ficin, cathepsin C and cathepsin H.The inhibitory activity of the chimeras for cathepsin B was extremely low level to compare with that of cystatin C.The inhibitory spectrums of S1C2C3 and S1C2S3 are different from those of cystatin S and cystatin C.The findings suggest that it is possible to design and produce artificial cystatins with new properties by exchanging exonic units of natural cystatins. Less
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