| Budget Amount *help |
¥19,000,000 (Direct Cost: ¥19,000,000)
Fiscal Year 1992: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1991: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1990: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Research Abstract |
By using a protein-production system with Bacillus brevis as a host, human proteins can be efficiently produced, although its efficiency is much lower than that of bacterial protein-production with some exception. Since it is presumed that the production efficiency depends on the structural change of the protein during its synthesis and secretion, we studied the enzymes con- trolling the formation of tertiary structure of proteins, i.e., peptidylprolyl cis-trans isomerase (PPI), protein disulfide isomerase (PDI), and proteases which discriminate the protein degradation according to its structure.
We found PPI activities from culture supernatant and cell extract of B. brevis, from which the enzyme was partially purified and its properties were analyzed. This enzyme is relatively heat stable and its activity was lowered by increasing salt concentrations of the reaction mixture. It was not sensitive to either cyclosporin A or FK506, an immunosuppressant. PDI like enzyme, i.e., protein disulfide bond forming enzyme (DSB), was found only in the culture supernant of B. brevis. We succeeded to clone the DSB gene by a shot-gun method. The nucleotide sequence of the gene revealed that the molecular weight of DSB is about 13,000 and it has the sequence of signal peptide and CysGlyTyr- Cys, which is the catalytic site of the PDI like enzyme. These secreted enzymes are presumed to play important roles in the formation of foreign proteins having correct conformation and fully biological activity.
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