| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
A new direct expression vector, ATG-TAG vector, was developed, and applied to the expression in Escherichia coli of hen lysozyme module combinations 1+2+3+4+5(hereafter abbreviated as Ml-5, and so on), Ml-4, Ml-3, M2-5 and M2-4. The expression of Mi-2 and Ml as fused proteins was not carried out. The repression of expression during uninduced period was observed when minimal media were used, but was not when rich media were used. Variation in IPTG concentration as well as in the culture temperature so far used did not lead to the expression into a soluble fraction. The primary structures of the expressed materials were as expected. These module combinations, when disulfide-bridges were opened, showed the CD spectra which indicated a decrease in alpha-helix and an increase in beta-sheet contents compared to authentic native lysozyme. This 'residual' structure showed a non-cooperative transition on GuHCl-induced denaturation. Glycerol, sorbitol, methanol and trifluoroethanol increased the
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secondary structure of the reduced module combinations. Renatured(reoxidized)module combinations contained, ' except for Ml-5 and M2-4, more than ten molecular species of different higher-order structure as monitored by RPHPLC. M2-5 showed an increase in higher-order structure when reoxidized in the presence of polyols or alcohols. Cooperative formation of tertiary structure, however, was not observed. on the other hand, the lysozyme derivatives which lacked one disulfide bond while retaining fulllength polypeptide were renatured efficiently in the presence of glycerol, and showed lytic activities and secondary structures comparable to those of authentic lysozyme. Another derivative which contained residues 21 to 129, and which could form three native disulfide bonds, did not show a 'correct' folding even in the presence of glycerol. Altogether, both amino-terminal(residues 1 to 20)and carboxyl-terminal region(108-129)are necessary for the correct higherorder structure formation of hen lysozyme. Less
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