| Project/Area Number |
01571214
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Kyushu University |
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Principal Investigator |
YAMADA Hidenori Kyushu University, Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (80037613)
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| Co-Investigator(Kenkyū-buntansha) |
UEDA Tadashi Kyushu University, Faculty of Pharmaceutical Sciences, Assistant professor, 薬学部, 助手 (90184928)
IMOTO Taiji Kyushu University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (90038282)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Gene engineering / Chicken lysozyme / Eschericia coli / Processing of N-terminal methionine / Deaminated / Renaturation / フォルディング / SS-SH交換反応 / 変異リゾチ-ム |
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| Research Abstract |
Expression of foreign proteins having disulfide bonds in schericia coli usually results in the formation of insoluble and denatured proteins called inclusion body. This greatly reduces the advantage of the expression system in E. coli. In order to improve the utility of the expression system in E. coli. In order to improve the utility of the expression system in E. coli, we investigated the solubilization, purity, processing of the N-terminal methionine residue, and renaturation of mutant chicken lysozymes expressod in E. coli, and the following results were obtained.
1. Lysozyme expressed in E. coli contained about 80 % of deaminated ones.
2. In the case of lysozyme with the extra methionine residue at the N-terminal (M-lysozyme), the lysozyme in which more than 4 residues were deaminated (at about 50 % of total lysozyme) could not be extracted with 10 % acetic acid from the inclusion body.
3. The M-lysozyme thus extracted was renaturated form the reduced form in the system of reduced an
… More
d oxidized glutathione, and it was found that the renaturation yield was only about 25 % and that the lysozyme in which more than 2 residues were deaminated was not renaturated but precipitated during the reaction.
4. Met-lysozyme without deamidized residue was renaturated in about 50 % yield.
5. New method to process the N-terminal methionine residue has been developed.
6. The resulting wild type lysozyme was renaturated in 80 % yield.
7. The mutant M-lysozyme in which Ala31 is replaced with Val could not be renaturated at all by the usual renaturation system was employed, but it was renaturated in about 10 % yield by the system containing 1 M urea.
All of these results described above indicate that the reactions and/or mutations which reduce the solubility of denatured lysozyme, such as diaminated, addition of the hydrophobic amino acid residue at the N-terminal and the replacement of a residue with more hydrophobic one, decrease the efficiency of the renaturation of lysozyme.
Thus, it is concluded that the use of E. Coli strain without deamidase, as well as the development of the method for the N-terminal methionine, is essential for the increase of the utility of the foreign protein expression system in E. Coli. Less
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