1993 Fiscal Year Final Research Report Summary
STRUCTURAL STUDY OF INTERACTION BETWEEN c-MYC PROTEIN AND ITS TARGET DNA OLIGOMER BY NMR
| Project/Area Number |
04452304
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Physical pharmacy
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| Research Institution | YOKOHAMA NATIONAL UNIVERSITY |
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Principal Investigator |
UESUGI Seiichi YOKOHAMA NATIONAL UNIVERSITY, FACULTY OF ENGINEERING, PROFESSOR, 工学部, 教授 (70028851)
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| Co-Investigator(Kenkyū-buntansha) |
KURIHARA Yasuyuki YOKOHAMA NATIONAL UNIVERSITY, FACULTY OF ENGINEERING, ASSISTANT PROFESSOR, 工学部, 助手 (80202050)
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| Project Period (FY) |
1992 – 1993
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| Keywords | c-MYC / NMR / MAX |
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| Research Abstract |
(1) Attempts to establish of a system for production of c-Myc protein in E.coli
DNA fragments encoding a part of c-Myc protein (342-439) were subcloned into two types of E.coli expression vectors (pRIT5 and pQE). However, no expression of the protein was demonstrated by SDS-PAGE and Western blotting by anti-c-Myc exon 3 serum. Then, we compared frequency of codon usage of E.coli and c-Myc. Both of Arg condons at 366 and 367 amino acid residue of c-Myc are AGG which is rarely found in E.coli. It is usually found that poor expression of recombinant protein is due to the continuous rare condons. So, we exchange the condons to relatively abundant ones to improve the expression. But, we failed to the expression of the protein.
(2) Establishment of production system of Max protein and structural analysis of the protein.
Full length of DNA fragment encoding Max (p22) protein was cloned into pQE E.coli expression vector. By using this plasmid, we succeeded to express the protein in E.coli. About 20mg of the purified protein was obtained from one litter culture of the E.coli. After the refolding the protein by stepwise reduction of urea, we carried out CD analysis. CD spectra showed highly alpha-helical nature of the protein. Also, addition of DNA oligomer induced drastic increase of the alpha-helix content.
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