| Project/Area Number |
12460038
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | University of Tsukuba |
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Principal Investigator |
HOSHINO Takayuki Institute of Applied Biochemistry, Professor, 応用生物化学系, 教授 (80219170)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2001: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 2000: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Keywords | Thermus thermophilus / Pyrococcus horikoshii / expression vector / restriction-modification system / amino acid biosynthesis / Pyrococcus horikoshii / expression vector / 分泌ベクター / α-amylase / 発見ベクター |
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| Research Abstract |
I have succeeded in expressing several P. horikoshii genes in T. thermophilus HB27 efficiently by replacing promoter sequences of the expression vector. The use of Pslp promoter was most effective. By using Pslp promoter, three OT3 genes were expressed at the higher levels than those expressed in E. coli. In particular, the mannnosidase gene, which was not expressed at all in E. coli, was clearly expressed in T. thermophilus.
In order to improve the expression levels of foreign genes, I tried to develop a high copy expression vector. The entire nucleotide sequences of the expression vector and its high-copy derivative were determined, and the sequences were compared. It was elucidated that integration of the fragment derived from host genome into the upstream region of a putative replication gene of the vector increased the copy number. I have constructed a new high copy expression vector, pET-Pslpm, as a shuttle vector available in both T. thermophilus and E. coli.
I have already shown that integration of foreign DNA fragments into T. thermophilus genome could be carried out efficiently. But it was necessary to methylate the donor DNA prior to introduce into T. thermophilus. I have cloned the methylase gene (MTthHB27) from T. thermophilus HB27. The cloned MTthHB27 gene was introduced into E. coli expression system. Heat-treated cell-free extract prepared from E. coli recombinant showed methylation activity.
Genes for restriction enzyme and modification enzyme are known to exist in tandem in various microorganisms. However, there was no possible restriction enzyme gene in the region nearby the MTthHB27. MTthHB8 and RTthHB8 genes were reported to be existing in tandem on HB8 genome. But their codon usage was far from Thermus-type. Thus, it was speculated that the restriction-modification system of HB8 was horizontally transferred from other organisms, and that HB27 has original Thermus-type one.
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