Abstract
We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb + background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.
| Original language | English |
|---|---|
| Pages (from-to) | 754-760 |
| Number of pages | 7 |
| Journal | Applied and Environmental Microbiology |
| Volume | 71 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Feb 2005 |
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ASJC Scopus subject areas
- Environmental Science(all)
- Biotechnology
- Microbiology
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Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo. / Mori, Koichi; Mukaihara, Takafumi; Uesugi, Yoshiko; Iwabuchi, Masaki; Hatanaka, Tadashi.
In: Applied and Environmental Microbiology, Vol. 71, No. 2, 02.2005, p. 754-760.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo
AU - Mori, Koichi
AU - Mukaihara, Takafumi
AU - Uesugi, Yoshiko
AU - Iwabuchi, Masaki
AU - Hatanaka, Tadashi
PY - 2005/2
Y1 - 2005/2
N2 - We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb + background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.
AB - We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb + background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.
UR - http://www.scopus.com/inward/record.url?scp=13544256354&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=13544256354&partnerID=8YFLogxK
U2 - 10.1128/AEM.71.2.754-760.2005
DO - 10.1128/AEM.71.2.754-760.2005
M3 - Article
C2 - 15691927
AN - SCOPUS:13544256354
VL - 71
SP - 754
EP - 760
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 2
ER -