2005年2月
Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo
Applied and Environmental Microbiology
- ,
- ,
- ,
- ,
- 巻
- 71
- 号
- 2
- 開始ページ
- 754
- 終了ページ
- 760
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1128/aem.71.2.754-760.2005
- 出版者・発行元
- American Society for Microbiology
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.
- リンク情報
- ID情報
-
- DOI : 10.1128/aem.71.2.754-760.2005
- ISSN : 0099-2240
- eISSN : 1098-5336
- ORCIDのPut Code : 102763865
- Web of Science ID : WOS:000227043400020