| RRC ID |
3245
|
| Author |
Mori K, Mukaihara T, Uesugi Y, Iwabuchi M, Hatanaka T.
|
| Title |
Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo.
|
| Journal |
Appl Environ Microbiol
|
| 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.
|
| Volume |
71(2)
|
| Pages |
754-60
|
| Published |
2005-2-1
|
| DOI |
10.1128/AEM.71.2.754-760.2005
|
| PII |
71/2/754
|
| PMID |
15691927
|
| PMC |
PMC546693
|
| MeSH |
Amino Acid Sequence
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Base Sequence
Enzyme Stability
Escherichia coli Proteins
Gene Library*
Hot Temperature
Molecular Sequence Data
Phospholipase D / genetics
Phospholipase D / metabolism*
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism*
Recombination, Genetic*
Ribosomal Protein S9
Sequence Analysis, DNA
Streptomyces / enzymology
Streptomyces / genetics*
|
| IF |
4.016
|
| Times Cited |
16
|
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
MICROBIOLOGY
|
| Resource |
| Prokaryotes E. coli |
|
