Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo.
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.
Mori
Koichi
K
Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Mukaihara
Takafumi
T
Uesugi
Yoshiko
Y
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D015723:Gene Library; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D000091243:Ribosomal Protein S9; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D010739:Phospholipase D