In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase.
We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding attB (TG1) and attP (TG1) sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of phi C31 integrase. In this report, we expressed TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP (TG1) and attB (TG1) sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB (TG1) and attP (TG1) sites irrespective of their substrate topology. The minimal sequences of attP (TG1) and attB (TG1) sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of the serine integrase.
Morita
Kentaro
K
Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa, 252-8510, Japan.
Yamamoto
Tomoyuki
T
Fusada
Naoki
N
Komatsu
Mamoru
M
Ikeda
Haruo
H
Hirano
Nobutaka
N
Takahashi
Hideo
H
D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D019426:Integrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D013379:Substrate Specificity
D019426:Integrases