Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage.
The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.
Yasukawa
H
H
Department of Microbiology, Faculty of Pharmaceutical Sciences, Kanazawa University, Ishikawa, Japan.
Kakikawa
M
M
Masamune
Y
Y
Taketo
A
A
Kodaira
K I
KI
D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism*; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007778:Lactobacillus / Q000821:virology; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding
D004269:DNA, Bacterial; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019426:Integrases