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) encode s an integrase gene int linked to a phage attachment site attP (Kakika wa 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 s equence. Amino-acid sequencing revealed that the N-terminal 20 amino-a cids of the purified Int protein completely coincided with those deduc ed 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 pr otected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interac tion of Int with the attP site, the reaction prerequisite for integrat ion and excision of the phi g1e genome into and/or out of the host chr omosome. (C) 1997 Elsevier Science B.V.