Purification and Expression of the Gene III Protein from Filamentous Phage φLf》.pdf

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 242, 113–117 (1998) ARTICLE NO. RC977932 Purification and Expression of the Gene III Protein from Filamentous Phage Lf Tzu-Jun Liu, Bih-Yuh You, 1 Nien-Tsung Lin, Ming-Te Yang, and Yi-Hsiung Tseng2 Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan Received November 14, 1997 host chromosome (3, 4), ii) having a replication initia- The gene III protein (pIII) from Lf, a filamentous tion protein (pII) that possesses sequence domains con- phage of Xanthomonas campestris pv. campestris, was served to the superfamily I Rep proteins of the rolling purified by gel filtration with FPLC. The gIII coding circle replicating replicons which does not include the region was amplified by PCR, which was then cloned proteins of other filamentous phages (5), and iii) having into pUC18 and expressed in Escherichia coli. The size the origin for viral strand replication being contained of both pIII, purified from phage particle and ex- pressed in E. coli, is similar to the value deduced from within the pII coding region instead of the intergenic the nucleotide sequence as shown by Western blot region (6). The complete nucleotide sequence of Lf has analysis. This is different from the case in Ff phages been determined and ten genes have been found on the (f1, fd, and M13), in which the size of pIII observed in viral strand (7). These genes having an organization SDS-polyacrylamide gel electrophoresis is substan- similar to the order of Ff phages (f1, fd and M13) genes, tially larger than the deduced value. Upo