Materials and methods Restriction endonucleases T DNA polyme

Materials and methods Restriction endonucleases, T4 DNA polymerase, Taq polymerase and a DNA ligation kit were purchased from Takara Shuzo, [α-32P]dCTP (3000 Ci/mmol) and a DNA sequencing kit were from Amersham, a Gene Clean II kit was from Bio 101, plasmid vector pET-3a was from Novagen, marker proteins for SDS-PAGE were from Bio-Rad, phenyl-Sepharose CL-4B was from Pharmacia and DEAE-HPLC column was from Tosoh. B. subtilis strain AC327 was used throughout this study. E. coli JM109 [23]and E. coli BL21(DE3) (Novagen) were used as hosts in the cloning and expression, respectively. The strains were grown in LB broth or on 2×YT plates [23]. Ampicillin (50 μg/ml) was added when required. B. subtilis PPase was expressed in E. coli under the inducible phage T7 promoter by making use of the pET system (Novagen). The open reading frame encoding B. subtilis PPase was amplified by polymerase chain reaction (PCR) using a 5′-sense oligonucleotide primer containing a restriction site for NdeI (5′-GCGGAAAAGATACTTATTTTCG-3′, the restriction site is underlined) and a 3′-reverse complement primer with a BamHI site (5′-AATTATTCAGCCATTGCGTCT-3′). The PCR product was digested with NdeI and BamHI, ligated into the vector pET-3a (Novagen), cut with the same restriction Benzethonium Chloride and transformed into E. coli JM109 and E. coli BL21(DE3) for DNA sequencing [24]and expression, respectively. The expression was induced for 3–6 h by 0.5 mM IPTG. The authentic PPase was purified from B. subtilis as described previously [21], whereas the recombinant enzyme was purified to homogeneity from E. coli by a simplified procedure including only phenyl-Sepharose CL-4B column chromatography and DEAE-HPLC [21]. About 26 mg of pure enzyme was obtained from 12 g of cell paste with a yield of 45%. To stabilize the enzyme, 1.5 mM MnCl2 was included in buffers at all purification steps. The purified enzyme gave a single band on SDS-PAGE gels stained with Coomassie brilliant blue [25]. The N-terminal sequence of the authentic enzyme was determined by an automatic Edman degradation method with an Applied Biosystem 477A/120A gas-phase sequenator. PPase activity was assayed at 37°C as described previously [26]. The assay medium contained 1 mM PPi, 2 mM MgCl2 and 20 mM Tris-HCl buffer (pH 7.3). One unit of activity corresponds to 1 μmol of PPi converted per minute. Protein concentration was determined with a Pierce BCA protein assay kit, with BSA as standard.
Results and discussion Soluble PPase isolated from B. subtilis was purified to homogeneity and its N-terminal residues 1–39 were determined by automatic Edman degradation method giving the sequence: 1-M-E-K-I-L-I-F-G-H-Q-N-P-D-T-D-T-I-X-S-A-I-A-Y-A-D-L-K-N-K-L-G-F-N-A-E-P-V-R-L-39, where X denotes an unidentified residue. By searching the Swiss-Prot database, this sequence was found to be identical with an N-terminal gene-deduced sequence of a hypothetical 34-kDa protein encoded by a non-identified open reading frame located in the COTF-TETB intergenic region of B. subtilis[27]. This open reading frame was amplified by PCR and its sequence was verified to be identical to that reported previously [27]. By its size, the hypothetical protein is similar to B. subtilis PPase (34–36 kDa [22]).