In lane 4, we analyzed the sample prepared from culture of the mutant strain (A. sobria 288 [asp−, amp−]) in NB (3.0). The pattern of the bands in lane 4 was considerably different from that in lane 3 because of the addition of

NaCl to the medium. Among these protein bands, we were interested in the protein indicated by the arrow in Figure 1. The density of the band in lane 3, which was our focus, was higher than that in lane 4. This suggests that production of this protein is down-regulated by NaCl around the bacteria. In addition, the existence of the protein band was not confirmed in lane 1, indicating that the protein was degraded by ASP and/or AMP. These properties of the protein are extremely interesting. We purified the protein of interest by salt outing with ammonium sulfate and successive column chromatography, Alectinib chemical structure as described in Materials and Methods. We detected the protein by SDS-PAGE. In fractionation with ammonium sulfate, the protein in the culture supernatant was efficiently recovered in the fraction of 30–50% saturated ammonium sulfate. We dissolved the recovered material in 10  mM phosphate Ulixertinib cell line buffer (pH 7.4) and dialyzed the solution against the buffer; thus, the prepared sample was designated the crude sample. We separated the crude sample by column chromatography using hydroxyapatite and Superdex.

Typical elution profiles of these columns are shown in Figures 2a and 2b. The fractions containing the target protein are shown by the double-headed arrows. In purification by Superdex, the protein was eluted as a single peak around fraction enough 14. To examine its purity, we analyzed it by SDS-PAGE. About 5 μg of purified protein was loaded onto the lane of SDS-polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie brilliant blue. As shown in Figure 2c, a single band appeared at the position of 75,000  daltons. The molecular size of the purified protein was measured by MALDI-TOFMS. As shown in Figure 2d, the main peak was 81,044.8  daltons. To clarify the characteristics of the protein indicated by the arrow in Fig. 1, we determined the N-terminal amino acid sequence of the purified

protein as described in Materials and Methods. The result showed that the sequence of the five amino acid residues from the amino terminus was GGDDN. This protein was thought to be about 81,000  daltons based on the measurement by MALDI-TOFMS (Fig. 2d). We therefore searched for a protein which fulfilled the following criteria: (i) the protein is a product of Aeromonas; (ii) the molecular size of the protein is about 81,000 daltons; and (iii) the amino terminal sequence is GGDDN. Blast search and literature search indicated that the protein may be a homologue of phospholipase A1 of A. hydrophila (GenBank accession number: AF092033) (11). As described above, we speculated that the purified protein was a homologue of phospholipase A1. Merino et  al. reported that E.