Ppsq 21 protein sequencer

N-terminal amino acid sequences of the purified POD were determined as follows: the purified preparation was subjected to SDS-PAGE30 (link), then the protein band with an estimated molecular weight of 30,000 in the gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) by a semi-dry electroblotting device (ATTO Co., Tokyo, Japan). The bands on the PVDF membrane were subjected to a PPSQ-21 protein sequencer (Shimadzu Co., Kyoto, Japan) to determine the N-terminal sequence. The gene encoding the protein whose N-terminal sequence was identical to that of the purified POD by a sequence alignment search on the draft data of the A. faecalis genome. Determination of the draft genome sequence is described in the Supplementary Materials.

The purified AJLec was reduced with tri-n-butylphosphine in 7 M guanidine-HCl, 10 mM EDTA, and 0.5 M Tris-HCl (pH 8.5) and pyridylethylated by treatment with 4-vinyl pyridine at 25 °C for 4 h in the dark. The resulting pyridylethylated AJLec was chemically cleaved at methionyl bonds with 1% CNBr in 70% (v/v) formic acid at 25 °C for 24 h by the method described by Gross^{31 (link)}. The peptides generated by CNBr cleavage were separated by reverse-phase HPLC using HITACHI model L-6200 and L-4200 liquid chromatographs on a Wakosil-II 5C18 AR column (4.6 × 100 mm, Wako Pure Chemical Industries Ltd., Osaka, Japan). The column was equilibrated with solvent A (0.1% trifluoroacetic acid; TFA), and the peptides were eluted at a flow rate of 1 mL/min using a linear gradient of 0–100% solvent B (acetonitrile/water/TFA, 80:20:0.1 [v/v/v]) at room temperature. Amino acid sequences of the separated peptides were determined using a PPSQ-21 protein sequencer (Shimadzu).