Xxx coli

Tris and HEPES were purchased from Research Organics.Glycylglycine, phosphoenolpyruvate mono(cyclohexylammonium) salt, , and HCl (99.999%, metal basis) were purchased from Alfa Aesar.The final preparation was concentrated by ultrafiltration to 0.6 mg/ml (Centriprep YM-10 concentrator), and the resulting solution was stored at −80 °C.The purification process of wild-type KDO 8-P phosphatase is summarized in Table I.The column was eluted at a flow rate of 2.0 ml/min using a linear gradient of 0–0.5 in buffer A was applied at a flow rate of 1.0 ml/min over a 60-min period.The fractions containing KDO 8-P phosphatase activity were pooled, dialyzed against 1 liter of buffer A overnight, and applied to a Mono Q (HR 5/5) column pre-equilibrated with buffer A.The p H of the supernatant was adjusted to 7.0 by 1 Tris-HCl (p H 7.4) in a final volume of 60 ml.

The sample was dialyzed against two liters of the same buffer overnight, and the resulting protein solution was applied to a Q-Sepharose column (1.2 × 21 cm) pre-equilibrated with buffer A.

High grade Spectra/Por® 7 dialysis tubing (10,000 molecular weight cut-off and metal-free) was obtained from VWR Scientific.

The Centriprep YM-10 Concentrators were purchased from Millipore. Mono Q (HR 5/5), phenyl Superose (HR 10/10), and Superose 12 (HR 10/30) chromatography columns were purchased from Amersham Biosciences.

Tris-HCl (p H 7.5) in a final volume of 4 ml at 37 °C for 2 h.

The reaction mixture was quenched by the addition of 0.45 ml 50% (w/v) trichloroacetic acid and centrifuged to remove precipitated protein.

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