A new, simple, rapid procedure for purification of Escherichia coli alkaline phosphatase

✍ Scribed by Milton J. chlesinger; Robert Olsen

• Publisher: Elsevier Science
• Year: 1970
• Tongue: English
• Weight: 952 KB
• Volume: 36
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(70)90334-9

✦ Synopsis

Escherichia coZi alkaline phosphatase is a heat-stable, mukimeCc:, metallo-enzyme localized to the periplasmic space of the bacterial cell (I-5). The protein's stability and its specific location have been c>xploited in procedures for purifying this enzyme; for example, after treating bacterial cells with either lysozyme (4) or with a. "cold osmoGc shock" (6) it, is possible to obtain cell-free ext8ract8s that contain alkaline phosphatase as 15 to 20% of the total protein.

We report here a new purification procedure which takes advantage of another property of this enzyme. Previous studies in our laboratory showed that E. coli alkaline phosphatase was dissociat'ed into it)s component subunits at pH 2.0, and t.hese denatured polypeptide chains could be completely refolded to an enaymically act'ive molecule indistinguishable from the native enzyme (7, 8). When bacterial cells containing alkaline phosphat'ase were treated at low pH, subunits of the enzyme were quantitively released into the medium. Subsequent reassociation and reactivation of these subunits provided an initial cell-free extract' that cont,ained almost 30% of the protein as alkaline phosphatase. Batch-wise adsorption and elution of the dialyzed extract on DEAE-cellulose yielded an enzyme preparation that was about 55% pure. The d&ails of this purification are described in t)his communication.

MATERIALS

All chemicals were reagent grade. We used E. coli strain CW3747 grown in a Tris medium (9) to obtain large amounts of enzyme, DEAE-cellulose was from Eastman and was washed with alkali and acid prior to use.

EXPERIMENTAL PROCEDURE

A culture of bacterial cells was harvested and washed with 10 mM Tris Cl, pH 7.4. The pellet of cells (they can be frozen at this stage) was rapidly resuspended in 2% the original volume with 0.05 hi HC1 and