Laboratory 5: Purification of Alkaline Phosphatase from E. coli

Introduction to Alkaline Phosphatase

  • Enzyme Overview: Alkaline phosphatases are non-specific enzymes that catalyze hydrolysis of phosphate monoesters to release inorganic phosphate.

  • Reaction: R-O-PO3H- + H2O → R-OH + H2PO4.

  • Classification: Orthophosphoric monoester phosphohydrolases (EC 3.1.3.1), active at basic pH.

  • Sources: Present in bacteria, fungi, and higher animals but absent in plants.

  • E. coli Specifics: Major alkaline phosphatase from E. coli is a bipartite enzyme (86,000 Da, composed of two subunits), requiring both for activity.

  • Physical Parameters:

  • pH Optimum: 8.0

  • Isoelectric Point (pI): 4.5

  • Cofactors: Contains two zinc atoms per enzyme.

  • Function: Extracts inorganic phosphate (Pi) from phosphorylated compounds

  • Hydrolysis occurs in the periplasmic space, where it is bound by Pi-binding protein and transferred to the Pst system for cytoplasmic transport.

  • Purification Strategy:

  • Exploits heat stability of alkaline phosphatase to selectively denature and remove other soluble proteins.

  • Utilizes ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose for further purification.

Week 1 - Preparation and Enzyme Extraction

  • Materials Needed:

  • Hydrated E. coli K-12 cells, lysozyme, DNase I, EDTA, MgSO4, dialysis tubing, and buffers.

  • Protocol:

  1. Prepare E. coli Cells:
    • Hydrate cells in sucrose for 48-72 hours at 4°C after washing and pelleting.
  2. Cell Lysis (all steps at room temperature except post-lysis):
    • Add lysozyme to hydrolyze cell wall.
    • Add DNase to prevent viscosity from liberated DNA.
    • Use EDTA for chelation of divalent cations (Ca2+, Mg2+) aiding in cell lysis.
    • Add MgSO4 to prevent prolonged EDTA exposure to alkaline phosphatase.
  3. Centrifugation (4°C):
    • Centrifuge at 9,500 x g to separate supernatant from pellet (Stage 1 Enzyme).
  4. Dialysis:
    • Prepare dialysis bag with ice-cold retentate to remove impurities and small molecules.

Week 2 - Heat Denaturation and Ammonium Sulfate Precipitation

  • Materials Needed:

  • Ammonium sulfate, 1N NaOH, dialysis buffer, and others as required.

  • Heat Treatment:

  1. Remove dialysis bag and squeeze out retentate into test tube.
  2. Heat at 80°C for 15 min and cool rapidly.
  3. Centrifuge and collect supernatant (Stage 2 Enzyme).
  • Ammonium Sulfate Precipitation:
  1. Calculate ammonium sulfate: 0.603 g/mL for 90% saturation.
  2. Add crystals slowly while stirring gently to avoid denaturation.
  3. Correct acidity with NaOH, allow precipitation.
  4. Centrifuge and discard supernatant, resuspend pellet in dialysis buffer.

Week 3 - Ion-Exchange Chromatography

  • Anion-Exchange Setup:

  • Pack DEAE cellulose, equilibrated with low-ionic-strength buffer (Buffer A).

  • Chromatography Steps:

  1. Apply Stage 3 Enzyme to column.
  2. Rinse with Buffer A and elute with Buffer B (higher NaCl) to collect enzyme fractions.
  3. Assay fractions for alkaline phosphatase activity (yellow color indicates presence).
  4. Pool active fractions for Stage 4 Enzyme.

Activity Assays and Protein Determination

  • Alkaline Phosphatase Activity Assay:

  • Monitor breakdown of pNPP to pNP spectrophotometrically at 410 nm.

  • Set up reaction mixtures and measure absorbance.

  • Perform enzyme blanks to correct for substrate degradation.

  • Protein Determination (Bradford Method):

  1. Prepare BSA standards of known concentration.
  2. Mix samples with Bradford reagent and measure at 595 nm.
  3. Construct standard curve for protein quantification.

Calculations and Data Analysis

  • Calculate absorptivity from pNP solution absorption.
  • Determine enzyme activity rate, specific activity, recovery percentages, and fold purification.
  • Present findings in a purification summary table and appropriate graphs comparing enzyme activity across purification stages.

References

  • Garen, A. & Levinthal, C. (1960) Alkaline Phosphatase Purification Study.
  • Neu, H.C. & Heppel, L.A. (1965) Enzyme Release Techniques.
  • Schlesinger, M.J. & Barrett, K. (1965) Alkaline Phosphatase in E. coli Study.