Purification or Removal of Calmodulin Binding Proteins: ATPases, Adenylate Cyclases, Protein Kinases, Phosphodiesterases, Neurotransmitters

Calmodulin Sepharose 4B

Calmodulin is a highly conserved regulatory protein found in all eukaryotic cells. This protein is involved in many cellular processes such as glycogen metabolism, cytoskeletal control, neurotransmission, phosphate activity, and control of NAD+/NADP+ ratios. Calmodulin Sepharose 4B provides a convenient method for the isolation of many of the calmodulin-binding proteins involved in these pathways.

Calmodulin binds proteins principally through interactions with hydrophobic sites on its surface. These sites are exposed after a conformational change induced by the action of Ca2+ on separate Ca2+-binding sites. The binding of enzymes can be enhanced if the enzyme substrate is present and enzyme-substrate-calmodulin-Ca2+ complexes are particularly stable.

Chromatography medium characteristics

The charactersistics of Calmodulin Sepharose 4B chromatography medium are shown in Table 3.7.

Table 3.7. Characteristics of Calmodulin Sepharose 4B chromatography medium

  Ligand density
(mg/ml)
Composition pH stability1 Average particle size (µm)
Calmodulin Sepharose 4B 0.9 to 1.3 Bovine testicular calmodulin coupled to Sepharose 4B by the CNBr method. Short term: 4 to 9 Long term: 4 to 9 90

1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. Long term refers  to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent  chromatographic performance.

Purification options

Calmodulin Sepharose 4B chromatography medium is available in chromatography media  packs for packing in columns, Table 3.8.

Table 3.8. Purification options for Calmodulin Sepharose 4B

  Binding capacity Maximum operating flow velocity(cm/h1) Comments
Calmodulin Sepharose 4B No data available 75 Supplied as a suspension ready for column packing.

1 See Appendix 4 to convert flow velocity (cm/h) to volumetric flow rate (ml/min). Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Performing a separation

Binding buffer: 50 mM Tris-HCl, 50 to 200 mM NaCl, 2 mM CaCl2, pH 7.5
Elution buffer: 50 mM Tris-HCl, 50 to 200 mM NaCl, 2 mM EGTA, pH 7.5
  1. Pack the column (see Appendix 3) and wash with at least 10 CV of binding buffer to remove preservative.
  2. Equilibrate the column with 10 CV of binding buffer.
  3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding).
  4. Wash with 5 to 10 CV of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm).
  5. Elute with 5 CV of elution buffer.

Remove proteases as quickly as possible from the sample as the calmodulin-binding sites on proteins are frequently very susceptible to protease action (see Purification or removal of serine proteases in this chapter).

Remove free calmodulin from the sample by HIC in the presence of Ca2+ on HiTrap® Phenyl FF (high sub) or by IEX on HiTrap® Q FF.

Since some nonspecific ionic interactions can occur, a low salt concentration (50 to 200 mM NaCl) is recommended to promote binding to the ligand while eliminating any nonspecific binding.

Use chelating agents to elute the proteins. Chelating agents strip Ca2+ from the calmodulin, reversing the conformational change that exposed the protein binding sites. Calcium ions can also be displaced by a high salt concentration, 1 M NaCl.

Cleaning

Alternative 1

Wash with 3 CV of 50 mM Tris-HCl, 1 M NaCl, 2 mM EGTA, pH 7.5 and re-equilibrate immediately with 5 to 10 CV of binding buffer.

Alternative 2

Wash with 3 CV of 100 mM ammonium carbonate buffer, 2 mM EGTA, pH 8.6 followed by 3 CV of 1 M NaCl, 2 mM CaCl2. Continue washing with 3 CV of 100 mM sodium acetate buffer, 2 mM CaCl2, pH 4.4 followed by 3 CV of binding buffer.

Remove severe contamination by washing with nonionic detergent such as 0.1% Tween™ 20 at 37°C for 1 min.

Chemical stability

Stable in all commonly used aqueous solutions.

Storage

Wash chromatography media and columns with 20% ethanol (use approximately 5 CV for packed media) and store at 4°C to 8°C.

Materials