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Selective Precipitation of Proteins

Rex E. Lovrien1,  Daumantas Matulis1

1University of Minnesota, St. Paul, Minnesota

Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 4.5
DOI: 
10.1002/0471140864.ps0405s07
Online Posting Date: 
May, 2001
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Daumantas Matulis

Abstract

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol 1: Selective Precipitation by Salting Out
  • Alternate Protocol 1: Selective Precipitation by Stepwise Salting Out
  • Basic Protocol 2: Selective Precipitation by Isoionic Precipitation: Column Method
  • Alternate Protocol 2: Selective Precipitation by Isoionic Precipitation: Dialysis Method
  • Basic Protocol 3: Selective Precipitation Using a Two-Carbon (C2) Organic Cosolvent
  • Basic Protocol 4: Selective Precipitation Using C4 and C5 Organic Cosolvents
  • Basic Protocol 5: Selective Precipitation Using Protein Exclusion and Crowding Agents and Osmolytes
  • Basic Protocol 6: Selective Precipitation Using Synthetic and Semisynthetic Polyelectrolytes
  • Basic Protocol 7: Selective Precipitation Using Metallic and Polyphenolic Heteropolyanions
  • Basic Protocol 8: Selective Precipitation Using Hydrophobic Ion Pairing (HIP) Entanglement Ligands
  • Basic Protocol 9: Selective Precipitation by Matrix-Stacking Ligand Coprecipitation
  • Basic Protocol 10: Selective Precipitation Using Di- and Trivalent Metal Cation Precipitants
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Selective Precipitation by Salting Out

 Materials
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • Appropriate pH buffer
  • Ammonium sulfate
  • Additional reagents and equipment for dialysis (unit 4.4 & appendix 3B)

Basic Protocol 2: Selective Precipitation by Isoionic Precipitation: Column Method

 Materials
  • Cation exchanger: Amberlite IR-120H+ resin (Sigma or Aldrich)
  • Anion exchanger: Amberlite IRA-400Cl– resin (Sigma or Aldrich)
  • ~0.5 and 1 M NaOH or KOH
  • 0.5 to 1 M HCl
  • 0.2 M acetic acid
  • 0.2 M ammonium hydroxide
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • Plastic beakers
  • Large Buchner funnel
  • Whatman no. 1 filter paper
  • Chromatography column of appropriate size

Basic Protocol 3: Selective Precipitation Using a Two-Carbon (C2) Organic Cosolvent

 Materials
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • Buffering system
  • Ethanol
  • Acetone
  • Centrifuge

Basic Protocol 4: Selective Precipitation Using C4 and C5 Organic Cosolvents

 Materials
  • Ammonium sulfate
  • Buffering system
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • C4 organic cosolvent (e.g., t-butanol, analytical grade)

Basic Protocol 5: Selective Precipitation Using Protein Exclusion and Crowding Agents and Osmolytes

 Materials
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • Appropriate pH buffer
  • Precipitating agent: neutral polymer or osmolyte

Basic Protocol 6: Selective Precipitation Using Synthetic and Semisynthetic Polyelectrolytes

 Materials
  • Water-soluble polyelectrolytes (Aldrich or Sigma)
  • Crude protein solution of interest
  • Test tubes
  • Glass or plastic cuvettes
  • Spectrophotometer

Basic Protocol 9: Selective Precipitation by Matrix-Stacking Ligand Coprecipitation

 Materials
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • Potential ligands (Fig. 4.5.10; also see Aldrich catalog)
  • Appropriate buffers
  • Dowex-1Cl– resin
  • Small test tubes or 2- to 3-ml plastic microcentrifuge tubes

Basic Protocol 10: Selective Precipitation Using Di- and Trivalent Metal Cation Precipitants

 Materials
  • Analytical Reagent (AR)-grade metal salts (generally as chloride or nitrate)
  • Crude protein solution of interest, particle-free (see discussion of Clarification in Strategic Planning)
  • Appropriate buffer(s)
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Figures

  • Figure 4.5.1
    First steps and options for clearing particulates—e.g., haze and cell walls—from crudes before precipitating proteins. Limited amounts of precipitating agents may be effective in removal of haze from proteins. However precipitating agents for haze removal should not reach lower threshold concentrations for starting precipitation of sought-for proteins. Abbreviation: MWCO, molecular-weight cutoff.

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  • Figure 4.5.2
    Two salting out protocols, single-stage and two-stage, starting from a particle-free aqueous crude preparation (e.g., cell or tissue extract or centrifuged fermentation broth).

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  • Figure 4.5.3
    Salting out—small-scale investigation. Maximum ammonium sulfate concentration ~4 M. Development of precipitate depends on the protein and unwanted materials which sometimes precipitate out in samples or fractions, apart from the sought-for protein. Other adjustable parameters are pH, temperature, and concentration of starting sample.

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  • Figure 4.5.4
    Deionization column, Dintzis design, described in Edsall and Wyman (1958). Passage through this column renders the protein salt-free and automatically adjusted to the protein's isoionic pH. This column is used for proteins that remain soluble at their isoionic point.

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  • Figure 4.5.5
    Deionization by dialysis. When proteins are insoluble and precipitate at their isoionic point, deionization is accomplished by placing dialysis tubing containing the protein sample in a slurry of mixed-bed resin exchangers (i.e., a mixture of IRA-400OH– and IR-120H+) and incubating with rocking. Proteins insoluble at their isoionic point precipitate inside the bag. The mixed-bed exchange resins remove free salts, forcing the protein to its isoionic pH. This method is considerably slower than the column method.

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  • Figure 4.5.6
    Procedure for precipitation of proteins from aqueous solutions using t-butanol. This figure applies to both large-scale precipitation and small-scale pilot experiments. The t-butanol layer develops when adequate salt is present in the aqueous solution. Both thet-butanol layer and the aqueous lower phase act as extraction solvents as well as partitioning systems.

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  • Figure 4.5.7
    Principal steps in protein coprecipitation with polyelectrolytes. “Metered” addition is slow addition (with good stirring) of aliquots of polyelectrolyte stock to the protein sample near the threshold of precipitate formation as seen by eye or spectrophotometric turbidity determination.

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  • Figure 4.5.8
    HIP coprecipitate. Strong anions (sulfate or sulfonate-bearing C10–C14 alkane tails, e.g., dodecyl sulfate) bind to protein cationic sites forming ligand-protein complexes. Complexes draw together via tail-tail hydrophobic entanglement, aggregate, and coprecipitate. At maximum coprecipitating efficiency with ~10–5 M protein, the stoichiometric ratio of bound ligand anions () is to cation side chains (math) in the protein molecule is close to 1:1.

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  • Figure 4.5.9
    Molecular structure-function basis for matrix-stacking ligand coprecipitation of proteins. Protein molecules initially soluble in water and bearing a positive net charge attract strong anion (sulfonate) ligands. Such ligands associate via organic tail/organic tail stacking interaction, reinforced by alkane R-groups. Ligand/protein complexes are thus drawn together and coprecipitate out of solution.

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  • Figure 4.5.10
    Anionic ligands synthesized with reinforcing alkane groups (Little Rock Orange and Jenelle's Orange) are very strong protein coprecipitants. Commercially available dye anions—Orange II, Crocein Orange G, and Orange ROF (available from Aldrich)—frequently are also efficient coprecipitating ligands for cationic proteins in pH ranges 2 to 4 units below the isoionic pH of the protein.

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  • Figure 4.5.11
    Sets of small test tubes or plastic microcentrifuge tubes, 2 to 3 ml in volume, may be used to to explore optimum conditions for matrix-ligand/protein mutual coprecipitation by varying pairs of important parameters. Five principal variables determining coprecipitation are: kind of ligand; ligand-protein ratio (y; often between 2 and 20); initial pH; temperature; and concentrations of auxiliary coprecipitating agents such as Zn2+ (10–3 to 10–4 M).

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  • Figure 4.5.12
    Calibration plot for zinc ion spectrophotometric analysis using pyridylazoresorcinol reagent.

    View Image

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