Protein Purification

Isolation of Proteins from Cells

  • Many different proteins exist within one cell.
  • Techniques are employed to eliminate contaminants and obtain a pure sample of the protein of interest.
  • Pre-purification step involves releasing the protein from cells and subcellular organelles.

Example of a Protein Purification Scheme: Purification of the Enzyme Xanthine Dehydrogenase from a Fungus

  • Table 5.1 Breakdown:
    • Crude extract:
      • Volume: 3,800 mL
      • Total Protein: 22,800 mg
      • Activity: 2,460
      • Specific Activity: 0.108
      • Percent Recovery: 100%
    • Salt precipitate:
      • Volume: 165 mL
      • Total Protein: 2,800 mg
      • Activity: 1,190
      • Specific Activity: 0.425
      • Percent Recovery: 48%
    • Ion-exchange chromatography:
      • Volume: 65 mL
      • Total Protein: 100 mg
      • Activity: 720
      • Specific Activity: 7.2
      • Percent Recovery: 29%
    • Molecular-sieve chromatography:
      • Volume: 40 mL
      • Total Protein: 14.5 mg
      • Activity: 555
      • Specific Activity: 38.3
      • Percent Recovery: 23%
    • Immunoaffinity chromatography:
      • Volume: 6 mL
      • Total Protein: 1.8 mg
      • Activity: 275
      • Specific Activity: 152.108
      • Percent Recovery: 11%

Techniques for Protein Isolation

  1. Sonication:
    • Sound waves break open cells.
  2. Freeze/Thaw:
    • Ruptures cells through cycles of freezing and thawing; detergents may be required for detaching membrane-bound proteins.
  3. Homogenizer:
    • A thick-walled test tube with a tight-fitting plunger is used to squeeze homogenate around the plunger, breaking open cells while leaving most organelles intact.
  4. Blender (in buffer):
    • Breaks open cells and subcellular organelles such as mitochondria and endoplasmic reticulum.

Isolation and Purification of Proteins from Cells

  • After lysing the cell, a complex mixture containing proteins, organelles, and unlysed cells, known as a "soup," is obtained.

Differential Centrifugation

  • A process where ruptured cells are centrifuged multiple times, increasing gravitational forces each time.
  • Varying speeds of spin result in the separation of particles based on their density.

Ammonium Sulfate Precipitation

  • Commonly employed for separation based on solubility (salting out).
  • Mechanism:
    • Ammonium sulfate is added to soluble protein solutions, which removes some water from proteins, creating ionic bonds with salts and causing loss of the solvation shell.
    • Reduced hydration increases protein interactions via hydrophobic interactions leading to protein precipitation.
  • Process:
    • Proteins are centrifuged down; increasing amounts of ammonium sulfate are added to the supernatant to precipitate different protein sets.
    • The target protein's precipitate is collected through centrifugation.

Column Chromatography

  • Definition: Originating from the Greek words "chroma" (color) and "graphein" (to write).
  • A small concentrated sample is applied to a column and diluted with an eluent. Components travel at distinct rates and are collected in fractions based on mobility differences.
    • Stationary Phase: Material packed in the column.
    • Mobile Phase: Eluent flows through the column, carrying the sample while interacting with stationary phase material.
    • Components with stronger interactions with stationary phase will be transported more slowly.

Column Chromatography Composition

  • The stationary phase consists of cross-linked gel particles, typically in bead form, including:
    1. Carbohydrate polymers (e.g., dextran (Sephadex) or agarose (Sepharose)).
    2. Polyacrylamide (Bio-Gel).
    • The cross-linked structure produces varying pore sizes based on the extent of cross-linking.

Size-Exclusion Chromatography (Gel-Filtration)

  • Function: Separates molecules based on size.
    • Larger molecules are excluded from gel pores and elute faster; smaller molecules have access to include the inner areas of the beads and elute slower.
  • Molecular Weight Estimation: Comparing elution volumes against size standards allows for molecular weight estimation.
    • Variables:
      • $V_0$ (void volume) signifies volume for excluded molecules.
      • $V_e$ (elution volume) signifies volume for molecules that access the bead interior.

Affinity Chromatography

  • Methodology: Separates proteins based on binding properties.
    • The polymer in the stationary phase is covalently linked to a specific ligand that binds the target protein.
    • Proteins that do not bind are eluted using a buffer, while the desired protein remains fixed in the column.
  • Elution of Target Protein: Achieved by adding a high concentration of soluble ligand, disrupting the binding between protein and stationary phase or by altering pH/ionic strength.

Ion-Exchange Chromatography

  • Ligands: Utilizes cation-exchange resins (negatively charged) and anion-exchange resins (positively charged).
  • Operation:
    1. The column is equilibrated with a suitable pH and ionic strength buffer.
    2. Proteins flow through and those with net positive charges stick to the negatively charged column.
    3. The eluent buffer's pH is altered or Na+ concentration is increased to elute bound proteins.

Performance Metrics in Protein Purification

  • Percent Recovery: Measures the amount of protein retained at each purification step, which decreases throughout purification.
  • Specific Activity: Expressed in units of activity/mg protein, reflecting protein purity at each stage; should increase with successful purification.

High-Performance Liquid Chromatography (HPLC)

  • A sophisticated chromatography method providing rapid and efficient purifications.
  • Types:
    • Reverse phase HPLC utilizes a nonpolar stationary phase with a polar mobile phase.
    • Allows for high-resolution separation with shorter timeframes than standard columns.

Example 5.1 - Protein Purification

  • The enzyme lactate dehydrogenase catalyzes the transformation between lactic acid and NAD+, producing NADH and pyruvate.
  • Monitoring Purification: Achieved by comparing specific activities at various steps; higher specific activity signifies purer samples.

Example 5.1 Results

  • Identification of effective purification steps can be assessed based on the fold purification calculations, which compares specific activity at each stage.
  • Notably, the affinity chromatography step yielded a significant fold purification of 12.4.
  • The impact of the gel filtration chromatography step resulted in a substantial loss (over 90%) of activity, establishing it as the most costly phase of purification.

Electrophoresis

  • A method for separating molecules according to their charge-to-size ratios.
  • Macromolecule mobility varies based on charge, shape, and size.
    • Media include agarose for nucleic acids and polyacrylamide predominantly for proteins.
    • Acrylamide provides more resistance to larger molecules compared to smaller counterparts.

Electrophoresis Techniques

  • Agarose and acrylamide are the common supporting media for gel electrophoresis;
  • SDS-PAGE Technique:
    • Samples treated with sodium dodecyl sulfate (SDS) denature proteins, breaking non-covalent interactions,
    • Resulting in negatively charged proteins that separate based on size: smaller proteins migrate faster in the gel, and the position of each band reflects specific proteins present.
    • Can also be utilized for estimating protein molecular weights when compared with standards.

Isoelectric Focusing

  • This technique enables separation based on isoelectric points.
    • A gel with a pH gradient is created alongside an electric field gradient.
    • Proteins migrate through the gel until they reach a point where they possess no charge (pI), at which they cease moving.

Determining the Primary Structure of a Protein

  • Tasks include:
    • Identifying amino acids present.
    • Determining N-terminal and C-terminal residues.
    • Fragmenting proteins into smaller pieces for subsequent sequence determination.

Edman Degradation

  • A methodology for assessing the amino acid sequence of peptides and proteins.
    • Efficiently deduces sequences containing 10 to 40 amino acids within approximately 30 minutes.
    • The need for identifying N-terminal and C-terminal ends has diminished due to its efficiency.

Peptide Cleavage Enzymes

  • Proteolytic Enzymes:
    • Trypsin: Cleaves at basic amino acid residues; C-terminal ends consist of charged residues.
    • Chymotrypsin: Targets amide bonds next to aromatic residues; aromatic residues appear at the peptide's C-terminal ends.
    • Chemical Reagent: Cyanogen bromide cleaves at methionine residues within proteins.

Protein Detection Techniques

  • ELISA: Measured reactions between proteins and antibodies, widely used for protein detection.
  • Western Blot: Proteins are first separated by gel electrophoresis. They are then transferred to nitrocellulose membranes for analysis.
  • Protein Chips: Small plates hosting thousands of proteins, also known as microarrays.

Proteomics

  • The comprehensive study of all the proteins within a cell, termed the proteome.
  • Types of Proteomics Studies:
    1. Structural: Provides detailed protein structure analyses.
    2. Expression: Assesses protein expression levels under various conditions.
    3. Interaction: Investigates protein interactions with other molecules.