Protein Purification I

Lecture Overview

Topic: Protein Purification I
Date: October 20, 2025
Required Reading: Lab manual, part 6, pp. 68-74
Recommended Video: Gel filtration video posted on CANVAS

Protein Purification Overview

Definition of Protein Purification

• Refers to strategies utilized to separate a specific protein from a mixture containing various proteins.

Purpose of Protein Purification

• Objectives:

  • Study protein activity and structure

  • Achieve purification in substantial quantities

Factors Influencing Purification Strategy

• Determined by the properties of the target protein, including:

  • Cellular localization (cytosolic vs. membrane-bound)

  • Size of the protein (large or small)

  • Interaction with other proteins or ligands in vivo

  • Hydrophobicity vs. hydrophilicity

Consideration of Potential Pitfalls

• Be aware of possible losses during purification, such as:

  • Loss of enzyme activity

  • Protein degradation

  • Decrease in yield

• Experimental design should address and mitigate these issues.

Sources of Proteins and Initial Steps in Purification

Sources of Proteins

• Common sources include:

  • Mammalian organs

  • Tissue culture cells

  • Yeast

  • Bacteria

  • Plants

Cellular Localization of Proteins

• Identify where the target protein is located in the cell:

  • Cytosol

  • Nucleus

  • Membrane-embedded structures

Techniques for Cell Disruption

• When targeting cytosolic proteins, use various methods to break open cells:

  • Grinding with a mortar and pestle

  • Freeze-thaw cycles (for nuclear extracts)

  • Blending

  • Press force application

  • Sonication

• For membrane-embedded proteins:

  • Isolate the insoluble/pelleted fraction and apply detergent to solubilize the protein.

Model Organism and Enzyme of Interest

Organism: Saccharomyces cerevisiae

• Commonly known as baker's yeast, utilized in winemaking, brewing, and baking since ancient times. It is a model organism in biological research.

Target Enzyme: Alcohol Dehydrogenase (ADH1)

• Important for fermentation; unlike humans, yeast ferments glucose to carbon dioxide (CO2) and ethanol.

• Biochemical Pathway of ADH1:

  • Pyruvate from glycolysis is converted to acetaldehyde and CO2.

  • Acetaldehyde is reduced to ethanol by ADH1.

Gene Information on ADH1

Database Reference: Saccharomyces Genome Database (SGD)

• Standard Name: ADH1

• Systematic Name: YOL086C

• Functional Description:

  • Alcohol dehydrogenase is involved in converting acetaldehyde to ethanol.

  • Active as homo- or heterotetramers and plays a crucial role in the glycolytic pathway.

  • Paralog: ADH5, arising from whole genome duplication.

Initial Protein Extraction Techniques

Homogenization

• Critical first step in the purification process, involving the controlled disruption of tissues and cells.

• Common procedures include:

  1. High-frequency sound (sonication)

  2. Use of mild detergent to disrupt membranes

  3. Application of high pressure to force cells through small openings

  4. Mechanical shearing using a rotating plunger against glass walls

Resulting Product: Homogenate

• The product of homogenization is referred to as a homogenate or extract, which comprises various cellular components such as enzymes, ribosomes, and metabolites.

Differential Centrifugation

Purpose

• To separate cellular components into distinct fractions based on size and density.

Procedure

• Centrifuge the cell homogenate:

  • Increasing centrifugal forces are used to separate components by size.

  • Each fraction can be assayed for the presence of the target protein.

Expected Results

• Example of typical centrifugation steps:

  • Crude extract: at 150,000 x g for 3 hours

  • Additional centrifugation steps at varied speeds (1000 x g for 10 min, 20,000 x g for 20 min, 80,000 x g for 60 min).

Centrifugation Techniques

Centrifuge Components

• Essential parts include a chamber for sedimenting material, a rotor (fixed-angle or swinging-arm), refrigeration capability, and a motor.

Outcomes of Centrifugation

1. Before Centrifugation: The homogenate contains all cell components.

2. After Centrifugation:

   • Supernatant: Contains smaller, less dense components.

   • Pellet: Contains larger, more dense components.

Protein Purification Techniques

Overview of Purification Techniques

• Salting out: Differential solubility method using high salt concentrations.

• Dialysis: Removes excess salt from solution.

• Column Chromatography:

  • Gel Filtration Chromatography: Separation based on size.

  • Ion-Exchange Chromatography: Separation based on charge.

  • Affinity Chromatography: Separation based on specific ligand interactions.

Importance of Multi-Property Exploitation

• The purification strategy typically exploits more than one property and involves a series of steps to enrich the target protein while measuring its concentration and activity.

Salting Out Technique

Methodology of Salting Out

1. Add ammonium sulfate to the protein solution at a concentration that induces precipitation of the target protein (e.g., 55% for the protein of interest).

2. Centrifuge to precipitate all proteins insoluble at this concentration.

3. Resuspend the pellet in a physiological buffer.

Example of Solubility Differences

• Fibrinogen precipitates out at 0.8 M concentration, while serum albumin precipitates at 2.4 M.

Dialysis in Purification

Concept of Dialysis

• Process whereby large molecules retained in a dialysis bag, and smaller molecules can diffuse out into the surrounding buffer.

• The dialysis buffer should be changed periodically to effectively remove small unwanted molecules.

Types of Dialysis Membranes

• Dialysis membranes come with different molecular weight cut-offs, allowing for selective diffusion of various sizes of compounds.

Column Chromatography

Definition and Functionality

• Utilizes a hollow tube packed with various matrices to separate proteins based on their physical and chemical properties.

• Types of chromatography include gel filtration (size), ion exchange (charge), and affinity (ligand affinity).

Key Definitions in Chromatography

• Mobile Phase: The moving solvent or effluent.

• Stationary Phase: The sorbent material through which separation occurs.

• Analytes: The molecules being analyzed or separated in the process.

Gel Filtration Chromatography

Process Description

• Also known as size exclusion chromatography, it separates proteins by size.

• Larger proteins exit the column first as they cannot enter the polymer matrix, while smaller proteins enter the beads, resulting in delayed elution.

Process Visual Representation

• A mixture of proteins is applied to a column filled with porous gel beads where differentiation occurs based on size.

Preparation for Practical Work in Enzyme Purification

Group Work Activities

Tasks for Group Members:

1. Pour and calibrate a gel filtration column (ref. pp. 77-79 of the manual).

2. Use the fraction collector to obtain 1 mL samples (ref. p. 79).

3. Homogenize yeast and separate soluble from insoluble materials (ref. p. 73).

4. Perform ammonium sulfate precipitation (ref. pp. 74-75).

Best Practices in Enzyme Purification

• Sample Handling:

  • Keep all samples on ice to reduce the risk of degradation and loss of activity.

  • Meticulously measure and record the volumes of generated samples.

• Sample Management:

  • Reserve all samples and assay them for enzyme activity and protein concentration in the following week.

  • Carefully mix and label all samples through the purification process.

Example of Quantification Protocol

Sample Data Table for a Hypothetical Purification Process