2.2 Separation and Purification of Biomolecules

Overview

  • Focus on physical biochemistry techniques for biomolecule separation and purification.

Why Purify a Protein?

  • Establish Biological Activity: Confirm unique protein activities like enzymatic or signaling capabilities.

  • Biochemical Investigations: Utilize purified proteins as tools for in-depth studies.

  • Study Protein Properties: Analyze mass, isoelectric point (pI), specific activity, etc.

Protein Purification Objectives

  • Main Goal: Isolate a specific protein from other cellular components.

    • Target protein may constitute between 0.001% - 20% of total protein.

  • Components Present:

    • Nucleic acids, carbohydrates, lipids, small molecules.

    • Proteins can exist in various states and locations (e.g., soluble, insoluble, membrane-bound).

Protein Purification Purposes

  1. Preparative Purification:

    • Generate large quantities of purified proteins for commercial uses, such as enzymes and biopharmaceuticals.

  2. Analytical Purification:

    • Produce small amounts of proteins for research, identification, and characterization.

Purification Strategy

  • Approach: Aim for minimal steps from organism to pure protein, preserving activity.

  • Individualized Process: Tailored for specific protein characteristics.

  • Common Techniques: Fractionate crude extracts to isolate the target protein in the appropriate fraction (pellet or supernatant).

  • Progress Monitoring: Use functional assays to track purification success.

Recognizing Target Protein

  • Monitoring Levels: Continuously assess the target protein against contaminating materials.

  • Assay Requirement: A viable assay that targets unique protein properties, usually assessing enzymatic activity.

  • Unit Definition for Activity: One unit quantifies catalytic activity related to turnover rates or binding amounts.

Lactate Dehydrogenase Assay

  • Assesses activity by measuring NADH absorbance at 340 nm.

  • As unit activity increases, protein purity typically increases.

Separation Processes for Protein Fractionation

Separation Process

Basis of Separation

Precipitation

Ammonium sulfate, polyethyleneimine (size, charge)

Chromatography

SEC, IEX, HIC, DNA affinity (size, charge, hydrophobicity)

Electrophoresis

PAGE, IEF (charge, size)

Centrifugation

Sucrose gradient (size, density)

Ultrafiltration

Size

Other Considerations

  • pH Stability: Ensure buffer solutions maintain protein stability.

  • Protease Inhibition: Remove proteases with pH or temperature adjustments and by using inhibitors.

  • Temperature Sensitivity: Most proteins denature above 25°C; some may be cold-labile.

Protein Sources for Purification

  • Traditional Sources: Bacteria, animal tissues, plant tissues.

  • Recombinant Proteins: Produced using overexpression vectors in systems like E. coli.

  • In Vitro Systems: Transcription/translation systems for protein synthesis.

Considerations for Protein Sources

  • Assess the difficulty in obtaining, growing, and handling the source, potential proteolytic activity, and presence of inhibitors.

Steps in Protein Purification

  1. Extraction: Homogenization followed by differential centrifugation.

  2. Protein Enrichment: Techniques like salt precipitation and isoelectric precipitation.

  3. Purification: Column chromatography followed by yield and activity determination.

Protein Purification Approaches

  • Low vs. High Abundance Proteins: Different strategies may be required based on protein levels within the source.

Protein Solubilization Methods

  • Cell Lysis: Mechanical methods based on tissue characteristics and protein location (e.g., osmotic lysis, lysozyme treatment).

  • Mechanical Disruption: Grinding, freezing/thawing, sonication, and homogenization.

Differential Centrifugation Steps

  • Sequence of centrifugal force applications to separate cellular components based on size and density (e.g., nuclei, organelles, soluble fractions).

Salting Out Technique

  • Principle: Different proteins precipitate at varying salt concentrations (ammonium sulfate commonly used).

  • Dialysis: Technique to remove excess salt post-salting out purification.

Kosmotropic vs. Chaotropic Agents

  • Kosmotropes, like ammonium sulfate, stabilize proteins, while chaotropes, like urea, can denature proteins.

Other Enrichment Methods

  • Isoelectric Precipitation: Adjusting pH to precipitate proteins at their isoelectric point.

  • Solvent Precipitation: Using organic solvents to precipitate proteins.

Dialysis Procedure

  • Function: Semi-permeable membranes allow for the removal of small molecules while retaining larger proteins.

Recombinant Protein Production

  • Reasons: To study function, analyze physical properties, and produce proteins for industrial or therapeutic use.

Steps in Recombinant Protein Purification

  1. Design and transform expression plasmid.

  2. Grow positive clones and induce expression.

  3. Lyse cells and isolate protein-containing fractions.

  4. Purify through column chromatography and assess purity via SDS-PAGE.

Protein Pharmaceuticals

  • Recombinant methods are preferred due to cost-effectiveness, safety, and higher yields compared to natural sources.

Vector Selection for Recombinant Expression

  • Ensure compatibility with the host and strong promoter sequences; affinity tags facilitate purification.

Key Vector Components

  • Origin of replication, selectable markers, and multi-cloning sites (MCS) are critical in plasmid design.

Successes in Bacterial Systems

  • Advantages: Quick growth and high yields.

  • Disadvantages: Difficulty with large proteins; absence of post-translational modifications.

Alternatives to Bacterial Production

  • Options include yeast, baculovirus, mammalian cell culture, and even plant systems.

Pichia pastoris as a Host

  • Methylotrophic yeast capable of high expression yields, especially for protein involving glycosylation.

Baculovirus Expression Systems

  • Advantages include proper post-translational modifications; however, set-up is time-consuming.

Mammalian Cell Line Expression

  • Usually utilizes vectors derived from viral genomes, often using CHO cells for production.

Protein Characterization Steps

  1. Determine molecular weight (MW) and isoelectric point (pI).

  2. Assess amino acid composition and sequence.

  3. Analyze three-dimensional structure and interactions.

  4. Establish function and post-translational modifications.

Protein Purification Parameters

  • Yield: Activity recovered vs. starting levels.

  • Purification Level: Increased purity quotient compared to initial extract.

Common Problems in Purification

  • Inactive proteins often arise due to improper folding; proper conditions are essential for correct protein maturation.