Lecture 6 - Affinity Chromatography

Overview of Important Concepts and Terms

• Lactate Dehydrogenase (LDH)

  • Catalyzes the NAD(H)-dependent interconversion of lactate and pyruvate.

  • Reaction: ( L\text{-lactate} + NAD^+ \rightarrow Pyruvate + NADH + H^+ )

Cori Cycle

• Functioning Under Low Oxygen Conditions:

  • In muscles, pyruvate from glycolysis cannot enter the TCA cycle due to oxygen depletion.

  • Therefore, 2 electrons from NADH are transferred, and pyruvate is converted to lactate.

• Reversal in Liver:

  • Lactate can be converted back to pyruvate, thus allowing gluconeogenesis.

Structural Biology of LDH

• Crystal Structure of LDH:

  • Chicken LDH:

  • Resolution: 2.8 Å

  • Molecular weight: 40 kDa (monomer); 160 kDa (tetramer)

  • Drosophila LDH:

  • Resolution: 2.0 Å

  • Molecular weight: 35.5 kDa (monomer); 142 kDa (tetramer)

Affinity Chromatography Basics

• Definition:

  • A method that separates proteins based on their specific biological interactions.

• Purification Methods:

  • Separation based on specific interaction of a protein with binding partners, which can include:

  • Small molecules (ligands)

  • Enzyme-substrate affinity

  • Antibody-antigen interaction

• Advantages:

  • Rapid purification, often a single-step process

  • High specificity, reducing non-specific interactions prevalent in ion-exchange chromatography

• Disadvantages:

  • Potentially expensive resin (e.g., Cibacron Blue costs $750/100 ml)

  • Unstable ligand coupling can lead to loss of function

  • Low or high enzyme affinity to the resin affects binding

Basic Steps of Affinity Chromatography

1. Binding: The target protein binds to the affinity medium.

2. Washing: Removal of non-specifically bound proteins.

3. Elution: Release of the target protein using an excess of free ligand.

Types of Affinity Chromatography

• Mono-Specific Ligands:

  • Substrate analogues (for enzymes)

  • Cofactor analogues (for enzymes)

  • Hormones (for receptors)

  • Antibodies (interactions with any protein)

• Group-Specific (Pseudo Affinity Chromatography):

  • Dyes (for nucleotide binding proteins)

  • Calmodulin (for Ca2+-binding proteins)

  • Lectins or concanavalin A (for glycoproteins)

  • Polysaccharides (growth factors)

  • Heparin (for growth factors)

Tagging of Recombinant Proteins

• Epitope Tagging:

  • Involves creating a specific target sequence for antibody binding.

• His-Tagging:

  • Utilizes immobilized metal ion affinity chromatography (IMAC) for purification.

Examples of Ligands and Their Target Proteins

• Ligands:

  • 5’ AMP, ATP

  • NAD, NADP

• Target Proteins:

  • Dehydrogenases for both ligands and others like Protein A and G, lectins, histones, etc.

• Additional Examples:

  • Cibacron Blue (bindings with antibodies, polysaccharides, and proteins).

Structural Models

• NADH and Cibacron Blue:

  • LDH is capable of binding both NADH and Cibacron Blue, indicating structural compatibility and potential interaction sites.

Practical Application: Purification of Chicken LDH

• Procedure:

  • Initial desalting using G-25 column to remove ammonium sulfate

  • Affinity chromatography:

    • Running desalted protein over Cibacron Blue column leads to LDH binding.

    • Washing with NAD+ removes unbound proteins.

    • Elution with NADH to retrieve LDH.

    • Reason for NAD+ wash and NADH elution is rooted in the reversible conversion of lactate and pyruvate.

Epitope Tag Characteristics

• Epitopes:

  • Typically 8-12 amino acids long

  • Highly charged to enhance binding specificity.

• Commercial Availability:

  • Monoclonal antibodies available that specifically bind to epitope tags with minimal cross-reactions.

• Expression Vectors:

  • Numerous specialized vectors with epitope tags commercially available.

• Sensitivity Increase:

  • Incorporating multiple epitope tags improves sensitivity in detection methods like immunochemistry.

Flag Epitope Tag

• Sequence:

  • Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, fused to the N-terminus of a target protein.

• Biological Function:

  • Cleaved by enterokinase protease, restoring native protein structure

• Commercial Monoclonal Antibodies:

  • Available for binding to the Flag-Tag and some require Ca2+ for optimal binding.

Epitope Tagging Process

1. Creation of Target:

   • Fusing the DNA sequence of the epitope to the DNA encoding the target protein.

2. Expression:

   • Introducing the DNA construct into organisms (bacteria, yeast, insect, or mammalian cells).

3. Localization and Interaction:

   • Localizing tagged protein for analysis of function and interactions.

4. Functional State Analysis:

   • Assaying tagged proteins for their activities, interactions, and phosphorylation states.

5. Characterization of Unknowns:

   • Analyzing proteins with unknown functions through expression studies and interaction assays.

Specific Activity

• Definition: Criterion of purity defined as enzyme activity per milligram of protein.

• Comparison:

  • When two samples have the same total activity but vary in protein content, specific activity helps indicate purity:

  • High specific activity means more active enzyme present relative to the total protein amount.

Protein Purification Table Example

Class Objectives/Questions for Lecture 6

• Understand the principle of affinity chromatography.

• Analyze advantages and disadvantages of affinity chromatography.

• List three different kinds of affinity chromatography methods.

• Describe basic steps involved in the affinity chromatography process.

• Discuss the covalent attachment of molecules during Flag-tag purification.

• Differentiate between loading/washing and elution buffers.

• Explain the use of cibacron blue as an affinity ligand for LDH purification.

• Consider potential applications of fusing affinity tags to recombinant proteins.

• Examine changes in total amount and specific activity across multiple purification steps.