Protein Purification and Analysis Techniques
Ion Exchange Chromatography: Salt Concentration
Ion exchange chromatography separates proteins based on their charge. The process involves:
Initial Wash: Using a low salt concentration to remove weakly binding proteins (competitive proteins).
Gradient Ramp: Gradually increasing the salt concentration to elute proteins based on their binding affinity.
Elution:
A shallow gradient is crucial to separate proteins with similar binding affinities, ensuring contaminants that bind more strongly remain on the column.
The target protein elutes at a specific salt concentration, indicated by an arrow on a chromatogram.
Column Cleaning: Maintaining a high salt concentration at the end to remove all remaining proteins and clean the column.
This technique relies on the charge properties of proteins.
SDS-PAGE and Protein Size Determination
Separation based on size uses SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). This technique, while not ideal for purification, is valuable for analytical purposes to determine protein size.
Predicting Protein Size: Protein size can be initially estimated from the nucleotide sequence and codon usage. However, post-translational modifications and oligomerization can affect the final size.
Quaternary Structure: The protein could form dimers, trimers, tetramers, or even octamers, leading to a larger complex than predicted from the gene sequence.
Gel filtration chromatography helps determine the oligomeric state of a protein.
Gel Filtration Chromatography (Size Exclusion Chromatography)
Gel filtration separates proteins based on their size using a resin with pores.
Resin Structure: The resin contains pores and a tortuous matrix of polymers within the beads.
Separation Principle:
Small Proteins: Access the pores, navigate a tortuous path, and elute later due to their increased path length.
Large Proteins: Cannot enter the pores and travel through the crevices between resin beads, eluting first.
Illustration: A resin bead with small pores is depicted, with small proteins entering the pores and large proteins flowing around the beads.
Counterintuitive Behavior: Larger proteins elute before smaller ones, which is opposite to what is expected in most other separation techniques.
Buffer Usage: This method requires a significant amount of buffer, leading to protein dilution.
Analytical Measurement: Useful for determining if a protein is a monomer, dimer, or larger oligomer.
Resin beads are available with different pore sizes to accommodate various protein size ranges, typically from 10,000 g/mol to 500,000-1,000,000 g/mol. This size range is much larger than typical small molecules, which are in the range of tens or hundreds of Daltons.
The size of pores can be customized based on the expected size of the protein of interest.
Buffer Control and Fraction Collection
During ion exchange chromatography, controlling buffer conditions is crucial, including:
Concentration Control: Buffer control affects the final protein concentration.
Fraction Collection: UV absorption monitors general protein elution. Knowing the expected elution point allows for aggressive fraction collection.
Sacrificing Peak Shoulders: Discarding the beginning and end fractions of the elution peak to collect only the most concentrated and pure fractions.
Gradient Control: The steepness of the salt gradient affects the speed of elution and the degree of purification.
Steeper Gradients: Result in faster elution but potentially reduce separation from closely binding contaminants.
Protein Storage and Buffer Optimization
Storage Conditions: Proteins are best stored in a concentrated form and in a buffer that maintains stability.
pH Considerations: Protein stability depends on the number of positive and negative charged side chains, with each protein having an isoelectric point.
Isoelectric Point: The pH at which the protein has no net charge, often leading to precipitation.
Optimal pH: Varies for each protein.
Salt Concentration: Some proteins require higher salt concentrations for stability.
Protease Inhibitors: Added to prevent proteases from degrading the purified protein.
Dialysis for Buffer Exchange
Dialysis is used to change the buffer composition after purification, as the initial purification buffer (e.g., high salt from ion exchange or large volume from gel filtration) may not be suitable for long-term storage.
Dialysis Bags: Proteins are placed in a dialysis bag with pores of a specific size.
Pore Size:
Large Proteins: Larger pore sizes are used.
Small Proteins: Smaller pore sizes are required to prevent protein loss.
The primary purpose of dialysis is to exchange the buffer, salt concentration, and remove contaminants. Small molecules freely move in and out of the bag until equilibrium is reached.
Procedure: Typically performed overnight, allowing the protein solution to equilibrate with the desired buffer.
Multiple Exchanges: Often, a second dialysis step is performed to ensure thorough equilibration.
Cell Lysis and Protein Extraction
Before purification, cells must be lysed to release the protein.
Methods: Sonication (using sound waves) disrupts the cell membrane.
E. coli: Is commonly used for protein expression due to its rapid growth and high protein production capacity.
Induction: Protein expression is induced using methods such as the lac promoter or T7 promoter.
Workflow: Grow E. coli, induce protein expression, lyse cells via sonication, purify using ion exchange chromatography, and perform dialysis.
Multiple dialysis exchanges are typically required, with an overnight exchange followed by one or more shorter exchanges (e.g., 1-2 hours) to ensure equilibrium.
Affinity Chromatography: Histidine Tag Purification
Affinity chromatography provides the highest purity but is more expensive.
Nickel Column: Uses a nickel column that binds to a polyhistidine tag (e.g., six histidine repeats) engineered onto the target protein.
Tagging: The histidine tag is fused to the protein's gene, resulting in a protein with the tag.
Procedure:
Lysate Application: The cell lysate is passed through the nickel column.
Binding: The His-tagged protein binds to the nickel resin.
Washing: Unbound proteins are washed away.
Elution: Imidazole (a histidine analog) is used to compete with the His-tag, eluting the protein.
To remove the imidazole after elution:
Dialysis: Is used to exchange the buffer and remove the high concentration of imidazole.
Cleavage of Histidine Tag
Protease Site: A protease recognition site is engineered between the His-tag and the protein.
Procedure:
Purification: The protein is first purified using the nickel column.
Cleavage: The protease cleaves the tag.
Second Affinity Step: The protein solution is passed through the nickel column again.
Outcome:
Target Protein: Flows through because it no longer has the His-tag.
Protease and Tag: Bind to the column if the protease also has a His-tag.
This two-step process can achieve very high purity, approaching 99% for well-expressed proteins.
Alternative Affinity Methods
Ligand Binding: Other proteins can be purified by using resins with ligands that selectively bind the target protein.
Considerations for Histidine Tagging
Protein Folding: Adding a tag can sometimes affect protein folding and function.
N or C Terminus: Tags are typically added to either the N-terminus or C-terminus.
Trial and Error: It is common to test both N- and C-terminal tags to determine which works best for a given protein.
Location of Tag: The success of tagging depends on the three-dimensional structure of the protein; exposed tags are more likely to be functional. AlphaFold can be used to predict the likelihood of successful tagging.
Tag Addition during PCR
PCR Primers: Primers with 5' extensions (up to 40-45 base pairs) can be used to add the His-tag sequence during PCR.
Analyzing Proteins: SDS-PAGE and Western Blotting
SDS-PAGE
SDS-PAGE separates proteins based on size using a gel matrix and SDS (sodium dodecyl sulfate), a detergent that denatures proteins and provides a uniform charge per size.
SDS Structure: SDS has a hydrophobic tail (12 carbons) and a negatively charged sulfate head group.
Mechanism: SDS unfolds proteins by interacting with hydrophobic amino acids and coats the protein with negative charges.
Illustration of SDS interacting with a protein, unfolding and coating it with negative charges.
Procedure:
Sample Preparation: Proteins are mixed with SDS.
Gel Electrophoresis: The sample is loaded onto a gel, and an electric field is applied.
Migration: Proteins migrate through the gel based on size, with smaller proteins moving faster.
Staining: The gel is stained with Coomassie blue to visualize the protein bands.
Coomassie Blue: Binds to charged amino acids, allowing visualization of protein bands.
Gel Matrix: The gel provides a tortuous path for protein migration.
Size Separation: The smallest proteins reach the bottom, while the largest remain at the top.
By comparing with the SDS-PAGE, in gel filtration the larger proteins elute faster because those resins have big pores. For the SDS-PAGE, there is no such thing as the pores, so small proteins wiggle their way faster than large ones and go to the bottom of the gel faster.
Western Blotting (Immunoblotting)
Western blotting is used to selectively visualize a specific protein using antibodies.
Procedure:
Transfer: Proteins from the SDS-PAGE gel are transferred to a nitrocellulose membrane using an electric field.
Blocking: The membrane is blocked with a protein-rich solution (e.g., milk powder) to prevent non-specific antibody binding.
Primary Antibody Incubation: The membrane is incubated with a primary antibody that specifically recognizes the target protein.
Washing: The membrane is washed to remove unbound antibody.
Secondary Antibody Incubation: The membrane is incubated with a secondary antibody that recognizes the primary antibody and is conjugated to an enzyme.
Washing: The membrane is washed to remove unbound secondary antibody.
Detection: The enzyme substrate is added, producing a detectable signal (e.g., chemiluminescence) at the location of the target protein.
Antibodies: The primary antibody selectively binds the protein of interest, then the secondary antibody is conjugated to an enzyme, catalyzing a reaction. To selectively visualize beta proteins.
Illustration showing the steps of a western blot: transfer, blocking, primary antibody binding, secondary antibody binding, and detection.
Benefits:
Selectivity: The technique allows for the selective visualization of a single protein in a complex mixture.
Size Determination: It provides information about the size of the protein.
Relative Quantification: It allows for relative quantification of the protein.
The western blot does not depend on the activity of the proteins like the Elijah method, but does depend on having an antibody to work with. Usually those antibodies are easy to order.