Protein purification is essential for studying individual proteins in detail, requiring relatively large amounts (milligrams) of pure proteins.
The main goal of protein purification is to obtain a pure, undenatured protein from a complex mixture that may contain hundreds or thousands of different proteins.
Techniques for Protein Purification
Various techniques are employed to separate proteins based on their distinct properties. These methods mainly include chromatography, categorized according to the following parameters:
Size: Separation based on the physical size of protein molecules.
Charge: Separation based on the electrostatic charge characteristics of proteins.
Hydrophobicity: Separation based on the hydrophobic (water-repelling) qualities of protein residues.
Specific Binding: Separation based on specific interactions between proteins and other molecules.
An essential component of any purification process is an assay to track the proteins through the purification stages.
Chromatography Overview
Common Steps in Chromatography:
Start with a crude extract of proteins.
Utilize a chromatography column filled with one of three types of resins:
Ion-exchange resin
Gel-exclusion resin
Affinity resin
The proteins are then collected after being subjected to specific conditions that may involve elution techniques involving increasing concentrations of a specific solution or competitive agents.
Types of Chromatography
1. Separation Based on Charge: Ion Exchange Chromatography
In ion exchange chromatography, the method capitalizes on the charge interactions:
Positively charged proteins adhere to negatively charged beads (resin).
Negatively charged proteins do not adhere and pass through the column.
Elution of positively charged proteins is achieved by increasing the salt concentration in the elution solution.
2. Separation Based on Size: Size Exclusion or Gel Filtration Chromatography
This technique uses beads with aqueous pores allowing size-based separation:
Smaller proteins can navigate through the pores, slowing their progress through the matrix.
Larger proteins are excluded from the pores, allowing them to elute more quickly from the column.
3. Separation Based on Affinity: Affinity Chromatography
The principle behind affinity chromatography is based on the specific interaction between a tagged protein and a binding target:
Tagging Method: You can create a specific tag that attaches to a protein, allowing it to bind tightly to a substance that non-tagged proteins do not usually bind to.
This is addressed through molecular cloning having the DNA encoding the targeted protein along with a region in the vector encoding a tag (e.g., His-tag - a sequence of six histidine residues).
Example: A protein may be denoted as:
My Protein: M-AA2-AA3-AA4-…AA_N-Stop
With His Tag: M-AA2-AA3-AA4-…AA_N-HHHHHH-Stop
His-tag and Ni2+ Affinity Column
The His-tag enables the tagged protein to bind to Ni2+ ions in an affinity chromatography setup.
A bacterial cell contains the plasmid encoding the His-tagged protein.
When using an Ni2+ affinity column, the His-tag chelates (binds to) the Ni2+ ions, facilitating separation from other proteins in the crude extract.
Once the tagged protein is captured, a competing eluent displaces the His-tagged protein from the Ni2+ ions, resulting in the release of the protein in a purified form.
Monitoring Protein Purification: SDS-PAGE
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is an analytical tool used to track protein purification:
The gel is stained with Coomassie Blue dye to visualize proteins.
Lanes on SDS-PAGE:
Lane 1: Molecular weight standards to determine the size of proteins.
Lane 2: Crude protein mixture before purification.
Lane 3: Purified protein post purification.
Overexpression in Protein Purification
Many cellular proteins exist at low concentrations, sometimes as little as 0.1% of the total protein content.
Cloning the gene encoding the protein intended for purification significantly enhances the expression level of that protein.
Plasmids can be utilized in multiple copies, thereby amplifying the gene encoding the target protein.
It is notably easier to purify proteins when they constitute a significant percentage of total cellular proteins (e.g., 10% compared to 0.1%).
Cloning facilitates the expression and purification of proteins from convenient organisms (e.g., human proteins expressed in E. coli).