Protein expression is the process by which cellular machinery is directed to manufacture a specific protein in large amounts. Following production, the target protein must be isolated from the complex mixture of cellular components through a process called purification.
This technique is used to produce therapeutic proteins like insulin and antibodies, and it allows researchers to study protein structure and function. Industrial applications also benefit from this technology, using purified enzymes in food manufacturing and environmental cleanup.
Gene Cloning and Host System Selection
The first step is gene cloning, where the gene for the protein of interest is inserted into a circular piece of DNA known as a plasmid. These vectors contain regulatory elements, such as a promoter, which acts as an “on” switch to initiate gene transcription.
Plasmids also carry a selection marker, often a gene conferring antibiotic resistance. When introduced into host cells, only cells that have taken up the plasmid will survive in a growth medium containing the antibiotic, ensuring production is focused on the desired protein.
The choice of a host system depends on the target protein’s properties. The most common host is the bacterium Escherichia coli because it grows rapidly and the process is straightforward, making it ideal for many simple proteins.
Some proteins require complex folding and modifications that E. coli cannot perform. For these, eukaryotic hosts like the yeast Pichia pastoris are a better option as they can perform necessary post-translational modifications. For the most complex proteins, such as therapeutic antibodies, insect or mammalian cell lines are used to produce proteins with the intricate structures needed for full biological activity.
Inducing Expression and Cell Lysis
After growing host cells to a suitable density, protein production is activated through induction. This is triggered by adding a specific chemical, such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) in many E. coli systems. The addition of IPTG allows the cell’s machinery to begin large-scale production of the target gene.
Optimizing induction conditions is important for maximizing the yield of functional protein. Factors like inducer concentration, culture temperature, and induction duration are controlled. Lowering the growth temperature after induction can slow protein synthesis, which helps the protein fold correctly and remain soluble rather than forming inactive aggregates called inclusion bodies.
Once enough protein is produced, the host cells are broken open, or lysed, to release it. The lysis method depends on the host cell and protein stability. Common mechanical methods include sonication, which uses high-frequency sound waves, and the French press, which uses high pressure.
Alternatively, chemical or enzymatic methods can be used, such as detergents to dissolve cell membranes or enzymes like lysozyme to break down bacterial cell walls. This step results in a crude lysate—a mixture containing the target protein and all other cellular components—which is the starting material for purification.
Protein Purification Chromatography
The next objective is to isolate the target protein from the crude lysate. This is achieved through a series of chromatography techniques, which separate molecules based on their distinct physical and chemical properties.
The most common initial step is affinity chromatography, which uses a specific binding interaction. The target protein is often engineered to include an affinity tag, such as a His-tag, which binds tightly to metal ions like nickel that are chemically attached to a resin in a column.
When the crude lysate is passed through the column, only the tagged protein binds to the resin while contaminants wash through. The bound protein is then eluted from the column by washing it with a solution containing a high concentration of a molecule like imidazole, which displaces the target protein.
Further purification is often needed to remove minor contaminants. Ion-exchange chromatography (IEX) is a common second step that separates proteins based on their net surface charge. By controlling the buffer’s pH, a protein can be made to bind to an oppositely charged resin, while other proteins are washed away. The bound proteins are then eluted by increasing the salt concentration of the buffer.
A final “polishing” step is often performed using size-exclusion chromatography (SEC), also known as gel filtration. This technique separates proteins based on their size. The column is packed with porous beads; smaller proteins enter the pores and take a longer path, while larger proteins cannot and thus elute first.
Purity Analysis and Quantification
After purification, the protein’s purity is assessed and its concentration is determined. The standard method for analyzing purity is Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). In this technique, the protein sample is treated with a detergent that denatures the proteins and gives them a uniform negative charge, allowing them to migrate through the gel based only on their size.
A successful purification appears as a single, prominent band on the gel at the correct molecular weight. To confirm the protein’s identity, Western blotting can be used, which employs a specific antibody that binds only to the target protein.
The final step is to measure the concentration of the purified protein to determine the overall yield. Spectroscopic methods like the Bradford or BCA assay are used for this purpose. These assays involve adding a reagent that results in a color change proportional to the amount of protein present, which is measured with a spectrophotometer to calculate the concentration against a standard curve.