Expression and purification of recombinant proteins is a fundamental technique in molecular biology, biotechnology, and biochemistry that involves producing and isolating a protein of interest in a host system, typically by introducing a gene encoding the protein into the host. Here’s an overview of the typical steps involved in this process:
Cloning of the Target Gene
Identify the gene of interest: The gene encoding the target protein must be identified and obtained (e.g., by PCR amplification from cDNA or genomic DNA).
Insert the gene into an expression vector: The gene is inserted into a plasmid (expression vector) that contains:
A promoter to drive transcription of the gene.
A ribosome binding site to enable translation.
A selectable marker gene (e.g., antibiotic resistance) to select cells that have taken up the plasmid.
An optional fusion tag (e.g., His-tag, GST-tag, FLAG-tag) to facilitate protein purification.
Subclone the gene into the vector using molecular techniques like restriction enzyme digestion and ligation or Gibson assembly.
Transformation into Host Cells
Choose a host system: The most commonly used hosts for recombinant protein expression include:
Bacteria (e.g., Escherichia coli): Often used for rapid and cost-effective expression of simple proteins.
Yeast (e.g., Pichia pastoris): Suitable for proteins requiring post-translational modifications.
Insect cells (e.g., baculovirus system): Used for proteins with more complex folding and modifications.
Mammalian cells (e.g., HEK293, CHO): Used for expressing proteins requiring human-like post-translational modifications.
Transform the host cells with the recombinant plasmid using methods like:
Chemical transformation (e.g., heat shock for E. coli).
Electroporation (application of an electrical field to introduce the plasmid into cells).
Expression of the Recombinant Protein
Induc protein expression:
Once the host cells have taken up the plasmid, the expression of the protein is induced, often by adding a chemical inducer like IPTG (for E. coli) if using an inducible promoter (e.g., lac promoter).
Optimize expression conditions:
Temperature: Lower temperatures (e.g., 18-30°C) can improve the folding and solubility of the expressed protein.
Inducer concentration: Optimize the inducer concentration for efficient protein expression.
Time: Monitor expression over time and harvest cells when protein expression peaks.
Cell Lysis
Harvest the cells: After protein expression, the host cells are harvested by centrifugation to pellet the cells.
Lyse the cells to release the protein:
Physical methods: Sonication, French press, or freeze-thaw cycles.
Chemical methods: Detergents (e.g., Triton X-100), lysozyme (for bacterial cells), or lytic enzymes.
Protease inhibitors are often added to prevent degradation of the protein during lysis.
Protein Purification
The purification method used depends on the properties of the protein and any tags that were added to the protein during cloning.
Common purification methods:
Affinity chromatography:
This is one of the most common purification methods when using tagged proteins (e.g., His-tag, GST-tag).
For His-tagged proteins, nickel or cobalt-based affinity chromatography (Ni-NTA or Co-NTA) is used to bind the His-tag. The protein is then eluted with imidazole or other suitable elution buffers.
For GST-tagged proteins, glutathione affinity chromatography is used to bind the GST tag, followed by elution with glutathione.
Ion-exchange chromatography:
Proteins are separated based on their charge. Proteins bind to the column based on their net charge at a given pH, and they are eluted by gradually changing the salt concentration or pH.
Cation exchange: For positively charged proteins.
Anion exchange: For negatively charged proteins.
Size-exclusion chromatography (SEC) (also called gel filtration):
Proteins are separated based on their size. Larger proteins elute first, while smaller proteins elute later. This method can also help in removing protein aggregates or contaminants based on size.
Hydrophobic interaction chromatography (HPIC):
This technique exploits the hydrophobic properties of proteins. Proteins are bound to the column at high salt concentrations and are eluted by decreasing the salt concentration.
Tag Removal (Optional)
If a fusion tag was added to the protein to facilitate purification, it may need to be removed for downstream applications. This is typically done using site-specific proteases like TEV protease or Factor Xa that recognize cleavage sites engineered between the protein and the tag.
After cleavage, the protein can be passed through another affinity column to separate the cleaved tag from the target protein.
Verification and Analysis of the Purified Protein
After purification, the protein is typically analyzed for purity, size, and concentration using methods like:
SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is used to check the molecular weight and purity of the protein.
Western blotting: If specific antibodies are available, Western blot can be used to confirm the presence of the target protein.
Mass spectrometry: To confirm the identity of the purified protein.
Concentration determination: Protein concentration can be measured using the Bradford assay, BCA assay, or absorbance at 280 nm (based on the protein’s tryptophan and tyrosine content).
Activity assays: Functional assays may be performed to ensure that the recombinant protein is active and correctly folded.
Storage of Purified Protein
The purified protein can be stored at 4°C for short-term storage or -80°C for long-term storage.
Proteins are often stored with stabilizing agents like glycerol, sucrose, or protease inhibitors to prevent degradation.
Flash freezing the protein in liquid nitrogen before storing at -80°C can preserve protein structure and activity.
Example Workflow for a His-tagged Protein in E. coli
- Cloning: Insert the gene for the protein of interest into an expression vector with a His-tag and transform into E. coli.
- Expression: Induce protein expression using IPTG.
- Cell lysis: Harvest the cells, lyse them using sonication, and collect the lysate.
- Purification: Use Ni-NTA affinity chromatography to bind the His-tagged protein, wash to remove contaminants, and elute the protein using imidazole.
- Verification: Analyze the purified protein using SDS-PAGE and determine its concentration.
- Storage: Store the purified protein at -80°C in an appropriate buffer.
Tips for Successful Recombinant Protein Production
Codon optimization: Ensure the gene of interest has been optimized for the expression host to improve translation efficiency.
Expression conditions: Carefully optimize conditions (temperature, inducer concentration, time) to maximize protein yield and minimize aggregation or misfolding.
Solubility: Use tags like maltose-binding protein (MBP) or thioredoxin (Trx) if solubility is an issue.
Buffer optimization: Optimize the composition of purification buffers (salt concentration, pH, detergents) to prevent aggregation or degradation of the protein.
By following these steps, recombinant proteins can be expressed and purified for various downstream applications such as structural biology, enzymology, drug discovery, or therapeutic development.