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The E. coli expression system holds its reputation as the most commonly employed and cost-effective method in protein expression. Its simplicity in structure, well-defined genetic background, ability to produce high yields of target proteins, and short cultivation period contribute to its widespread use and popularity.
Why pick the E.coli expression system?
- Cost-effective: E. coli is relatively inexpensive to culture compared to other expression systems, making it cost-effective for large-scale protein production.
- High Yield: E. coli can produce high quantities of recombinant proteins, often comprising a significant proportion of total cellular protein, which is advantageous for applications requiring large amounts of protein.
- Speed: E. coli has a rapid growth rate and short generation time, allowing for quick protein production compared to other systems. This speed can be particularly beneficial for high-throughput applications.
- Simple Culture Conditions: E. coli can be cultured in simple media under standard laboratory conditions, requiring minimal specialized equipment or resources.
- Compatibility with Fusion Tags: Fusion tags can be easily attached to target proteins to aid in purification, solubility, or detection, and E. coli is amenable to a wide range of fusion tag systems.
- Versatility: E. coli can express a broad range of proteins, including complex eukaryotic proteins, membrane proteins, and enzymes, making it a versatile system for various research and industrial applications.
Service Process
1. Plasmid construction
Process
- Codon optimization; gene synthesis
- Restriction digestion of PCR products; Ligation to expression vector
- Transform TOP 10 E.coli competent cells
- Obtain the correct recombinant plasmid
Features
Multiple vectors optimisation, more options for customers - Simultaneously optimise multiple vectors, to determine which has the highest yield, to shorten lead time.
Lead Time: 15-20 business days
2. Transformation and strain screening
Process
- Transform the recombinant plasmid to host cells.
- Choose a single colony for small-scale induced expression; Confirm protein expression using SDS-PAGE. Preserve the superior colony and optimise expression conditions.
Features
Optimising conditions across multiple parameters and selecting from various hosts. During preliminary testing, temperature and IPTG concentrations are fine-tuned to achieve the most suitable culture conditions.
Simultaneously, multiple hosts are transformed to identify the bacteria yielding the highest output.
Lead Time: 5 business days
3. Target protein expression and purification
Process
- 1-10 L large-scale expression.
- Protein purification
Features
Multi-condition purification methods (optional) - Determine the optimal purification method, using different chromatographic conditions.
Lead Time: 12-15 business days
4. Inclusion body renaturation
Process
- Refolding is necessary if the target protein is in the form of inclusion bodies.
Features
Different buffering conditions are utilised to swiftly evaluate the most effective refolding buffer formulation. Refolding proteins to attain purity levels surpassing 90% is accomplished through techniques like dilution renaturation, dialysis renaturation, column chromatography renaturation, and others.
Lead Time: 5 business days
5. Optional additional services
Process
- Endotoxin removal, lyophilisation or filter-sterilisation.
- Tag removal (Additional charge)
Features
Customers have the flexibility to select from a range of additional services tailored to their specific requirements.
Lead Time: 3-5 Business Days
6. Quality control
Process
- The purity, concentration and further tests are performed.
Features
Detailed COA report is provided.