Protein Expression in E. coli

Overview Capabilities Advantages Services Process FAQs

Overview

We specialize in recombinant protein expression through Escherichia coli. As one of the most popular host systems, Escherichia coli is preferred because it has a quick production time, is cheap, easy to use, and is highly scalable. Our services range from plasmid development and cloning, expression optimisation to protein purification and quality control.

Protein folding and secretion in E. coli(HP Sørensen, et al.,2005)

We offer a broad range of plasmid vectors and Escherichia coli strains to match the requirements of different proteins such as BL21 (DE3), Rosetta, etc., to get the desired protein expressed successfully. We also employ high-tech techniques, including fusion tags and chaperone co-expression, to increase protein solubility and stability.

Our group is highly experienced and can tailor expression system and processing environment to protein requirements in order to produce high-quality and high-efficiency protein expression. Whether it's small molecule proteins or large molecule proteins, we can offer customized solutions to enable our customers to achieve advances in drug discovery and development as well as biological research.

Capabilities

The following are the E. coli (E. coli) products provided by our company coli) protein expression service detailed flow table:

Step	Description	Turnaround	Remarks
1. Gene synthesis and codon optimization (optional)	The gene encoding the target protein was synthesized and codon optimized to improve the expression of E. Expression efficiency in coli.	1-2 weeks	Choose whether to perform codon optimization as needed.
2. vector construction	Clone the optimized genes into appropriate expression vectors, such as pET series or pGEX series.	2-3 weeks	Including plasmid sequencing and large-scale plasmid preparation.
3. transforming a host cell	The constructed expression plasmid was transformed into efficient E. coli expression strains such as BL21 (DE3), Rosetta(DE3), etc.	1 week	Positive clones were screened and amplified.
4. Small amount expression and condition optimization	Carry out small-scale expression experiments and optimize culture conditions, such as temperature, IPTG concentration, etc., to improve protein expression levels.	2-4 weeks	Including SDS-PAGE to detect protein expression.
5. Mass expression and purification	Mass expression was carried out under optimized conditions, and the protein was purified by affinity chromatography, ion exchange chromatography and other methods.	2-4 weeks	Provide purified protein samples and purification reports.
6. Protein testing and analysis	The purified protein was subjected to SDS-PAGE, UV analysis, Western blot and other tests to ensure the protein quantity and purity.	non-fixed period	Provide corresponding testing services according to customer needs.
7. project delivery	After completing all experimental steps, deliver the purified protein samples and experimental reports to the customer and issue an invoice.	non-fixed period	Delivery is completed according to the time agreed in the contract.

The exact time may vary based on the complexity of the project and customer needs.

Advantages

E. Coli protein expression system is very competitive with other recombinant protein expression systems because it's quick, inexpensive, high yield, easy genetic manipulation and easy scale up production.

Affordable

The E Coli expression system is renowned for its low cost, medium and operation, suitable for mass production.

Rapid growth and very short production time

Since E Coli has a rapid growth rate, it can achieve a very high cell density quickly, which makes the protein production speedier.

Super-yield

E Coli can readily release recombinant protein, typically achieving the production of hundreds of milligrams per liter of medium.

Tech maturity and diversity

The technology for genetic manipulation of E Coli is extremely mature, and allows the selection of multiple expression vectors and strains, each suited to different protein properties.

Scalable production

E Easy to scale production : The coli expression system is extensible, which means that it can be gradually scaled up from laboratory experiments to industrial production at various scales.

Anti Pollution Effective

The expression of E Coli has high anti pollution efficacy and prevents contamination during production.

Broad applications

E Coli has many applications in scientific research, diagnosis, industrial enzymes and therapeutic proteins, and is suitable for a wide range of protein expression.

Different optimization options

By optimizing expression parameters and methods, including fusion tags, chaperone coexpression and others, proteins are much more solubile and stable.

Services Process

This is the cooperation workflow of E.coli protein expression service project:

Signing the contract

First, you need to talk to the customer regarding the technical plan, identify the technical service elements, and prepare the quote. When everyone is satisfied, a contract is executed.

Project implementation

Design of plasmids: Design plasmids that encode the target protein as per the requirements of the customer.
transformed into E coli: Convert the plasmid to a suitable E. coli expression strain (BL21 (DE3), Rosetta, etc.)
Screening positive clones: scouting positive clones that are positive for the target protein through SDS-PAGE and other techniques and increasing their number.
Expression induction: Establish the recombinant bacteria in a shake flask or fermentation tank and promote expression of the target protein.
Protein purification: The expressed protein is purified using affinity chromatography, ion exchange, hydrophobic chromatography, etc.

Project delivery

Qualitative analysis: SDS-PAGE and Western blots were used to check the expression and purification property of the protein.
Reporting: Write an experimental report detailing plasmid building, transfection, expression, purification, and verification.
Deliver samples: Distribute purified protein samples to customers based on customer's need and provide experimental reports.

Service extension

If customers require additional services, including protein renaturation or special processing, technical assistance and charges can be offered as well.

Signing the contract

Project implementation

Project delivery

Service extension

FAQs

Here is the section on Frequently Asked Questions (FAQs) for E.coli protein expression:

Q: How to select the appropriate E coli strain for protein expression?

A: E coli strains vary in many ways. The BL21 (DE3) strain, for instance, is most commonly employed for IPTG expression, while the Rosetta strain is appropriate for rare codon-rich proteins. Selecting appropriate strains can dramatically enhance protein expression efficiency and permeability.

Q: How to maximize protein expression in E. coli?

A: Optimizations of expression conditions include temperature, IPTG concentration, and induction time. For the most part, 37°C is a suitable expression temperature, but the exact conditions must be adjusted depending on the target protein to avoid degradation of the protein.

In addition, expression systems with the T7 promoter use T7 RNA polymerase, typically co-sold with the BL21 (DE3) strain.

Q: What about inclusion bodies produced during articulation?

A: Inclusion bodies are a general problem in protein expression, and can be overcome by dissolution and renaturation techniques. Which renaturation process to choose depends on the sequence and structure of the protein.

Q: What are the strengths and weaknesses of the E. coli expression system?

A: E. Coli's expression benefits include high growth rate, high yield, a fast culture, and the genome can be altered. But it doesn't permit the expression of proteins that need glycosylation changes and generate endotoxins and inclusion bodies.

Q: How to circumvent commonly encountered challenges in protein expression?

A: Low translation initiation efficiency and low protein solubility are two common challenges. You filter different E coli strains, molecular chaperones, fusion tags and so forth to overcome these issues.

Q: How to select the appropriate expression vector?

A: The most popular E coli expression vectors are the pET vectors, which target the T7 promoter and encode for T7 RNA polymerase. Moreover, different promoters can be used on demand such as the lac promoter or the araBAD promoter.

Q: How to purify proteins?

A: The protein purification method is different depending on the structure and proteins. The most commonly adopted purification methods are affinity chromatography, ion exchange chromatography and gel filtration.

Q: How to encode and store expressed proteins?

A: The expressing protein needs to be stored at proper temperatures to keep it functioning and stable. Store at low temperatures and keep it from being thawed over and over again.

Please note that all services are for research use only. Not intended for any clinical use.

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