Protein Expression in Bacterial System

E.coli is the preferential one of bacterial systems for recombinant protein production and large scale fermentation as its advantages including fast rate of reproduction, ease of culture, and rich knowledge about its genetics. Bacterial?protein expression service offered by DetaiBio provide a relatively simple, fast, and inexpensive platform for native and heterologous recombinant protein production. Contact us for quotations and more information.

Service Highlights

• No protein delivered, no billing!
• Customizable service process!

Service Content

Provide	Deliverables	Timeline	Price
Plasmids or sequences of target protein	Cloning vector QC data Final report >90% purity 3-5 mg soluble proteins	4-6 weeks	Inquire

Notes:

1. The recombinant protein produced by DetaiBio will carry the His-tag (its size is only 0.84 kDa) preferentially. We can also provide other tag options, including GST (about 26 kDa), SUMO (11.2 kDa) and MBP (42.5 kDa). In addition, we can provide tag-free proteins with high quality.
2. Endotoxin control runs through the entire experimental procedure, and the final level of endotoxin can less than 1 EU/μg. We can also offer endotoxin removal service which can reduces the endotoxin level to 0.01-0.1 EU/μg.
3. Methods of QC detection are SDS-PAGE and western blot without charge. Other detection methods (additional charges) are optional, such as mass spectrometry (MS) or high performance liquid chromatography (HPLC).
4. Target protein can be labeled by biotin, isotope or fluorescien if required.
5. Quantity of target protein can be customized. The purity of target protein is more than 90% in our standard package. Our professional team has ability to provide protein with a high purity (95%) if you required.
6. Large scale fermentation is available.

For more detailed information, please contact us.

Service Process

1. Codon Optimization

Rare codon may cause the failure of protein expression and affect the secondary structure of mRNA. After performing thousands of projects, we can analyze and optimize the codon of the sequence of target protein perfectly by using our unique bio-informatics software Maxcodon^TM.

2. Gene Synthesis & Vector Construction

We can start the gene synthesis after codon optimization. Meanwhile, dozens selections of expression vectors can be selected, including pET series (pET28a, pET30a, pET43.1 a, etc.), pGEX4T-1, pBAD24, pGADT7, pQE-30, etc. If you do not know how to select, we are happy to assist you.

3. Competent Cell & Pilot Expression Test

According to the property of target protein, DetaiBio can also help to choose the most suitable competent cells, which include several kinds, such as BL21 (DE3), OrigamiB (DE3), Rosetta (DE3), BL21 CodonPlus (DE3), ESL, etc. Then we perform pilot expression tests and explore the best expression conditions.

4. Scale Up & Protein Purification

According to expression level and customer’s specific needs, we scale up the protein expression with suitable level using the optimized conditions. We can purify the target protein with major protein purification methods (such as affinity chromatography, size-exclusion chromatography, and hydrophobic interaction chromatography) using advanced AKTA purification equipment.

5. QC

Target protein will be detected by SDS-PAGE and western blot. Then target protein will be delivered with the QC report.

Supplementary

1. Why did my protein not express?

If your proteins did not express in e.coli system, the most possible reason is that its gene is from eukaryotes. Eukaryotic genes generally have signal or leader sequences and intervening sequences, which can influence the result of protein expression in bacteral system. In addition, E.coli cells lack the post-translational modification system of eukaryotic proteins, which may result in failure of eukaryotic proteins expression that require glycosylated phosphorylation.

Solution:

You can replace the host bacteria,such as BL21 Condon plus (DE3) or Rosetta, which is specially designed to express eukaryotic proteins.

2. Why did the target protein have a low expression level?

Several points need your attention, including: translation initiation site, GC content, secondary structure of mRNA, codon, plasmid vector, the size of target protein and gene mutation.

Solution:

You can analyze the gene sequence of target protein and optimize the culture conditions (such as medium composition, temperature, induction time, concentration of inducer, and induction time).