Optimize your chance to produce your recombinant protein efficiently! Our service of protein production in E. coli includes up to 32 protein expression tests in standard to guarantee that even the most difficult-to-express proteins have a high probability to be produced.

DATA REPORT

Get free access to genuine, representative data
from a E.coli Expression & purification test project

An ideal example of the insights you can expect from this type of report

This comprehensive report includes:

  • A full set of detailed data
  • Advanced tests and results
  • Key insights at every step of the process

Download the Data Report

What are the 32 tests included in our
E.coli protein expression service?

Recombinant protein production is highly technical and requires strong expertise as one production protocol cannot fit all product types. With more than 16 years of experience and 1500+ proteins expressed, ProteoGenix developed an in-house protocol including up to 32 expression conditions tests in its package of “Protein production in E. coli” to meet the best your requirements. The technical possibilities are almost infinite and such a long lasting experience is decisive.

Varying Expression Conditions Include:

  • E. coli strains
  • Temperature
  • Induction time
  • Protein extraction method
  • Purification method
E. coli protein production tests
High yield protein expression

You need to produce a protein in E. coli or want more information about our capacities of protein expression in E. coli? Our account manager is at your disposal to answer to your questions.
For difficult to express proteins we also have a dedicated high throughput screening service that allow to increase the probability to find the most optimal conditions. The more conditions are tested, the higher the probability to ensure optimal solubility, functionality, productivity…
All conditions are varied from codon optimizations, vectors with different promoters, protein sequences, purification tags down to the purification method itself, tag clivage conditions, refolding method and formulation. The advantage of protein expression in E. coli is the infinite range of solution and technology and ProteoGenix has a wide experience of all these technologies thanks to the large variety of project carried out until now.

Flowchart of our protein expression in E. coli

Expression vector construction

  • Gene synthesis including codon optimization for E. coli production.
  • Gene subcloning in a high producing expression vector.

E. coli strain transformation

  • Transformation of the plasmid into various bacterial strains.

Test of 32 protein expression conditions

  • Evaluation of 32 protein expression conditions (temperature, induction time, fractions).
  • Determination of the best protein production conditions.
  • Protein purification tests.

Protein expression scale up and purification

  • 1L protein expression and purification with optimized conditions previously determined.

QC analysis

  • SDS/PAGE analysis and WB.
  • Quantitative analysis by Bradford method.
Step Content Timeline Deliverables
Gene synthesis
  • Gene design including
  • codon optimization
  • Gene synthesis
  • Subcloning in expression
  • vector
3 to 4 weeks
  • Gene optimized for the desired expression system
  • Gene cloned in a plasmid
Small scale protein expression evaluation
  • Plasmid transformation
  • Test of 32 protein expression
  • conditions
  • Purification tests
  • QC analysis
2 to 3 weeks
  • Detailed reportincluding optimal protein production protocol
Protein production scale up
  • 1L protein expression
  • Protein purification
  • QC analysis
2 to 3 weeks
  • Purified protein from 1L culture Detailed report

Options available: 

  • Tag elimination via enzymatic cleavage
  • Additional purification steps (IEC, SEC…)
  • Endotoxin assay and removal
  • Scale up production / Fermentation

Case study: protein expression in E. coli

Aim Of The Project

One of our customers requested a recombinant protein production in E. coli. This project included also gene synthesis and subcloning in an expression vector (information included in the PDF report).

Protein Expression Tests

The aim of the protein expression tests is to determine the optimal conditions for protein expression by evaluation of:

  • E. coli strain used for protein production
  • Induction strategy
  • Temperature
  • Induction time
  • Best protein extraction condition (native or denatured)

More details about the protocol are provided in the complete PDF report.

E. coli protein expression tests

Figure 1 : Expression tests of the target protein. Coomassie blue staining Reducing-PAGE analysis of NPE and DPE. MW. Molecular weight marker. Ø. Non-induced bacteria culture (negative control). Induction with IPTG 1mM.

The protein is expressed at high level in all strains tested, and is mainly localized in NPE (Figure 1). The optimal conditions for native expression are indicated in the complete PDF report.

Small Scale Protein Purification Tests

A small-scale (200ml culture) purification test was performed in native conditions. Starting material: NPE prepared after optimal native expression. Details about the protein purification protocol are available in the complete PDF report.

E. coli protein expression tests
Strain No. Purity* Yield(mg/L)**
XXX No.1 90% 146

Table 1: Production and purification yields Final buffer: TBS pH8.0. **Estimates based on the 200ml-culture tests. *Based on the 2µg loadings in Figures 2B (green arrows)

Protein Production testimonial

Karina ARREGUIN,
Augusta University

“ ProteoGenix assisted me with the generation of a peptide and a protein. They were very helpful throughout the entire process, and provided invaluable information and recommendations based on their expertise. Based on this experience, I look forward to future collaborations with ProteoGenix ”

M. Napieralski Rudolf
Therawis Diagnostics GmbH

“ ProteoGenix is for us a reliable partner for the production of recombinant proteins in E.coli and always provided an excellent service, with regular updates, quick response to our queries and delivery of product in high quality. We appreciate the collaborative attitude and professionalism of ProteoGenix.”

Protein production in E. coli: pros and cons

E. coli is a well-characterized organism for protein production and was the first host expressing an approved therapeutic recombinant protein.

Until the middle of the 90’s, E. coli was considered as the best choice for biopharmaceutical production due to several advantages such as:

  • Fast growth kinetics,
  • Possibility to achieve high-cell density cultures,
  • Fast and easy plasmid transformation.

 

These advantages make E. coli the best choice if you are looking for a fast and cost effective protein expression solution.

However, protein production in E. coli may not be the most relevant solution for the expression of complex protein structures including post-translational modifications or biotherapeutic productions due to high levels of endotoxin (which can be removed thanks to our endotoxin removal service).

To get more information about our protein production in E.coli platform, please feel free to contact our dedicated account manager.

Exploiting inclusion bodies for high
yield protein expression in E. coli

Inclusion bodies are often considered as a bottleneck in recombinant protein production but they are often related to high protein expression levels. Several approaches have been developed to limit their formation but only irregular results have been obtained because protein production is product dependent. It is for instance possible to produce at low temperature using specific strains. However, inclusion bodies can present several advantages:

  • Fast growth kinetics,
  • Possibility to achieve high-cell density cultures,
  • Fast and easy plasmid transformation.

Thus, many microbiologists developed protein refolding protocols allowing the exploitation of these inclusion bodies as a source of recombinant proteins.

At ProteoGenix, we developed a unique protein refolding platform tailored to best exploit recombinant protein expression and meet your requirements. To get more information about it, please feel free to contact our dedicated account managers.