Cell-based expression feasibility

Expression feasibility in microbial systems

Each protein requires specific conditions for expression and purification, and these conditions are little predictable.
The expression feasibility step allows to rapidly identify suitable conditions for expressing your protein and evaluate production and purification performances.

1. Cloning

Your gene is cloned in appropriate Proteus' vectors to allow the subsequent expression profiling (in a single or multiple hosts).
A tag can be added in the construct to facilitate the purification of the protein after production, as well as a proteolytic cleavage site.

2. Expression profiling

Because each protein requires specific conditions for expression, Protéus has developed an optimised approach for rapid identification of the optimal expression conditions.
A variety of parameters known to influence protein expression are tested in parallel, such as: host system, construction, temperature, induction, tags, intra-cellular, periplasmic or secreted expression, helpers...

Building on our extensive experience in expressing hundreds of proteins, Protéus' Expression Profiling brings reliable results to rapidly focus on the best expression system for scaleable production of your proteins.

A panel of expression profiling services (multi-host or single host) is available. Please see the services below, or enquire.

3. Analysis

The expression levels are then compared by SDS-PAGE analysis with Coomassie blue staining.
Optionally, the solubility can also be analysed.

4. Small-scale validation

A small-scale fermentation is then performed is a 50mL shake flask for the two best conditions. Provided that a tag has been added to the construct, the protein is purified by affinity chromatography. The global yield after production and purification is then evaluated by SDS-PAGE analysis.

The remaining amount of purified protein can then be transferred to you for validation.

Optionally, Protéus can also provide MALDI analysis of your protein.

• Rapid screening of expression possibilities for selected hosts
• Define the optimal expression host and condition to produce active proteins

• A whole range of expression hosts and conditions
• A choice of profiling sets to fit with your objectives: single host, multi-host, extensive E. coli profiling...
• High success rate (over 80% of proteins tested have been successfully expressed)
• Rapid answer on feasibility of expression
• A first approach to purification

A report including :

Results of sequencing controls after cloning
Results of SDS-PAGE analysis and estimation of expression yield
Summarised data sheet for SDS-PAGE interpretation
Conclusions and perpectives

Delivery of the quantity of proteins obtained from fermentation in Erlen 50 mL

Prod’line™ Multi-host Expression Profiling was performed on a 55kDa human protein. The expression yield was then measured in the soluble fractions.
E. coli clearly appeared as the most suitable host for expressing this protein. Unexpectedly, a single condition appeared to express much higher level of soluble, functional protein than all other conditions.

The best-performing condition was then applied by Protéus to scale-up the expression in a 2L fermentor. A yield over 1g/L was obtained, confirming the suitability of the selected condition for the protein.