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Recombinant protein expression and purification custom services in E. Coli
Step 0: Gene synthesis and codons optimization
This gene synthesis step is very important because it allows us to optimize rare codons for E. Coli expression system and therefore significantly increases in some cases production yields.
Step I: cDNA cloning into an expression vector
This step consists in integrating the cDNA into a bacterial expression vector containing a tag and inducing the expression of the fusion protein of interest and allowing the protein purification on affinity column via the tag. The tag can be removed.
Step II: Recombinant protein expression tests:
- Transformation in different bacterial strains
- Bacterial cultures in different conditions (medium types, additives)
- Synthesis induction (kinetic of 5 hours)
- Affinity purification with a specific resin
- Acrylamide gel (SDS-PAGE) and/or Western blot analysis
NB: We perform several runs of tests in order to identify optimal conditions:
1) of culture for the protein production
2) for protein extraction (soluble or in the inclusion bodies) necessary for its folding
3) for the protein purification
4) for the production yield
In order to identify the optimal conditions for expression of recombinant proteins, we need to modify a large number of parameters and we are involved in all stages of codons optimization, choice of vector, tag, culture medium, additives, bacterial strains, induction temperatures, IPTG concentrations, purification technique types, renaturation / refolding (dialysis buffer change) … This is part of the strength of our organization.
Step III: Recombinant protein production and purification:
- Bacterial culture: 1 or more liters
- Growing conditions according to protocol established during step II
- Preparation of bacterial lysates according to protocol established during step II
- Affinity chromatography purification according to protocol established during step II
- Optional: tag removal by enzymatic cleavage
NB: the production step is carried out according to customer specifications based on results obtained at the end of step II.
Turnaround time :
- Cloning : 2 weeks
- Expression evaluation : 10 days
- Production and purification : 5 days