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Custom expression and purification of recombinant protein in yeast
Step 0: Gene synthesis and codons optimization
This gene synthesis step is very important because it allows us to optimize rare codons for yeast 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 yeast 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. It is possible to remove the tag.
Then, we perform a maxi-prep prior to the linearization of the DNA for transformation in yeast.
Step II: Recombinant protein expression tests:
- Transformation of linear DNA in suitable yeast strain
- Confirmation of positive transformants by PCR
- Confirmation of the expression
- Affinity purification on specific resin
- Analysis by acrylamide gel / SDS-PAGE (lysates) and / or Western blot
Step III: Recombinant protein production and purification:
- Yeast culture: 1 to several liters
- One-step purification or more to reach a purity equal to or greater than 90%
- Quality control by acrylamide gel (SDS-PAGE) and / or Western blot
NB: the production step is carried out according to the customer requirements based on the results obtained at the end of step II. It is then possible to perform small to very large scale productions.
Turnaround time :
- Cloning : 2 weeks
- Expression evaluation : 2 weeks
- Production and purification : 2 weeks