The yeast expression system is one of the standard systems for the eukaryotic expression of proteins. It has many advantages eukaryotic expression system, such as protein processing and folding, post-translational modifications, etc. And the operation of yeast protein expression is relatively simple compared to mammals.
Lifeasible's yeast expression system is an essential component of the recombinant protein expression service platform. We provide gene sequence optimization, multi-vector selection construction, multiple strain transformation, large-scale fermentation, etc. We can deliver high-purity proteins that can be solubilized for expression.
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| Target gene synthesis |
- Finding the cDNA sequence of the target protein from GenBank.
- Optimize the cDNA for codon and mRNA secondary structure according to the selected expression system.
- Synthesize the designed gene sequence.
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| Target gene subcloning |
- Culture and extract the expression vector containing the target gene.
- Subclone the target gene into a suitable expression vector.
- Sequencing to verify the accuracy of the constructed expression vector.
- Expression vectors (pPICZa-based) are available.
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| Yeast expression strain electrotransformation |
- Enzymatic linearization of the expression vector.
- Electrotransfer the expression vector into an efficient yeast expression strain.
- Select at least 5 positive clones by PCR or expression level of the product identification.
- Expression strains of different phenotypes are available.
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| Yeast expression strain screening |
- The 5 positive clones are amplified in the BMGY medium and then replaced in the BMMY medium with methanol to induce expression of the target protein.
- The expression of the target protein in the culture supernatant was detected by SDS-PAGE electrophoresis, and suitable monoclonal strains were selected for the expression of the target protein.
- Suppose the expression is not detected in the culture supernatant. In that case,t, the target protein expression is detected again by concentrating the supernatant 20-fold for action or by the Western-blot method (the customer needs to provide relevant antibodies).
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| Expression optimization of yeast expression strain |
- Twenty positive clones were selected for routine expression screening, and the expression conditions were optimized for the best-expressed strains.
- For the selected monoclonal strains, we optimize the culture and induction conditions (medium, density, methanol concentration, induction time, etc.) to improve the target protein expression.
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| Target protein expression and purification |
- Shake flask culture of 1L recombinant yeast to induce the expression of the target protein.
- The expressed recombinant proteins were purified by various chromatographic methods such as affinity, ionic, hydrophobic, and gel filtration.
- The results of each purification step are examined by SDS-PAGE electrophoresis, and the final product quality is controlled.
- Validate the correctness of the target protein by the Western blot.
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| Target protein reprocessing and detailed detection |
- Further processing such as endotoxin removal, decontamination, and lyophilization.
- Detailed testing of the quality of the target protein by HPLC and other methods.
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| Large-scale preparation |
Scale-up production according to small pilot process, 5L-30L high-density fermentation, and preparation of qualified protein products required by customers. |
General service flow
Lifeasible has a complete yeast expression platform and can design different experimental protocols based on protein characteristics or customer needs. Our high-throughput screening platform allows us to quickly and efficiently obtain optimal expression conditions. We can produce large-scale fermentation. Feel free to contact us for the latest protocols.
For research or industrial raw materials, not for personal medical use!
