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Yeast protein expression system is a highly economical eukaryotic expression system that do both secretion expression and intracellular expression. The exogenous gene expressed by Yeast expression system has a certain post-translational processing capacity, the expressed exogenous protein has a certain degree of folding and glycosylation modification, it’s more stable than prokaryotic expressed proteins, particularly suitable for the expression of eukaryotic genes and preparation of functional proteins. Yeast secretion expression can secrete the expressed exogenous protein into the extracellular matrix, so that it is easy to obtain a high purity protein. Yeast expression system has many advantages which make its research and application more and more widely.
Risk-free: We do NOT charge if we cannot deliver the protein.
Note: This risk-free custom protein service is only suitable for proteins within 800aa. We will charge according to different steps if you need to express proteins more than 800aa.
| Steps | Project | Process | Cusabio Features | Lead Time | ||
|---|---|---|---|---|---|---|
| 1 | Expression vector construction |
Codon optimization; gene synthesis | Multiple vectors optimization, More options for customers
In order to improve the success rate of expression and achieve higher yield, in addition to conventional N-terminal fusion protein expression, we also provide protein with C-terminal fusion label, in greater degree to ensure the activity while ensuring the purity. |
15-20 business days | ||
| The PCR amplification products are ligated to the expression vectors e.g. pPic9k, pPic3.5k, pPiczαA, etc. | ||||||
| Transform ligation mixtures into E. coli strain | ||||||
| Obtain the correct recombinant plasmid | ||||||
| 2 | Transformation and strain identification  |
Prepare the recombinant plasmid in large quantities | High copy screening
Conduct multiple screening through unique screening markers of different vectors, and the highest expression level strain was gradually obtained. |
PickRight Technology
The high expression level strain was obtained directly after transformation, and the time was shorten by 5-10 business days compared with the traditional screening. (Theoretically this technology is mainly recommended for the production of less than 5 mg/L protein expression) |
10-13 business days | |
| Linearization of recombinant plasmid | ||||||
| Transform to GS115, X33, KM71 and other hosts by electroporation | ||||||
| PCR analysis is recommended to verify successful transformants | ||||||
| Use geneticin G418, Zeocion and other antibiotics for multiple copies screening to obtain high copy | ||||||
| 3 | Small test, scale up expression and purification |
Small scale expression screening (20-40 strains) | 7-12 business days/15-25 business days (Featured Purification) | |||
| Determine strain and optimize expression conditions | ||||||
| Scale up culture | ||||||
| Protein purification | Multiple purification methods (optional)
Explore different chromatographic conditions including ion exchange, hydrophobic and others by using AKTA, and then determine the optimal purification method. |
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| 4 | Additional services (Optional) |
Charge | Tag removal service | Flexible additional services
Customers can flexibly choose from a variety of additional services to their specific needs, e.g. Endotoxin removal, Filter-sterilization, Tag removal, Lyophilization, etc. Some are complimentary, and some require additional charge. |
3 business days | |
| Free | Filter-sterilization; Endotoxin removal; Lyophilization
(Note: Lyophilization and filter-sterilization can not be met simultaneously) |
2 business days | ||||
| 5 | Quality Control |
Testing of purity, concentration, etc. QC report is provided. | Detailed COA Report
Detailed product data sheet and COA are provided for each project. |
3-5 business days | ||
| Total lead time | 35-50 business days | |||||
Case 1 High Purity Protein
Difficulty: Yeast intracellular expression, many impurities can be observed in the lysate protein (lane 1), the target protein was not obvious, after the yeast system-specific chromatography system purification, less miscellaneous band was observed with SDS-PAGE detection, the purity reached more than 95% (lane 6-7).
Features: Yeast secretion expression, the expressed protein was directly secreted into the medium, basically no impurities, the purity is as high as 90% or more, the purification is mainly for removing pigment and other residues in the medium, leaving only the target protein in the appropriate buffer.
Case 2: Large Molecule Weight Protein Expression
Difficulty: More than 700 amino acids, Mw: 81 kDa, we chose secretory vector for expression in order to get a higher purity, finally the protein was successful expressed and secreted into the culture medium.
Case 3: Small Molecule Weight Protein Expression
Difficulty: 62 amino acids, Mw: 7 kDa, the molecular weight is very small, relatively difficult to concentrate and detect the protein. The expression, purification, collection were very successful from the SDS-PAGE detection result.
Difficulty: www 36 amino acids, Mw: 4 kDa, and this customer required to remove the tag, it’s rather difficult to remove the tag as the molecular weight itself is very small, but we have successfully remove the tag after a series processing, and WB detection result showed the target protein didn’t contain tag, so finally high-purity, low-molecular-weight and untagged protein was obtained.
Case 4: N-linked glycosylation modification (Yeast expression system unique characteristics)
Features: Close to the modification of native proteins, especially glycosylation in the Yeast expression system is particularly evident by SDS-PAGE detection, it showed diffuse band and a large molecular weight, after digested by Endo H, the band was shaped and the size is consistent with the theoretical value.
Case 5: pPic9k-SUMO Vector
Unique SUMO tag fusion protein
Characteristic expression systems
pPic9k-JT Plasmid+GS115 strain efficient expression
Carrys AOX1 strong promoter, containing alpha secreting factor which is able to do high efficient secretion expression
Seamless cloning, no restriction enzyme needed
Multiple linearization sites：SalⅠ, SacⅠ, BglⅡ
Can do Amp and Kan double-resistant screening to select positive strains during the prokaryotic stage
Can do His+ and G418 screening to select high expression level strains during the eukaryotic stage
Modified cloning sites, its cloning is not limited by the potential endonuclease in the target gene
The vector has his tag, thus making it easy for cloning and purification
pPic9k-SUMO Plasmid+GS115 strain efficient expression
Carry AOX1 strong promoter, containing alpha secreting factor which is able to do high efficient secretion expression
Contain SUMO fusion protein; possessing a strong ability to promote expression
Seamless cloning, no restriction enzyme needed.
Contain the EK cleavage site, can obtain untagged protein after enzyme digestion.
Multiple linearization sites：SalⅠ, SacⅠ
Can do Amp and Kan double-resistant screening to select positive strains during the prokaryotic stage
Can do His+ and G418 screening to select high expression level strains during the eukaryotic stage
Modified cloning sites, its cloning is not limited by the potential endonuclease in the target gene
The vector has his tag, thus making it easy for cloning and purification
Specialized CRO Services
