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Home > Protein Expression > Yeast Expression

Yeast Expression

In industrial microorganisms, Yeast P. Pichia expression system is a widely used system with the advantages of short production period, low cost and easy to be operated similar as prokaryotic expression. At the same time, it has the ability of eukaryotic cells to modify proteins with spatially fold and glycosylatation, which greatly improve the solubility of protein and provides a powerful tool for studying the physiological activity of the protein. There are many factors which will influence the experiment results during the yeast protein expression such as induction time, induction temperature, copy number of the target gene, culture conditions and identification methods. Any small negligence will make a few months of hard work be lost. HongTide has extensive experience on maximizing the expression potential of yeast strains, which can improve expression success rate and expression level.
Service Procedures and Specification
1. Programme analysis
Timeline
Deliverables

  • Optimization of codon

  • Fusion of tag

  • Purification methods

  • Optional host cells

1-2 days

  • Programme analysis report

2. Expression System System Selection (Please skip this step if you can provide an expression strain.)

2.1 Optional expression vector


GAP promoter vector: pYE-GAPα;
Commercial Carrier: Secretory Expression: pPICZαA, pPIC9K,
Intracellular expression: pPICZA, pPIC3.5K.

2.2 Optional expression strain


GS115, X33 strain;
SMD116 protease-deleting strain.
3. Pastoris expression vector construction
Timeline
Deliverables
A. Gene Optimization
    Subcloning design
    Gene synthesizing
B. Subcloning to vector?
C. plasmid extraction & linearization-10ug
Gene synthesis: about 2 weeks
Subcloning: 1-2 weeks
Linearization: 2-3 days
1. Gene Synthesis Report
2. Sequencing verification report
3. E. coli DH5a containing the expression plasmid
4. Expression plasmid
5. Linearization detection image
4. Pichia pastoris protein expression/identification
Timeline
Deliverables
D. Electric shock conversion
E. Positive clone screening (PCR method)??
F. High copy strain screening (optional)
G. Expression test (5-10 strain)
H. Expression analysis and identification
I. Fix plants, optimize expression conditions
Electric conversion and identification: 1-2 weeks
High Copy Filter: 2-3 weeks (optional)
Express test and identification: 1-2 weeks
6. Positive expression strain
7. PCR identified positive strain map
8. High copy positive strain (optional)
9. Small test expression process report
10. WB results
11. Summary of small test expressions
5. Pichia pastoris protein purification/identification
Timeline
Deliverables
J. Scale up 2-5 liters
K. Expression of supernatant concentration
L. Ni column low pressure affinity purification
M. PAGE analysis and purification process
N. Identification of purified protein by WB
O. PFM detection (optional)
P. MS detection (optional)
Amplification culture: 2-3 weeks
Protein purification: 1-2 weeks
WB: 1 week
PMF: 2-3 weeks
MS: 1-2 weeks
12. Protein Purification Process Report
13. Protein purification original picture
14. Finished protein
15. Western Blot (tag antibody)
16. Protein fingerprint (optional)
17. Protein MS result (optional)

Supplementary explanation

1. Pichia pastoris cells have higher requirements for codon preference, and it is recommended to perform gene optimization before expression to improve success rate.
2. The expression levels of different proteins vary greatly. We will try our best to provide the protein more than mg level to customers.
3. Even if the order fails, we will provide a full set of experimental procedures and original photos, and return the expression strain and expression plasmid to the customer.