1. Programme analysis
Timeline
Deliverables
Optimization of codon
Fusion of tag
Purification methods
Optional host cells
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.