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Expression of yeast protein
Our company has Successfully builded a complete variety of protein expression technology platform: including the prokaryotic protein expression and purification, expression and purification of yeastprotein, expression and purification of protein which from insect cells, expression and purification of proteins from mammalian cells. According to customer needs, we can offer one-stop services which comprise gene synthesis, vector, gene expression, protein purification, detection and other . Our company has established a strict quality control system and a set of perfect customer service processes, confidentiality system to ensure that customers would get the quality products and services Our team has very rich experience in optimizing gene codon and expression vector design and construction, integration of objects and tags selection, specific protein purification system selection, protein production process optimization and so on. we canmeet the needs of individual customers.
Yeast protein expression
Recombinant protein expression and purification technology service (Pichia P. expression system)
The expression system includes: prokaryotic expression system (Escherichia coli expression system and yeast expression system (P. Pichia pastoris expression ) . Our company has established strict product quality control system ,a set of perfect customer service process, security system to ensure to provide our customers with quality products and service. Our services charges according to the steps,you can choose any of the following steps to start the experiment or only choose one of the single step, please contact us.
Yeast expression system -- step 1 subclone of target gene into yeast expression vector:
We can amplify and extract the expression plasmid containing the target gene was.
subclone of target gene into yeast expression vector
The accuracy of plasmid was verified by sequencing.
We can provide expression vector: pPICZ alpha and pPIC9K
To provide customers: the correct construction of the recombinant expression plasmid, the recombinant plasmid containing DH5 strain and sequencing report.
Service cycle: 1 weeks
Yeast expression system - step 2 Pichia P. expression strain electrical transformation:
Enzyme expression plasmid.
The expression plasmid was transformed into a highly efficient Pichia P. expression strain by electroporation.
We can remove the Tags by the application of Protease, and then target proteins are obtained .
At least 5 positive clones were identified by PCR.
We can provide expression strains: X33, GS115, KM71 and SMD1168.
To provide customers: PCR positive clones and PCR results report.
Time: 1 weeks
Yeast expression system - step 3 Pichia P. expression strain screening:
The 5 positive clones were amplified in BMGY culture medium, and then were changed to BMMY culture medium, and the expression of target protein was induced by methanol.
The expression of target protein in the supernatant was detected by SDS-PAGE electrophoresis, and the suitable monoclonal strain was selected for the expression of the target protein.
If no expression was detected in the culture supernatant, the expression of the target protein was detected by the concentration of the supernatant 20 times, or by the Western-blot method.
To provide customers: any customer needs positive clones and expression screening results report.
Time: 2 weeks
Yeast expression system -- step 4 Pichia P. expression optimization:
20 positive clones were selected for routine expression screening, and the optimal expression conditions were optimized.
Optimization of culture and induction conditions (culture medium, cell density, methanol concentration, induction time, etc.), and the expression of target protein was improved.
To provide customers: any three customers need positive clones strains and optimize the expression of the results of the report.
Time: 2 weeks
Yeast expression system -- step 5 expression and purification of target protein:
1L recombinant yeast was cultured in shake flask , induce the expression of target protein.
By AffinityChromatography, Ion Chromatography , Gel chromatography and so on ,the expressed recombinant protein was purified .
We will check the results of each test and control the quality of the final product through SDS-PAGE electrophoresis .
Through the Western-blot , we verify target protein is right or not .
Provide customers: purified target protein 1~10mg and purification report. purified target protein , report ,tag (Tag) or the final protein which has removal of the tag (Tag) . the purity will be more than 90%.
Time: 2~3 weeks
• Yeast expression system -- step 6: remachiningand detection of target protein
The protein product with high biological activity is studied to renature.
Endotoxin removal, sterilization, freeze-drying and further processing.
The quality of target protein was detected by HPLC, mass spectrometry and other methods.
Service content:
1 complex research:
Using up to 18 different types of complex buffer system, a minimum of target protein was renaturated with different refolding buffer system for customer to detect. According to customer requirements, a small quantity of refolded protein could be preparated according to the renaturation condition of one kind of sample. We can provide the renaturation buffer and renaturation process specific formula.
Other: sterilization, freeze-drying and further processing.
With the quality of detection of target proteins.
2 endotoxin removal
3 filtration
4 freeze dried
5.HPLC detection
6 mass spectrometry
Provide customer: final products and related experiment and test report.
Time: 1~2 weeks
Yeast expression system - step 7 preparation
Provide customers: qualified target protein and service report.
Time: negotiation
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