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Yeast protein expression
Yeast is a single cell low eukaryote, which not only has the advantages of rapid growth, easy culture and simple operation of prokaryote cells, but also has the functions of protein processing and modification of eukaryotes.As the most studied eukaryote, it has been applied to heterologous protein expression for a long time.Compared with prokaryotic expression system, yeast is easy to express proteins with good activity due to the existence of eukaryotic protein modification system; compared with other eukaryotic expression systems such as insects, animal and plant cells, it has advantages of rapidity, simplicity and low cost.Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris and Yarrowia lipolytica are the main hosts of protein expression, among which Saccharomyces cerevisiae and Pichia pastoris are the most widely used.
Yeast protein expression
Expression and purification of recombinant protein (P. Pichia expression system)
Recombinant proteins have many applications, including antigen preparation, protein interaction, enzyme analysis, and drug target research.The selection of appropriate protein expression system has an important impact on the application of downstream proteins.Protein function, yield and cost are the priority factors.
Trigoats Co., Ltd. has established a strict product quality system, a complete set of perfect customer service process and confidentiality system to ensure to provide customers with high-quality products and mature services.
Technical services are charged by steps. You can choose to provide services from any of the following steps or only choose a single step. Please contact us for detailed price.
•Yeast expression system step 1. Subcloning the target gene into yeast expression vector:
The vector plasmid containing the target gene was amplified and extracted.
The target gene was subcloned into a suitable expression plasmid.
The accuracy of the constructed plasmid was verified by sequencing.
Expression vectors: pPICZ α and pPIC9K
To provide customers with: correctly constructed recombinant expression plasmid, DH5 α strain containing recombinant plasmid and sequencing report.
Service period: 1 week
•Yeast expression system step 2. P. Pichia expression strain electrotransformation:
The expression plasmid was tangent.
The expression plasmid was transformed into P. Pichia expression strain by electricity.
We use protease to remove the unnecessary tag, and then purify the target protein (optional, when constructing the expression vector, we need to add the appropriate protease cutting site).
At least 5 positive clones were identified by PCR.
The expression strains are x33, GS115, km71 and smd1168.
Provide customers: PCR positive clone and PCR result report.
Time: 1 week
• Yeast expression system step 3. P. Pichia expression strain screening:
Five positive clones were amplified in bmgy medium, then transferred to BMMY medium, and methanol was added to induce the expression of the target protein.
SDS-PAGE was used to detect the expression of the target protein in the culture supernatant, and suitable monoclonal strains were selected for the expression of the target protein.
If no expression can be detected in the culture supernatant, the target protein expression can be detected by concentrating the supernatant 20 times, or by Western blot method (the customer needs to provide relevant antibodies).
Provide customers: report of positive clone strains and expression screening results required by any customer.
Time: 2 weeks
•Yeast expression system step 4. P. Pichia expression optimization:
20 positive clones were selected for routine expression screening, and the best expression conditions were optimized.
For the selected monoclonal strains, the culture and induction conditions (medium, cell density, methanol concentration, induction time, etc.) were optimized to improve the expression of the target protein.
Provide customers: report on the results of positive clone strains and optimized expression conditions required by any three customers.
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•Yeast expression system step 5. Expression and purification of target protein:
1 l recombinant yeast was cultured in shake flask to induce the expression of target protein.
The recombinant proteins were purified by affinity chromatography, ion exchange, hydrophobicity and gel filtration.
The results of each step were detected by SDS-PAGE electrophoresis and the quality of the final product was controlled.
Western blot was used to verify the correctness of the target protein.
Provide customers: purified target protein 1-10mg and purification report.We can provide the final protein containing purified tag or excised purified tag according to the customer's requirements, with the purity up to 90%.
Time: 2-3 weeks
•Yeast expression system step 6. Target protein reprocessing and detailed detection
The refolding of purified protein with high biological activity was studied.
Endotoxin removal, sterilization, freeze-drying and other further processing.
The quality of the target protein was determined by HPLC and MS.
Service content:
1. Renaturation study:
By using up to 18 different refolding buffer systems, small amount of refolding test was carried out on the target protein to provide samples for customers to detect after refolding.
A small amount of refolding protein can be prepared according to the refolding conditions of one sample according to the customer's requirements.
It can provide the specific formula and process of refolding buffer.
2. Endotoxin removal
3. Filtration and sterilization
4. freeze-drying
5.HPLC detection
6. Mass spectrometry
Provide customers: final products after processing and relevant test and inspection reports.
Time: 1-2 weeks
•Yeast expression system step 7. Large scale preparation
Provide customers: qualified target protein and service report.
Time: negotiation
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