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Mammalian cells have the ability to produce the most complete post-translational modifications and to secrete functioning proteins that are correctly folded and contain the most complete tertiary protein structure. Mammalian cells are widely used in the production of recombinant proteins, antibodies, virus, viral-subunit proteins, and gene-therapy vectors. Large-scale and fast mammalian transient system for protein expression is of great concerns to researchers conducting drug discovery projects.
Phase I: Subcloning into Expression Vector
Phase II: (CMV Based): Expression in Target Cells
Phase III: (Retrovirus): Expression in Target Cells
Phase IV: Protein Extraction & Purification
Phase I: Subcloning into Expression Vector
Description:
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Amplification/isolation of the gene of interest out of a customer-supplied vector and subcloning it into a CMV promoter based high-level expression vector or a Phoenix retroviral vector
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Verification of the subcloned gene by restriction digest and sequencing
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Preparation of the recombinant vector DNA
Required Materials:
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5 ug of plasmid containing gene of interest plus complete vector sequence in electronic format
Deliverables:
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20 ug of expression-ready recombinant CMV based mammalian expression plasmid containing the gene of interest or 20 ug of Phoenix retroviral vector (ready for use) containing the gene of interest
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Final sequencing data
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Detailed research report
Duration:
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2 weeks; If insert > 2kb, an additional week per 1kb.
Price (USD):
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$960; If insert > 2kb, an additional $350 per 1kb.
Phase II: (CMV Based): Expression in Target Cells
Description:
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Transfection of recombinant vector into target cells (107 cells. Choose from 293, CHO, COS-7, and NIH 3T3) for transient expression
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Harvest and lysis of cells (usually after 2-3 days)
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Test for recombinant protein by western blot (customer must provide appropriate antibodies), if desired
Required Materials:
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20 ug of ready for use CMV based recombinant mammalian expression plasmid containing the gene of interest plus complete vector sequence in electronic format.
Deliverables:
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Cell pellet from 107 cells after transient expression
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Western Blot data (if desired)
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Detailed research report
Duration:
Price (USD):
Optional: For His or GST tagged protein, you may choose to use our antibodies in stock for an additional charge of $25.
Phase III (Retrovirus): Expression in Target Cells
Description:
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Transfection of recombinant vector into amphotropic or ecotropic packaging cells (107 cells) for transient expression
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Retrovirus isolation and titration
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Infection of target cells for transient recombinant protein expression (107 cells)
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Harvest and lysis of cells (usually after 2-3 days)
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Test for recombinant protein by SDS-PAGE and Western blot (customer must provide appropriate antibodies), if desired
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Excess of retroviral stock may be provided to customer
Required Materials:
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20 ug of ready for use retrovirus expression plasmid containing the gene of interest plus the complete vector sequence in electronic format.
Deliverables:
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Cell pellets from 1 liter fermentation
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SDS-PAGE or Western Blot data
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Detailed research report
Duration:
Price (USD):
Optional:
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For His or GST tagged protein, you may choose to use our antibodies in stock for an additional charge of $25
Phase IV: Protein Extraction & Purification
Description:
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Cell pellet harvested after transient expression will be lysed with our optimized reagents and protocol
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Protein purification using appropriate affinity resin for 6xHis or GST-tagged recombinant proteins
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Purification can be performed either under native or denatured conditions (as desired by the customer)
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SDS Page and Western blotting will be performed for testing protein expression
Required Materials:
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Cell pellet harvested after transient expression.
Deliverables:
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Protein solution in standard or customer-specified buffer
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SDS-PAGE or Western Blot data
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Detailed research report
Duration:
Price (USD):
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$1420/L, discounts offered on larger volumes
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