Large Scale Transient Expression Services

Phase 1: Construct Design and Confirmation

Kemp Proteins will use sequence files from clients. Kemp Proteins will design a cloning strategy based on optimal expression vector for stable cell line generation. The design will include Kozac and signal sequences for the production of soluble secreted protein in appropriate cells. The cloning strategy will be submitted to the client for approval. The completed sequences will be submitted to an outside vendor for gene synthesis and cloning into the expression vectors. Each vector will be checked for the proper insert size by PCR prior to amplification and purification.

• Estimated Time for Completion: 3-4 weeks
• Kemp Proteins Deliverables: QC data for each completed construct.

Phase 2: Test Expression and Purification Development

Kemp Proteins will amplify and purify a sufficient amount of each expression vector to perform test expressions at the 250 mL scale and larger scale expressions. The 250mL test expressions will be performed in shake-flask cultures of cells using Kemp Proteins’ protocols and protocols obtained from the literature or client. The cell pellet and culture supernatant from individual 250 mL cultures will be collected at 48, 72 and 96h. post-infection and analyzed by Western Blot and SDS PAGE (Coomassie Stain). The bulk supernatant and/or cell pellet from each test expression timepoint will be run over chromatography resin(s) selected by Kemp Proteins to obtain a purified preparation of materials. The purified protein will be concentrated and examined for the presence of target protein by SDS PAGE and Western Blot. Kemp Proteins will submit samples to an outside vendor for particle analysis by TEM. The samples will be negatively stained, imaged and high-definition TIFF files will be provided as the deliverable.

• Estimated Time for Completion: 4-5 weeks
• Kemp Proteins Deliverables: A data summary for the test expression including growth conditions,
• SDS PAGE, Western Blot and TEM data. A complete purification protocol.

Phase 3: 10-L Expression and Purification

Kemp Proteins will perform a 10-liter expression using cells and culture conditions identified in Phase 2 to produce a pilot batch of material. Kemp Proteins will harvest the cell culture supernatant and/or cell pellet containing the protein and purify it using methods developed in Phase 2. The purification will include an ion-exchange capture step followed by a size exclusion purification step. Purity of the final product will be assessed using Coomassie stained SDS PAGE (non-reducing) and Western Blot. Kemp Proteins will submit samples to an outside vendor for particle analysis by TEM. The samples will be negatively stained, imaged and high-definition TIFF files will be provided. The protein concentration of the final product will be determined using the BCA assay. The final buffer will be PBS pH 7.4 and the protein will be stored in aliquots at -80C prior to shipment on dry ice.

• Estimated Time for Completion: 3-4 weeks
• Kemp Proteins Deliverables: The purified protein and a complete batch record for the production and purification of the protein

Phase 4: 100-L Expression and Purification

Kemp Proteins will perform a 100-liter expression using appropriate cells and culture conditions identified in Phase 3 to produce a pilot batch of materials. Kemp Proteins will harvest the cell culture supernatant and/or cell pellet containing the protein and purify it using methods developed in Phase 3. The purification will include an ion-exchange capture step followed by a size exclusion purification step. Purity of the final product will be assessed using Coomassie stained SDS PAGE (non-reducing) and Western Blot.  Kemp Proteins will submit samples to an outside vendor for particle analysis by TEM.  The samples will be negatively stained, imaged and high-definition TIFF files will be provided. The protein concentration of the final product will be determined using the BCA assay. The final buffer will be PBS pH 7.4 and the protein will be stored in aliquots at -80C prior to shipment on dry ice.

• Estimated Time for Completion: 5-7 weeks
• Kemp Proteins Deliverables: The purified protein and a complete batch record for the production and purification of the protein.

Use our quick quote form to request a quote for your next large scale transient expression project.

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