Immunogen expression, purification and verificationBesides immunogen design, gene synthesis (amplification) or subcloning, our full services for immunogen include optimization of construct containing appropriate N-terminal or C-terminal tags, transfection or transformation of the recombinant construct into appropriate protein expression system, rigorous testing to identify cells expressing the target protein, large-scale cell culture (production), and final recombinant protein purification using appropriate tag-specific affinity columns and FPLC if necessary. Besides SDS-PAGE verification, we strongly suggest to verify the expressed immunogen in other assays if customer has tools, such as ELISA with a known antibody.
We use a number of protein expression systems, including bacterial cells and mammalian cells, whichever is best for the type of antibody production.
HighlightsOptimized vector design—we will suggest the most effective design and expression system to ensure the production of antigen that is effective for antibody production
Protein immunogen production—use our expression system to generate and prepare protein immunogen for antibody production project
Expression systems and affinity tags—choose from bacterial, insect or mammalian protein expression systems and incorporate popular affinity tags for protein purification
Protein and antibodies to keep—any purified protein antigen remaining after completion of the antibody production project will be returned to you
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Details and additional information
Protein expression for antigen production
We usually use bacterial protein expression system as default, unless mammalian cell expression is necessary with one of the following considerations: post-translational modifications (PTM) are needed for protein specificity or the customer specifically needs the mammalian system.
General procedure
Our default protein expression using bacterial system, which typically takes six weeks to complete, is as follows:
We usually use bacterial protein expression system as default, unless mammalian cell expression is necessary with one of the following considerations: post-translational modifications (PTM) are needed for protein specificity or the customer specifically needs the mammalian system.
General procedure
Our default protein expression using bacterial system, which typically takes six weeks to complete, is as follows:
- Optimization if necessary—To avoid any ambiguity, we may need to optimize the nucleotide sequences with your agreement for best application in target animal species to elicit antibodies.
- Construction and seamless cloning—We can obtain sequence from a plasmid you supply or mutate sequence from supplied plasmid as you request, or synthesize the gene (extra cost). For plasmid you supply, we need vector and plasmid sequences, a vector map, and any relevant information, like antibiotic resistance. For mutagenesis, except plasmid info, we also need mutation sites. Be noted, mutated construct may loss stability and cause protein expression failure. We also offer seamless cloning to clone complicated fragments into a vector (extra cost).
- Transformation and selection—We transform recombinant construct into a high-efficiency expression bacterial strain, select positive colonies, induce the high expression of the target protein and verify the expressed protein.
- Delivery—The process typically produces 3 to 5mg of protein, which is sufficient for antibody production projects. If desired, we can supply a small sample of the protein and/or cell stock for you to test before animal immunization. Any protein remaining from the antibody production project is delivered to you upon completion.
- Optimization if necessary—Codon-optimization of the nucleotide sequences may be necessary for mammalian expression system.
- Construction and seamless cloning--We can obtain sequence from a plasmid you supply or mutate sequence from supplied plasmid as you request, or synthesize the gene (extra cost). For plasmid you supply, we need vector and plasmid sequences, a vector map, and any relevant information, like antibiotic resistance, to subclone to a vector containing signal peptide and purification tag. For mutagenesis, except plasmid info, we also need mutation sites. Be noted, mutated construct may loss stability and cause protein expression failure. We also offer seamless cloning to clone complicated fragments into a vector (extra cost).
- Transfection—After the construct is sequenced, we will transfect it into insect or mammalian cell line to express antigen protein.
- Purification—Expressed protein will be released outside of cells and purified by tag. Additional purification by FPLC might be necessary.
- Delivery and verification—Purified protein will be verified in SDS-PAGE or Native blue gel; Further verification using customer's tools can be requested. We will deliver a small sample for you to test before animal immunization.