At Intact Genomics, we are experts in leading your projects related to gene cloning, protein expression and purification from start to finish. We can assist you in the optimization of the key conditions that will aid your team in GMP production. For optimal results for your project, we utilize our state-of-the-art facility and equipment including the most advanced ÄKTA Avant 150 chromatography system and temperature humidity-controlled walk-in cold room. Our team can custom-purify significant quantities of proteins for various research purposes to meet customers’ needs.
- End-to-end service – complete your project from design, clone, express, purify, and QC test to provide a deliverable that is a complete solution to your protein expression and purification needs
- Utilizing novel technology including our seamless cloning with IG’s Quick10™ Cloning Kit, we simplify your gene design with the only requirement being 15bp homologous sequence of gene ends with an expression vector, speeding up your project
- Planning and production of large amounts of endotoxin-free proteins
- Validation of purification strategies via FPLC
- Dedicated cold room for protein purification
- Analysis of purified protein samples by both reverse phase and size exclusion high-performance liquid chromatography
- Limulus amebocyte lysate (LAL) testing to determine endotoxin concentrations
- Digestion by tobacco etch virus (TEV) protease to remove tags
- Endotoxin removal
- Expertise in membrane protein purification
Protein Expression and Purification Service
1. Analysis and process development
Each target protein will require analysis to determine physical characteristics that can be exploited in order to purify it from bulk cell lysate. While our expression platforms will include affinity tags that are capable of separating out most proteins, it is possible that a given protein will require several methods of chromatography to reach a high enough standard of quality.
Once an initial strategy has been determined, a detailed procedure will be written in accordance with GMP guidelines so clients will easily be able to scale successful purification strategies accordingly. If a particular resin is required for the purification of the target protein, it should be ordered at this stage, prior to beginning the cloning work. Any variation from the proposed approach will be documented and communicated with the client.
2. Construct design, cloning, and expression optimization
Each target will be either amplified from genomic DNA/plasmids or a synthesized gene with ends that allow for simple seamless assembly into our expression vectors or your own expression vector. The constructs will be transformed into an expression strain, and the expression of the target protein will be optimized by testing the conditions of expression (temperature, media, inductions conditions), as well as the most ideal tags (type of affinity tag and position). Tags can be designed with a tobacco etch virus (TEV) cleavage site as a linker in case the removal of the tag is necessary for protein function. Once the ideal expression conditions have been found, we can scale up to a 6L batch size. After expression, the cells will undergo lysis by lysozyme and sonication. Proteins that localize to inclusion bodies will be tested for their ability to resolubilize for purification
3. Protein Purification
In most cases the first step for protein purification will be affinity chromatography, using the affinity tag present in the construct (e.g., His6-Tag, Maltose Binding Protein, Glutathione S-Transferase, etc.). By starting the purification with the affinity tag, subsequent purifications will remove the eluent which may interfere with downstream processes. The subsequent purifications will depend on the physical characteristics of each protein, but potential strategies that we currently have material for include ion exchange, hydrophobic interaction, and size-exclusion chromatography. Purified protein will be >95% pure as determined by SDS-PAGE and Coomassie staining.
4. TEV digestion and purification
Intact Genomics has previously generated TEV protease successfully and will generate in-house TEV protease for the cleavage of affinity tags from purified protein. At the client’s request, the target protein will be digested overnight, and further purified to separate the affinity tag from the mature protein.
In addition to chromatograms and standard SDS-PAGE analysis for each purification, Intact Genomics can develop analysis strategies for each protein target in coordination with the client. Depending on the future applications in client hands, the protein can be tested for endotoxin concentrations via LAL cartridges or for nuclease activity, to help determine continued contamination by low-abundance/high-activity proteins.
Get started by simply emailing email@example.com with details on your project!