Protein Production for Assay Development and Structural Studies
The availability of sufficient protein of good quality can be rate limiting for many drug discovery programs, as a lack of target protein supply can hamper the ability to establish hit screening assays and to complete structural studies. Domainex's biology team is able to clone and express drug targets of interest and can produce protein at the required levels for ongoing biochemical compound screening, using a range of approaches.
Domainex's Proprietary Technology - Combinatorial Domain Hunting (CDH)
In situations where standard molecular biology approaches will not be suitable or time is crucial, Domainex's proprietary Combinatorial Domain Hunting (CDH) approach will enable the fast identification of suitable protein constructs.
CDH is the unique, proprietary technology for quickly identifying soluble, highly expressible constructs of drug target proteins, that are able to bind ligand and are suitable for use in structural studies. This results in the ability to develop drug target binding or activity assays and to complete compound screening within exceptionally fast timelines.
CDH combines a method for the production of unbiased finely-sampled gene-fragment libraries, with a screening protocol that provides 'holistic' readout of solubility, ligand binding and yield for thousands of protein fragments.
CDH has an established pedigree: over 50 genes have been studied to date, including projects for many of the 'Top 20' global pharmaceutical companies (click her to view UCB press release on successful use of CDH technology). The entire process takes only 3-4 months from target to binding assay and can enable significant advancement in early discovery phase programs.
In brief, the CDH process consists of following key steps:
- Firstly, we synthesise a recoded version of the drug target gene using our proprietary algorithms, which optimises the gene for expression in E. coli and for our fragmentation technology.
- To generate random fragments of the target gene we carry out a modified PCR in the presence of deoxyuridine so that uracil is partially incorporated in place of thymine. We then use a mixture of enzymes which we have designed to recognise sites where uracil is present and create blunt-ended double-strand breaks in the DNA at these points. Electrophoresis of the fragmented DNA allows us to select libraries that correspond to the approximate size of the desired piece of protein.
- These DNA libraries are cloned into proprietary vectors that allow expression of all potential open reading frames. The final construct also includes a C-terminal poly-histidine tag sequence.
- Typically a library of 20,000-100,000 E. coli clones is plated, grown, and probed for the expression of soluble poly-histidine fusion protein. Typically about 750 colonies expressing high-levels of protein are selected and grown as larger cultures. Soluble extracts from these cultures are partially purified, and we are then able to confirm the solubility and stability of each protein fragment, and its level of expression.
- At this stage we are also able to identify which of these protein fragments are able to bind to a known ligand (which could be a small-molecule probe, or a peptide). A number of clones expressing functional (i.e. ligand-binding) recombinant protein which is of the correct size, soluble, and antigenically correct will typically be identified. These clones are end-sequenced to confirm their identity and location on the parental gene sequence.
- Using the ligand binding protein constructs, binding or activity assays for use in hit screening are optimized.
Following the identification of positive clones that express significant amounts of soluble drug target protein, protein can also be purified for use in x-ray crystallography studies.
For more information regarding Domainex’s CDH drug discovery technology platform, please contact us at firstname.lastname@example.org.
Relevant publications relating to this technology can be found by clicking here.