Tools for fragment based drug discovery

Protein Production

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Protein expression is done in bacteria (E. coli). Expression in coli is typically less expensive, faster, more amenable to rapid mutagenesis and enables the greatest flexibility in isotope labeling for advanced NMR studies. A number of removable tags for affinity purification and immobilization are available. The choice of tag depends on the nature of the protein and the studies to be performed. Recombinant proteins are always extensive characterized for functionality as a critical part of a Go/No Go decision point. An array of biochemical assays, including enzyme activity and ligand binding, as well as biophysical assays is applied to assess the functionality of the proteins.

When protocols for expressing proteins are available, expression constructs can be rapidly generated. Where a customer’s target is not previously structural biology enabled, we will design an approach to rapidly assess the optimal protein construct. Typically this involves generating multiple versions of the protein with variable N- and C-termini and possibly investigating fusion partners as well. We can readily probe large numbers of clones (20,000-100,000) for the expression of soluble protein in E. coli and produce suitable protein variants of challenging targets that were not previously amenable to structural studies.

Simple and sensitive 1D 1H NMR can then be used to make a preliminary assessment of the multimeric state of the protein in solution as well as the degree of folding. This unique approach allows us to quickly find proteins that are suitable for biochemical, biophysical, NMR and crystallography studies.

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