Peak Proteins is CRO that specialises in the production of bespoke protein reagents, both soluble and membrane proteins, and protein structure determination using primarily x-ray. When we were recently tasked with producing a full-length transcription factor with no solved structure and an AlphaFold prediction of ‘spaghetti’, we knew this was going to be a challenging target. Heavily truncated forms of the protein had previously been produced (and even crystallised!) but expressing the full-length protein, with all its disordered regions, was vital to study its dimerisation and establish assays to inhibit this protein:protein interaction.
The client had previously tried expressing the protein with different fusion partners at the N or C termini in both insect and Expi293 cell systems at other CROs but a number of problems had been encountered which included low yields and protein degradation. Even when success was achieved at small scale, it proved impossible to repeat or scale up. Our approach was to try all three expression systems at our disposal, E.coli, insect and HEK293 cells but with carefully chosen fusion partners and evaluate their suitability for protein production. Our early work showed that the protein could be successfully expressed at significant levels in our highly optimised insect and E.coli systems. Through careful evaluation and troubleshooting at each stage, we were able to generate mg quantities of the protein and also produce in vivo biotinylated protein in insect cells for downstream studies, as confirmed by intact mass spectrometry analysis. We will present data showing the steps taken and our biophysical characterisation of the transcription factor, along with key learnings from the project.
