Cancer drug discovery campaigns frequently require stocks of high-quality recombinant proteins for assay development, fragment screening, binding studies, and structural biology. Protein engineering and production at CSIRO Manufacturing Parkville includes construct design and utilisation of bacterial, insect cell, and mammalian expression systems to over-express such proteins. For the Cancer Therapeutics CRC, a wide variety of targets have been produced as the first steps in the drug discovery pipeline, including WD repeat-containing protein 5 (WDR5), Stimulator of Interferon Genes protein (STING), Ectonucleotide pyrophosphatase 1 (ENPP1), and the histone acetyltransferase KAT5. Results presented will describe protein engineering approaches taken to obtain consistent and high-quality preparations of these proteins, and their validation using biophysical techniques such as mass spectrometry and crystallography.