Choice of fusion proteins, expression host, and analytics solves difficult‐to‐produce protein challenges in discovery research

Abstract High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein‐based discovery research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts—91% of which are successfully expressed and purified—are discussed. Mammalian expression combined with the use of solubility‐promoting fusion proteins is deemed suitable for most targets. Furthermore, cases utilizing stable cell line generation and choice of fusion protein for higher yield and quality of difficult‐to‐produce proteins (Leucine‐rich repeat‐containing G‐protein coupled receptor 4 (LGR4) and Neurturin) are presented and discussed. In the case of Neurturin, choice of fusion protein impacted the target binding 80‐fold. These results highlight the need for exploration of construct designs and careful Quality Control (QC) of difficult‐to‐produce protein reagents.