Identification of correct expression construct boundaries resulting in high-yield protein production can be time- and labour intensive especially if the protein of interest has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using members of a structurally uncharacterized, eukaryotic receptor family as examples we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify expression constructs that can be produced with high yields and moderate costs. Furthermore, we show that HDX-MS can also serve as a valuable tool in validating AlphaFold structure predictions.
