Cell-free protein production of a gamma secretase homolog

Cleavage of the transmembrane domain (TMD) of amyloid-beta precursor protein (APP) by gamma-secretase, an intramembrane aspartyl protease, generates A beta peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of gamma-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of gamma-secretase with its substrates APP and Notch, the mechanism of gamma-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma.The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to gamma-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 C-degrees, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.