Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy

PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression and purification of uniformly 13C- and 15N-labeled PAPf39 peptide, through expression as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH.

► A protocol was developed to prepare pure isotopically labeled PAPf39 peptide for NMR.
► PAPf39 peptide was purified by intein mediated self-cleavage and TEV protease cleavage.
► Recombinant PAPf39 showed amyloid fibril formation.
► Chemical shift for backbone atoms were assigned.