Lytic phages, such as lambdoid enterobacteriophage φ21, infect bacterial hosts in order to exploit their biosynthetic machinery for reproduction. The subsequent release of virions is ensured by a complex interplay of effectors resulting in the lysis of the host cell. This process is orchestrated by a small, helical, amphiphilic membrane protein called pinholin, via time-specific formation of homo-oligomeric pores in the host cell membrane – the eponymous pinholes.¹ While translocation of a transmembrane domain to the membrane surface (flipping) has been proposed to convert pinholin into its active, pore-forming state ^2,3, sufficient experimental verification is lacking.
Here, we aimed to produce sufficient amounts of pinholin for future solid-state NMR- and cryogenic transmission electron microscopy-based structural investigation.
To overcome its intrinsic cytotoxicity, we opted to trap the protein in its non-flipped, inactive conformation by means of intramolecular cyclisation, utilising the SpyTag and SpyCatcher fusion tags.⁴ After tag cleavage and chromatographic purification, functionality and correct fold of the protein were verified via a fluorescence-based vesicle leakage assay and circular dichroism spectroscopy, respectively.
Cyclisation of pinholin was succesful and significantly reduced its toxicity. Upon optimisation of the fusion construct and expression conditions, milligram-amounts of functional and correctly folded pinholin could be purified to apparent electrophoretic homogeneity.
In summary, we could establish a method for the biosynthetic production of large amounts of functional pinholin, which can be used in future experiments to elucidate the structural basis of its activation mechanism.
1. Pang, T. et al. PNAS 110, E2054–E2063 (2013)
2. Steger, L. M. E. et al. PNAS 117, 29637–29646 (2020)
3. Pang, T. et al. PNAS 106, 18966–18971 (2009)
4. Schoene, C. et al. Methods Enzymol 580, 149–167 (2016)