Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high‐quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent “antifreezes”. Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid‐phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants.

Highlights d eCIS loci are widely distributed among bacteria genomes d eCIS loci encode phage-tail-like proteinaceous machines d eCIS superfamily is grouped into six families with distinct genetic features

Photorhabdus is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small molecule effectors. Among these, the Photorhabdus Virulence Cassettes (PVCs) represent an elegant self-contained delivery mechanism for diverse protein toxins. Importantly, these self-contained nanosyringes overcome host cell membrane barriers, and act independently, at a distance from the bacteria itself. In this study, we demonstrate that Pnf, a PVC needle complex associated toxin, is a Rho-GTPase, which acts via deamidation and transglutamination to disrupt the cytoskeleton. TEM and Western blots have shown a physical association between Pnf and its cognate PVC delivery mechanism. We demonstrate that for Pnf to exert its effect, translocation across the cell membrane is absolutely essential.

Tissue engineering,g ene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture,b ut current methods to cryopreserve cells in this format can give lowc ell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as am inimum (bio)synthetic mimic of antifreezep roteins that is accessible by solution, solid-phase,a nd recombinant methods.W ed emonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines.P olyproline mayb e av ersatile additive in the emerging field of macromolecular cryoprotectants.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.

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