Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

Improving the robustness of engineered bacteria to nutrient stress using programmed proteolysis

The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and the freeing of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-messenger RNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately seven times more efficient. Based on these results, we then show how proteolysis can be used to improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.

Intracellular phase separation of globular proteins facilitated by short cationic peptides

Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We add to this toolbox by designing short, highly charged peptide tags that provide several key advantages for engineering protein phase separation. Herein, we report the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

Modular Assembly of Phosphite Dehydrogenase and Phenylacetone Monooxygenase for Tuning Cofactor Regeneration

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD–RIAD) system. These peptides allow self-assembly based on specific protein–protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer–Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD–PTDHRIDD and PAMORIAD/RIAD–PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH–PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO–PTDH complex less PTDH was required for the same performance when compared with the PTDH–PAMO fusion enzyme.

Solubility Controlling Peptide Tags of Opposite Charges Generate a Bivalent Immune Response Against Dengue ED3 Serotypes 3 and 4

We previously demonstrated that a protein’s immunogenicity could be substantially increased by attaching a hydrophobic solubility controlling peptide tag (SCP-tag) producing small sub-visible aggregates. Here, we report the oligomerization of Dengue envelop protein domain 3 (ED3), and consequently, its immunogenicity increase by mixing ED3s attached with SCP-tags of opposite charges at equimolar concentration. We used ED3 of serotype 3 (D3ED3) and serotype 4 (D4ED3), which are, respectively, moderately and poorly immunogenic, and their SCP tagged variants constructed by attaching either a C-termini 5-Aspartic acid (C5D) or a 5-Lysine (C5K) tag. Light scattering indicated that the isolated tagged ED3s remained monomeric, but mixing the C5D and C5K tagged ED3s at equimolar concentration generated sub-visible aggregates or oligomers of ~500 nm through electrostatic interaction. In addition, the oligomerized ED3s remained in a native-like state, as assessed by fluorescence spectroscopy and circular dichroism. The in vivo immunogenicity of the D3ED3 and D4ED3 oligomers generated by the charged tags increased by 5 and 16 fold, respectively. Furthermore, injection of heterotypic ED3 oligomers (D3C5D+D4C5K) induced an immune response against both D3ED3 and D4ED3 in 3 of 4 responsive mice, and the IgG titer of the bivalent anti-D3C5D-D4C5K sera was over 100 times higher than that generated by co-injecting the untagged D3ED3 and D4ED3 (D3+D4). Altogether, these observations suggest that SCP-tags could be used as a platform for producing a long-sought tetravalent dengue vaccine.

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

ABSTRACT Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.