Self-assembled protein vesicles as vaccine delivery platform to enhance antigen-specific immune responses.

Self-assembling protein nanoparticles are beneficial platforms for enhancing the often weak and short-lived immune responses elicited by subunit vaccines. Their benefits include multivalency, similar sizes as pathogens and control of antigen orientation. Previously, the design, preparation, and characterization of self-assembling protein vesicles presenting fluorescent proteins and enzymes on the outer vesicle surface have been reported. Here, a full-size model antigen protein, ovalbumin (OVA), was genetically fused to the recombinant vesicle building blocks and incorporated into protein vesicles via self-assembly. Characterization of OVA protein vesicles showed room temperature stability and tunable size. Immunization of mice with OVA protein vesicles induced strong antigen-specific humoral and cellular immune responses. This work demonstrates the potential of protein vesicles as a modular platform for delivering full-size antigen proteins that can be extended to pathogen antigens to induce antigen specific immune responses.

Dual Antibacterial Properties of Copper-Coated Nanotextured Stainless Steel.

Bacterial adhesion to stainless steel, an alloy commonly used in shared settings, numerous medical devices, and food and beverage sectors, can give rise to serious infections, ultimately leading to morbidity, mortality, and significant healthcare expenses. In this study, Cu-coated nanotextured stainless steel (nSS) fabrication have been demonstrated using electrochemical technique and its potential as an antibiotic-free biocidal surface against Gram-positive and negative bacteria. As nanotexture and Cu combine for dual methods of killing, this material should not contribute to drug-resistant bacteria as antibiotic use does. This approach involves applying a Cu coating on nanotextured stainless steel, resulting in an antibacterial activity within 30 min. Comprehensive characterization of the surface revealing that the Cu coating consists of metallic Cu and oxidized states (Cu2+ and Cu+), has been performed by this study. Cu-coated nSS induces a remarkable reduction of 97% in Gram-negative Escherichia coli and 99% Gram-positive Staphylococcus epidermidis bacteria. This material has potential to be used to create effective, scalable, and sustainable solutions to prevent bacterial infections caused by surface contamination without contributing to antibiotic resistance.

Self-Assembled Recombinant Elastin and Globular Protein Vesicles with Tunable Properties for Diverse Applications.

Vesicles are self-assembled structures comprised of a membrane-like exterior surrounding a hollow lumen with applications in drug delivery, artificial cells, and micro-bioreactors. Lipid or polymer vesicles are the most common and are made of lipids or polymers, respectively. They are highly useful structures for many applications but it can be challenging to decorate them with proteins or encapsulate proteins in them, owing to the use of organic solvent in their formation and the large size of proteins relative to lipid or polymer molecules. By utilization of recombinant fusion proteins to make vesicles, specific protein domains can be directly incorporated while also imparting tunability and stability. Protein vesicle assembly relies on the design and use of self-assembling amphiphilic proteins. A specific protein vesicle platform made in purely aqueous conditions of a globular, functional protein fused to a glutamate-rich leucine zipper (ZE) and a thermoresponsive elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) is discussed here. The hydrophobic conformational change of the ELP above its transition temperature drives assembly, and strong ZE/ZR binding enables incorporation of the desired functional protein. Mixing the soluble proteins on ice induces zipper binding, and then warming above the ELP transition temperature (Tt) triggers the transition to and growth of protein-rich coacervates and, finally, reorganization of proteins into vesicles. Vesicle size is tunable based on salt concentration, rate of heating, protein concentration, size of the globular protein, molar ratio of the proteins, and the ELP sequence. Increasing the salt concentration decreases vesicle size by decreasing the Tt, resulting in a shorter coacervation transition stage. Likewise, directly changing the heating rate also changes this time and increasing protein concentration increases coalescence. Increasing globular protein size decreases the size of the vesicle due to steric hindrance. By changing the ELP sequence, which consists of (VPGXG)n, through the guest residue (X) or number of repeats (n), Tt is changed, affecting size. Additionally, the chemical nature of X variation has endowed vesicles with stimuli responsiveness and stability at physiological conditions.Protein vesicles have been used for biocatalysis, biomacromolecular drug delivery, and vaccine applications. Photo-cross-linkable vesicles were used to deliver small molecule cargo to cancer cells in vitro and antigen to immune cells in vivo. pH-responsive vesicles effectively delivered functional protein cargo, including cytochrome C, to the cytosol of cancer cells in vitro, using hydrophobic ion pairing to improve cargo distribution in the vesicles and release. The globular protein used to make the vesicles can be varied to achieve different functions. For example, enzyme vesicles exhibit biocatalysis, and antigen vesicles induce antibody and cellular immune responses after vaccination in mice. Collectively, the development and engineering of the protein vesicle platform has employed amphiphilic self-assembly strategies and rational protein engineering to control physical, chemical, and biological properties for biotechnology and nanomedicine applications.

Development of Self-Assembled Protein Nanocage Spatially Functionalized with HA Stalk as a Broadly Cross-Reactive Influenza Vaccine Platform.

There remains a need for the development of a universal influenza vaccine, as current seasonal influenza vaccines exhibit limited protection against mismatched, mutated, or pandemic influenza viruses. A desirable approach to developing an effective universal influenza vaccine is the incorporation of highly conserved antigens in a multivalent scaffold that enhances their immunogenicity. Here, we develop a broadly cross-reactive influenza vaccine by functionalizing self-assembled protein nanocages (SAPNs) with multiple copies of the hemagglutinin stalk on the outer surface and matrix protein 2 ectodomain on the inner surface. SAPNs were generated by engineering short coiled coils, and the design was simulated by MD GROMACS. Due to the short sequences, off-target immune responses against empty SAPN scaffolds were not seen in immunized mice. Vaccination with the multivalent SAPNs induces high levels of broadly cross-reactive antibodies of only external antigens, demonstrating tight spatial control over the designed antigen placement. This work demonstrates the use of SAPNs as a potential influenza vaccine.

Dual-Antigen Subunit Vaccine Nanoparticles for Scrub Typhus.

Orientia tsutsugamushi is the causative pathogen of scrub typhus, an acute febrile disease prevalent in the Asia-Pacific region that is spread to people through chigger bites. Despite the emerging threat, there is no currently available vaccine against O. tsutsugamushi. Here, we developed dual-antigen subunit vaccine nanoparticles using recombinant 47 kD and 56 kD proteins, which are immunogenic outer membrane antigens of O. tsutsugamushi. The biocompatible protein vaccine nanoparticles were formed via desolvation of r56 or r47E antigens with acetone, coating with an additional layer of the 56 kD protein, and stabilization with reducible homobifunctional DTSSP and heterobifunctional SDAD crosslinkers. The dual-antigen subunit vaccine nanoparticles significantly improved antigen-specific antibody responses in vaccinated mice. Most importantly, the dual-antigen nanoparticles coated with an additional layer of the 56 kD protein were markedly more immunogenic than soluble antigens or single-antigen nanoparticles in the context of cellular immune responses. Given the significance of cellular immune responses for protection against O. tsutsugamushi, these results demonstrate the potent immunogenicity of dual-layered antigen nanoparticles and their potential as a promising strategy for developing vaccines against scrub typhus.

Chemical and biological conjugation strategies for the development of multivalent protein vaccine nanoparticles.

The development of subunit vaccine platforms has been of considerable interest due to their good safety profile and ability to be adapted to new antigens, compared to other vaccine typess. Nevertheless, subunit vaccines often lack sufficient immunogenicity to fully protect against infectious diseases. A wide variety of subunit vaccines have been developed to enhance antigen immunogenicity by increasing antigen multivalency, as well as stability and delivery properties, via presentation of antigens on protein nanoparticles. Increasing multivalency can be an effective approach to provide a potent humoral immune response by more strongly engaging and clustering B cell receptors (BCRs) to induce activation, as well as increased uptake by antigen presenting cells and their subsequent T cell activation. Proper orientation of antigen on protein nanoparticles is also considered a crucial factor for enhanced BCR engagement and subsequent immune responses. Therefore, various strategies have been reported to decorate highly repetitive surfaces of protein nanoparticle scaffolds with multiple copies of antigens, arrange antigens in proper orientation, or combinations thereof. In this review, we describe different chemical bioconjugation methods, approaches for genetic fusion of recombinant antigens, biological affinity tags, and enzymatic conjugation methods to effectively present antigens on the surface of protein nanoparticle vaccine scaffolds.

Rational design of elastin-like polypeptide fusion proteins to tune self-assembly and properties of protein vesicles.

Protein vesicles made from bioactive proteins have potential value in drug delivery, biocatalysis, and as artificial cells. As the proteins are produced recombinantly, the ability to precisely tune the protein sequence provides control not possible with polymeric vesicles. The tunability and biocompatibility motivated this work to develop protein vesicles using rationally designed protein building blocks to investigate how protein sequence influences vesicle self-assembly and properties. We have reported an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) and functional, globular proteins fused to a glutamate-rich leucine zipper (ZE) that self-assemble into protein vesicles when warmed from 4 to 25 °C due to the hydrophobic transition of ELP. Previously, we demonstrated the ability to tune vesicle properties by changing protein and salt concentration, ZE : ZR ratio, and warming rate. However, there is a limit to the properties that can be achieved via assembly conditions. In order to access a wider range of vesicle diameter and stability profiles, this work investigated how modifiying the hydrophobicity and length of the ELP sequence influenced self-assembly and the final properties of protein vesicles using mCherry as a model globular protein. The results showed that both transition temperature and diameter of protein vesicles were inversely correlated to the ELP guest residue hydrophobicity and the number of ELP pentapeptide repeats. Additionally, sequence manipulation enabled assembly of vesicles with properties not accessible by changes to assembly conditions. For example, introduction of tyrosine at 5 guest residue positions in ELP enabled formation of nanoscale vesicles stable at physiological salt concentration. This work yields design guidelines for modifying the ELP sequence to manipulate protein vesicle transition temperature, size and stability to achieve desired properties for particular biofunctional applications.

ISCOMs/MPLA-Adjuvanted SDAD Protein Nanoparticles Induce Improved Mucosal Immune Responses and Cross-Protection in Mice.

The epidemics caused by the influenza virus are a serious threat to public health and the economy. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. In this study, a novel type of core/shell protein nanoparticle consisting of influenza nucleoprotein (NP) as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by SDAD hetero-bifunctional crosslinking is exploited. Immune-stimulating complexes (ISCOMs)/monophosphoryl lipid A (MPLA) adjuvants further boost the NP/NA-M2e SDAD protein nanoparticle-induced immune responses when administered intramuscularly. The ISCOMs/MPLA-adjuvanted protein nanoparticles are delivered through the intranasal route to validate the application as mucosal vaccines. ISCOMs/MPLA-adjuvanted nanoparticles induce significantly strengthened antigen-specific antibody responses, cytokine-secreting splenocytes in the systemic compartment, and higher levels of antigen-specific IgA and IgG in the local mucosa. Meanwhile, significantly expanded lung resident memory (RM) T and B cells (TRM /BRM ) and alveolar macrophages population are observed in ISCOMs/MPLA-adjuvanted nanoparticle-immunized mice with a 100% survival rate after homogeneous and heterogeneous H3N2 viral challenges. Taken together, ISCOMs/MPLA-adjuvanted protein nanoparticles could improve strong systemic and mucosal immune responses conferring protection in different immunization routes.

Effect of Antigen Structure in Subunit Vaccine Nanoparticles on Humoral Immune Responses.

Subunit vaccines offer numerous attractive features, including good safety profiles and well-defined components with highly characterized properties because they do not contain whole pathogens. However, vaccine platforms based on one or few selected antigens are often poorly immunogenic. Several advances have been made in improving the effectiveness of subunit vaccines, including nanoparticle formulation and/or co-administration with adjuvants. Desolvation of antigens into nanoparticles is one approach that has been successful in eliciting protective immune responses. Despite this advance, damage to the antigen structure by desolvation can compromise the recognition of conformational antigens by B cells and the subsequent humoral response. Here, we used ovalbumin as a model antigen to demonstrate enhanced efficacy of subunit vaccines by preserving antigen structures in nanoparticles. An altered antigen structure due to desolvation was first validated by GROMACS and circular dichroism. Desolvant-free nanoparticles with a stable ovalbumin structure were successfully synthesized by directly cross-linking ovalbumin or using ammonium sulfate to form nanoclusters. Alternatively, desolvated OVA nanoparticles were coated with a layer of OVA after desolvation. Vaccination with salt-precipitated nanoparticles increased OVA-specific IgG titers 4.2- and 22-fold compared to the desolvated and coated nanoparticles, respectively. In addition, enhanced affinity maturation by both salt precipitated and coated nanoparticles was displayed in contrast to desolvated nanoparticles. These results demonstrate both that salt-precipitated antigen nanoparticles are a potential new vaccine platform with significantly improved humoral immunity and a functional value of preserving antigen structures in vaccine nanoparticle design.

Surface Engineering of Protein Nanoparticles Modulates Transport, Adsorption, and Uptake in Mucus.

Protein nanoparticles have been demonstrated as effective carriers for protein antigens and therapeutics due to properties endowed by their protein composition. They exhibit high protein to carrier yields, biocompatibility, and heterogeneous surface properties. While protein nanoparticles have been delivered via multiple routes, including intranasal, their interactions with mucosal barriers have not been well studied or modified. Biological barriers associated with intranasal delivery consist of viscoelastic mucus that hinders material transport through surface interactions and the underlying epithelium. Herein, we altered protein nanoparticle surface properties and characterized interactions with nasal mucus and the subsequent effects on diffusion, cellular uptake, and immune cell maturation. Ovalbumin protein nanoparticles were used, serving as a model vaccine nanoparticle. Unmodified ovalbumin protein nanoparticles were compared to cationic ovalbumin particles functionalized with amine groups, neutral particles functionalized with polyethylene glycol, and zwitterionic particles coated layer-by-layer (LBL) with chitosan and oligonucleotides. Transport analysis indicated rapid diffusion of polyethylene glycol and LBL-modified ovalbumin nanoparticles in porcine nasal mucus, while cationic particles were mucoadhesive. Cellular uptake in the presence of mucus by epithelial and dendritic cells was highest for particles containing positive charges, both LBL and amine-functionalized. These particles also exhibited the most diverse adsorbed protein corona from nasal fluids. The corona impacted both dendritic cell uptake and maturation, with polyethylene glycol and LBL modifications improving CD86 expression. Altogether, surface modifications on protein-based nanocarriers are shown to facilitate distinctive physical and cellular behavior associated with mucosal delivery.

Pourbaix Diagram of Astatine Revisited: Experimental Investigations.

The Pourbaix diagram of an element displays its stable chemical forms with respect to the redox potential and pH of the solution, whose knowledge is fundamental for understanding and anticipating the chemistry of the element in a specified solution. Unlike most halogens, the Pourbaix diagram in the aqueous phase for astatine (At, Z = 85) is still under construction. In particular, the predominant domains of two astatine species assumed to exist under alkaline conditions, At- and AtO(OH)2-, need to be refined. Through high-performance ion-exchange chromatography, electromobility measurements, and competition experiments, the existence of At- and AtO(OH)2- has been confirmed and the associated standard potential has been determined for the first time (0.86 ± 0.05 V vs the standard hydrogen electrode). On the basis of these results, a revised version of astatine's Pourbaix diagram is proposed, covering the three oxidation states of astatine that exist in the thermodynamic stability range of water: At(-I), At(I), and At(III) (as At-, At+, AtO+, AtO(OH), and AtO(OH)2-).

Self-Assembly of Functional Protein Nanosheets from Thermoresponsive Bolaamphiphiles.

Nanosheets are two-dimensional materials, less than 100 nm thick, that can be used for separations, biosensing, and biocatalysis. Nanosheets can be made from inorganic and organic materials such as graphene, polymers, and proteins. Here, we report the self-assembly of nanosheets under aqueous conditions from functional proteins. The nanosheets are synthesized from two fusion proteins held together by high-affinity interactions of two leucine zippers to form bolaamphiphiles. The hydrophobic domain, ZR-ELP-ZR, contains the thermoresponsive elastin-like peptide (ELP) flanked by arginine-rich leucine zippers (ZR), each of which binds the hydrophilic fusion protein, globule-ZE, via the glutamate-rich leucine zipper (ZE) fused to a functional, globular protein. Nanosheets form when the proteins are mixed at 4 °C in aqueous solutions and then heated to 25 °C as the container is rotated end-over-end causing expansion and contraction of the air-water interface. The nanosheets are robust with respect to the choice of globular protein and can incorporate small fluorescent proteins that are less than 30 kDa as well as large enzymes, such as 80 kDa malate synthase G. Upon incorporation into nanosheets, enzymes retain more than 70% of their original activity, demonstrating the potential of protein nanosheets to be used for biosensing or biocatalytic applications.

Protein Vesicles with pH-Responsive Disassembly.

Protein biomaterials offer several advantages over those made from other components because their amino acid sequence can be precisely controlled with genetic engineering to produce a diverse set of material building blocks. In this work, three different elastin-like polypeptide (ELP) sequences were designed to synthesize pH-responsive protein vesicles. ELPs undergo a thermally induced hydrophobic transition that enables self-assembly of different kinds of protein biomaterials. The transition can be tuned by the composition of the guest residue, X, within the ELP pentapeptide repeat unit, VPGXG. When the guest residue is substituted with an ionizable amino acid, such as histidine, the ELP undergoes a pH-dependent hydrophobic phase transition. We used pH-responsive ELPs with different levels of histidine substitution, in combination with leucine zippers and globular, functional proteins, to fabricate protein vesicles. We demonstrate pH-dependent self-assembly, diameter, and disassembly of the vesicles using a combination of turbidimetry, dynamic light scattering, microscopy, and small angle X-ray scattering. As the ELP transition is dependent on the sequence, the vesicle properties also depend on the histidine content in the ELP building blocks. These results demonstrate the tunability of protein vesicles endowed with pH responsiveness, which expands their potential in drug-delivery applications.

Self-assembling nanocarriers from engineered proteins: Design, functionalization, and application for drug delivery.

Self-assembling proteins are valuable building blocks for constructing drug nanocarriers due to their self-assembly behavior, monodispersity, biocompatibility, and biodegradability. Genetic and chemical modifications allow for modular design of protein nanocarriers with effective drug encapsulation, targetability, stimuli responsiveness, and in vivo half-life. Protein nanocarriers have been developed to deliver various therapeutic molecules including small molecules, proteins, and nucleic acids with proven in vitro and in vivo efficacy. This article reviews recent advances in protein nanocarriers that are not derived from natural protein nanostructures, such as protein cages or virus like particles. The protein nanocarriers described here are self-assembled from rationally or de novo designed recombinant proteins, as well as recombinant proteins complexed with other biomolecules, presenting properties that are unique from those of natural protein carriers. Design, functionalization, and therapeutic application of protein nanocarriers will be discussed.

Protein Mediated Enzyme Immobilization.

Enzyme immobilization is an essential technology for commercializing biocatalysis. It imparts stability, recoverability, and other valuable features that improve the effectiveness of biocatalysts. While many avenues to join an enzyme to solid phases exist, protein-mediated immobilization is rapidly developing and has many advantages. Protein-mediated immobilization allows for the binding interaction to be genetically coded, can be used to create artificial multienzyme cascades, and enables modular designs that expand the variety of enzymes immobilized. By designing around binding interactions between protein domains, they can be integrated into functional materials for protein immobilization. These materials are framed within the context of biocatalytic performance, immobilization efficiency, and stability of the materials. In this review, supports composed entirely of protein are discussed first, with systems such as cellulosomes and protein cages being discussed alongside newer technologies like spore-based biocatalysts and forizymes. Protein-composite materials such as polymersomes and protein-inorganic supraparticles are then discussed to demonstrate how protein-mediated strategies are applied to many classes of solid materials. Critical analysis and future directions of protein-based immobilization are then discussed, with a particular focus on both computational and design strategies to advance this area of research and make it more broadly applicable to many classes of enzymes.

Coiled coil exposure and histidine tags drive function of an intracellular protein drug carrier.

In recent years, protein engineering efforts have yielded a diverse set of binding proteins that hold promise for various therapeutic applications. Despite this, their inability to reach intracellular targets limits their applications to cell surface or soluble targets. To address this challenge, we previously reported a protein carrier that binds antibodies and delivers them to therapeutic targets inside cancer cells. This carrier, known as the Hex carrier, is comprised of a self-assembling coiled coil hexamer at the core, with each alpha helix fused to a linker, an antibody binding domain, and a six Histidine-tag (His-tag). In this work, we designed different versions of the carrier to determine the role of each building block in cytosolic protein delivery. We found that increasing exposure of the Hex coiled coil on the carriers, through molecular design or removing antibodies, increased internalization, pointing to a role of the coiled coil in promoting endocytosis. We observed a clear increase in endosomal disruption events when His-tags were present on the carrier relative to when they were removed, due to an endosomal buffering effect. Finally, we found that the antibody binding domains of the Hex carrier could be replaced with monomeric ultra-stable GFP for intracellular delivery and endosomal escape. Our results demonstrate that the Hex coiled coil, in conjunction with His-tags, could be a generalizable vehicle for delivering small and large proteins to intracellular targets. This work also highlights new biological applications for oligomeric coiled coils and shows the direct and quantifiable impact of histidine residues on endosomal disruption. These findings could inform the design of future drug delivery vehicles in applications beyond intracellular protein delivery.

An expanded halogen bonding scale using astatine.

As a non-covalent interaction, halogen bonding is now acknowledged to be useful in all fields where the control of intermolecular recognition plays a pivotal role. Halogen-bond basicity scales allow quantification of the halogen bonding of referential donors with organic functional groups from a thermodynamic point of view. Herein we present the pK BAtI basicity scale to provide the community an overview of halogen-bond acceptor strength towards astatine, the most potent halogen-bond donor element. This experimental scale is erected on the basis of complexation constants measured between astatine monoiodide (AtI) and sixteen selected Lewis bases. It spans over 6 log units and culminates with a value of 5.69 ± 0.32 for N,N,N',N'-tetramethylthiourea. On this scale, the carbon π-bases are the weakest acceptors, the oxygen derivatives cover almost two-thirds of the scale, and sulphur bases exhibit the highest AtI basicity. Regarding the applications of 211At in targeted radionuclide therapy, stronger labelling of carrier agents could be envisaged on the basis of the pK BAtI scale.

Photocrosslinked, Tunable Protein Vesicles for Drug Delivery Applications.

Recombinant proteins have emerged as promising building blocks for vesicle self-assembly because of their versatility through genetic manipulation and biocompatibility. Vesicles composed of thermally responsive elastin-like polypeptide (ELP) fusion proteins encapsulate cargo during assembly. However, vesicle stability in physiological environments remains a significant challenge for biofunctional applications. Here, incorporation of an unnatural amino acid, para-azido phenylalanine, into the ELP domain is reported to enable photocrosslinking of protein vesicles and tuning of vesicle size and swelling. The size of the vesicles can be tuned by changing ELP hydrophobicity and ionic strength. Protein vesicles are assessed for their ability to encapsulate doxorubicin and dually deliver doxorubicin and fluorescent protein in vitro as a proof of concept. The resulting photocrosslinkable vesicles made from full-sized, functional proteins show high potential in drug delivery applications, especially for small molecule/protein combination therapies or targeted therapies.

Protein and Peptide Nanocluster Vaccines.

Recombinant protein- and peptide-based vaccines can deliver large amounts of specific antigens for tailored immune responses. One class of these are protein and peptide nanoclusters (PNCs), which are made entirely from the crosslinked antigen. PNCs leverage the inherent immunogenicity of nanoparticulate antigens while minimizing the use of excipients normally used to create them. In this chapter, we discuss PNC fabrication methods, immunostimulatory properties of nanoclusters observed in vitro and in vivo, and protective benefits of PNC vaccines against influenza and cancer mouse models. We conclude with an outlook on future studies of PNCs and PNC design strategies, as well as their use in future vaccine formulations.

Protein Vesicles Self-Assembled from Functional Globular Proteins with Different Charge and Size.

Protein vesicles can be synthesized by mixing two fusion proteins: an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) with a globular, soluble protein fused to a glutamate-rich leucine zipper (ZE). Currently, only fluorescent proteins have been incorporated into vesicles; however, for protein vesicles to be useful for biocatalysis, drug delivery, or biosensing, vesicles must assemble from functional proteins that span an array of properties and functionalities. In this work, the globular protein was systematically changed to determine the effects of the surface charge and size on the self-assembly of protein vesicles. The formation of microphases, which included vesicles, coacervates, and hybrid structures, was monitored at different assembly conditions to determine the phase space for each globular protein. The results show that the protein surface charge has a small effect on vesicle self-assembly. However, increasing the size of the globular protein decreases the vesicle size and increases the stability at lower ZE/ZR molar ratios. The phase diagrams created can be used as guidelines to incorporate new functional proteins into vesicles. Furthermore, this work reports catalytically active enzyme vesicles, demonstrating the potential for the application of vesicles as biocatalysts or biosensors.