Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone.

Reflectins, a recently identified protein family that is enriched in aromatic and sulphur-containing amino acids, are used by certain cephalopods to manage and manipulate incident light in their environment. These proteins are the predominant constituent of nanoscaled photonic structures that function in static and adaptive colouration, extending visual performance and intra-species communication. Our investigation into recombinantly expressed reflectin has revealed unanticipated self-assembling and behavioural properties, and we demonstrate that reflectin can be easily processed into thin films, photonic grating structures and fibres. Our findings represent a key step in our understanding of the property-function relationships of this unique family of reflective proteins.

Self-assembled particles of genetically engineered human L subunit ferritin expressing a silver-binding peptide were used as nanocontainers for the synthesis of silver nanoparticles. The inner cavity of the self-assembled protein cage displays a dodecapeptide that is capable of reducing silver ions to metallic silver. This chimeric protein cage when incubated in the presence of silver nitrate exhibits the growth of a silver nanocrystal within its cavity. Our studies indicate that it is possible to design chimeric cages, using specific peptide templates, for the growth of other inorganic nanoparticles.

Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the lightorgan iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh-and calciuminduced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.

Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago, the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials. Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront, but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines. Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness. Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein.We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligooxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,Ndimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; CMC, critical micelle concentration; DGS-NTA, 1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl]; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidylglycerol; DSP, dithiobis[succinimidyl proprionate; FM, fluorescein maleimide; FRET, F-rster resonance energy transfer; Ipa, invasion plasmid antigen; Ipg, invasion plasmid gene; LDAO, N,N-dimethyldodecylamine N-oxide; Mxi, major exporter of Ipas; OPOE, n-Octyl-oligo-oxyethylene; SATA, N-succinimidyl-S-acetylthioacetate; SEC, size exclusion chromatography; SRB, sulforhodamine B; T m , thermal unfolding midpoint; TCEP, (tris (2-carboxyethyl)phosphine; TRITC DHPE, (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine); T3SS, type-III secretion system; T3SA, type III secretion apparatus.Additional Supporting Information may be found in the online version of this article. that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.

The development of vaccines containing adjuvants has the potential to enhance antibody and cellular immune responses, broaden protective immunity against heterogeneous pathogen strains, enable antigen dose sparing, and facilitate efficacy in immunocompromised populations. Nevertheless, the structural interplay between antigen and adjuvant components is often not taken into account in the published literature. Interactions between antigen and adjuvant formulations should be well characterized to enable optimum vaccine stability and efficacy. This review focuses on the importance of characterizing antigen-adjuvant interactions by summarizing findings involving widely used adjuvant formulation platforms, such as aluminum salts, emulsions, lipid vesicles, and polymer-based particles. Emphasis is placed on the physicochemical basis of antigen-adjuvant associations and the appropriate analytical tools for their characterization, as well as discussing the effects of these interactions on vaccine potency.

BackgroundAdjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE).Materials and methodsIn this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations.ResultsWe identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies.ConclusionThese efforts will aid in the development of a platform formulation for use with other similar vaccines.