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Cry Protein Crystals: A Novel Platform for Protein Delivery
PLOS ONE
Abstract and Figures
Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.
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... Abe et al. from the Ueno group have observed crystallization of polyhedrin in insect cells (S. frugiperda and B. mori), 27 recapitulating earlier observations by Mori et al. 43 Similar in vivo crystallization approaches have been used for generating Cry3 crystals. 44 More recent work by the Ueno group describes a cell-free system for generating nanocrystals, by coincubating template mRNA with wheat germ extract; 45 although these crystallization reactions were only performed at millilitre scales, this approach, similar to in vivo crystallization, obviates the need for downstream purification of the protein. ...
... Therefore, it is often necessary to chemically crosslink the crystals following growth and washing, to ensure that they retain their structural integrity following solvent exchange. Some rare protein crystals, such as crystals derived from the insecticidal, Bacillus thuringiensis-derived Cry toxins 44,49 do not require additional downstream cross-linking. S-layer proteins, which confer structural rigidity to the outer membrane of certain prokaryotic species, have also been shown to form highly stable two-dimensional porous crystals on the surface of living cells. ...
Large-pore protein crystals (LPCs) are an emerging class of biomaterials. The inherent diversity of proteins translates to a diversity of crystal lattice structures, many of which display large pores and...
... 37 The latter is consistent with our observation thus far that the maximum size of subunits that have been successfully fused to the Cry3Aa protein to produce active fusion crystals in vivo is less than 50 kDa. 35,38,39 As one approach to directly immobilize larger enzymes, we decided to explore the use of Cry1Ab, another crystal-forming protein from the Cry protein family, 40 as the immobilization platform. Similar to Cry3Aa, Cry1Ab also contains three core domains (the N-terminal α-helical domain, the variable domain, and the conserved domain) but is much larger than Cry3Aa (130 vs 73 kDa) due to the presence of a C-terminal crystallization domain. ...
... 26,[28][29][30][31][32][33][34][35][36][37][38] The high porosity and stability of in-cell protein crystals permit the accumulation of foreign synthetic molecules and proteins. 24,[39][40][41][42][43] These advantageous properties will promote the collection of diffraction data for the target molecules in the crystals. While the structures of metal complexes immobilized in the pores have been determined, 44,45 the structures of exogenous proteins and peptides have not yet been reported because procedures for fixation of the proteins and peptides have not been established. ...
Protein crystals can be utilized as porous scaffolds to capture exogenous molecules. Immobilization of target proteins using protein crystals is expected to facilitate X-ray structure analysis of proteins that are difficult to be crystallized. One of the advantages of scaffold-assisted structure determination is the analysis of metastable structures that are not observed in solution. However, efforts to fix target proteins within the pores of scaffold protein crystals have been limited due to the lack of strategies to control protein-protein interactions formed in the crystals. In this study, we analyze the metastable structure of the miniprotein, CLN025, which forms a β-hairpin structure in solution, using a polyhedra crystal (PhC), an in-cell protein crystal. CLN025 is successfully fixed within the PhC scaffold by replacing the original loop region. X-ray crystal structure analysis and molecular dynamics (MD) simulation reveal that CLN025 is fixed as a helical structure in a metastable state by non-covalent interactions in the scaffold crystal. These results indicate that modulation of intermolecular interactions can trap various protein conformations in the engineered PhC and provides a new strategy for scaffold-assisted structure determination.
... These protein building blocks assemble into larger, stable and highly ordered structures that can be engineered to have a diameter ranging from 20 to 100 nm and possess unique physical properties (López-Sagaseta et al., 2016). Protein cages on the other hand include viral capsids, ferritins, and heat shock proteins, while protein-based complexes include Cry3Aa fusion protein platform (well known for the delivery of reporter proteins such as GFP in mammalian cells), silk proteins, and bovine serum albumin (Nair et al., 2015;Qin et al., 2019). ...
A worldwide estimate of over one million STIs are acquired daily and there is a desperate need for effective preventive as well as therapeutic measures to curtail this global health burden. Vaccines have been the most effective means for the control and potential eradication of infectious diseases; however, the development of vaccines against STIs has been a daunting task requiring extensive research for the development of safe and efficacious formulations. Nanoparticle-based vaccines represent a promising platform as they offer benefits such as targeted antigen presentation and delivery, co-localized antigen-adjuvant combinations for enhanced immunogenicity, and can be designed to be biologically inert. Here we discuss promising types of nanoparticles along with outcomes from nanoparticle-based vaccine preclinical studies against non-viral STIs including chlamydia, syphilis, gonorrhea, and recommendations for future nanoparticle-based vaccines against STIs.
Helicobacter pylori (H. pylori) causes infection in the stomach and is a major factor for gastric carcinogenesis. The application of antimicrobial peptides (AMPs) as an alternative treatment to traditional antibiotics is limited by their facile degradation in the stomach, their poor penetration of the gastric mucosa, and the cost of peptide production. Here, the design and characterization of a genetically encoded H. pylori‐responsive microbicidal protein crystal Cry3Aa‐MIIA‐AMP‐P17 is described. This designed crystal exhibits preferential binding to H. pylori, and when activated, promotes the targeted release of the AMP at the H. pylori infection site. Significantly, when the activated Cry3Aa‐MIIA‐AMP‐P17 crystals are orally delivered to infected mice, the Cry3Aa crystal framework protects its cargo AMP against degradation, resulting in enhanced in vivo efficacy against H. pylori infection. Notably, in contrast to antibiotics, treatment with the activated crystals results in minimal perturbation of the mouse gut microbiota. These results demonstrate that engineered Cry3Aa crystals can serve as an effective platform for the oral delivery of therapeutic peptides to treat gastrointestinal diseases.
The Orange Carotenoid Protein (OCP), involved in energy-quenching of the cyanobacterial light-harvesting antennae, is a two-domain protein functionalized by a non-covalently bound keto-carotenoid. To elicit its photoprotection function, the protein must be photoactivated by a blue-green photon, triggering transition from an orange inactive “dark” state (OCPO) to a red photoactive “light” state (OCPR). The photoactivation mechanism, which spans 13 decades in time, involves structural rearrangements at the level of both the photoexcited carotenoid and the protein scaffold. During my thesis, I investigated rhe OCP photoactivation mechanism using variety of biochemistry and structural biology methods.Firstly, through combination of spectroscopy and steady-state and time-resolved (TR) X-ray scattering, we examined the large-scale motions involved in the photoactivation and recovery of OCP. We demonstrated that oligomerization occurs at both the OCPO and OCPR level, and that it partakes in the regulation of the protein photoactivation and thermal recovery. Secondly, by using conventional X-ray crystallography, we determined the previously-uncharacterized crystal structure of Planktothrix aghardii OCP. Structural analysis pointed to the influence of protein flexibility on the photoactivation and recovery rates, and on the energy-quenching activity of this variant. In an effort to characterize early stages of OCP photoactivation by means of TR crystallography, we investigated in vitro and in vivo crystallization methods to produce sub-micron-sized OCP crystals. Specifically, we attempted in vivo crystallization in the crystalliferous bacterium Bacillus thuringiensis (Bt), endeavoring to obtain crystals of fusions of OCP with Cyt1Aa and Cry11Aa Bt toxins. While unsuccessful in terms of production of OCP crystals, this work enabled to shed light on the structure and bioactivation cascade of the two toxins, and to investigate the molecular mechanisms at the basis of their crystallization.
Crystalline materials are increasingly being used as platforms for encapsulating proteins to create stable, functional materials. However, the uptake efficiencies and stimuli-responsiveness of crystalline frameworks are limited by their rigidities. We have recently reported a new form of materials, polymer-integrated crystals (PIX), which combine the structural order of protein crystals with the dynamic, stimuli-responsive properties of synthetic polymers. Here we show that the crystallinity, flexibility, and chemical tunability of PIX can be exploited to encapsulate guest proteins with high loading efficiencies (up to 46% w/w). The electrostatic host-guest interactions enable reversible, pH-controlled uptake/release of guest proteins as well as the mutual stabilization of the host and the guest, thus creating a uniquely synergistic platform toward the development of functional biomaterials and the controlled delivery of biological macromolecules.
The production of Cry3Aa enzyme fusion crystals in Bacillus thuringiensis provides a direct method to immobilize individual enzymes and thereby improve their stability and recyclability. Nevertheless, many reactions require multiple enzymes to produce a desired product; thus a general strategy was developed to extend our Cry3Aa technology to multienzyme coimmobilization. Here, we report the direct production of particles comprising a modified Cry3Aa (Cry3Aa*) fused to SpyCatcher002 (Cry3Aa*SpyCat2) for coimmobilization of model enzymes MenF, MenD, and MenH associated with the biosynthesis of menaquinone. The resultant coimmobilized particles showed improved reaction rates compared to free enzymes presumably due to the higher local enzyme substrate concentrations and enhanced enzyme coupling made possible by colocalization. Furthermore, coimmobilization of these enzymes on Cry3Aa*SpyCat2 led to increased thermal stability and recyclability of the overall multienzyme system. These characteristics together with its overall simplicity of production highlight the benefits of Cry3Aa*SpyCat2 crystals as a platform for enzyme coimmobilization.
Protein crystals have attracted a great deal of attention as solid biomaterials because they have porous structures created by regular assemblies of proteins. The lattice structures of protein crystals are controlled by designing molecular interfacial interactions via covalent bonds and non-covalent bonds. Protein crystals have been functionalized as templates to immobilize foreign molecules such as metal nanoparticles, metal complexes, and proteins. These hybrid crystals are used as functional materials for catalytic reactions and structural analysis. Furthermore, in-cell protein crystals have been studied extensively, providing progress in rapid protein crystallization and crystallography. This review highlights recent advances in crystal engineering for protein crystallization and generation of solid functional materials both in vitro and within cells.
The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern. Thus, there has been great interest in developing efficient strategies to directly deliver proteins into cells as an alternative that can avoid such safety concerns while achieving a comparable therapeutic effect. Nevertheless, intracellular delivery of protein therapeutics remains a challenge. Our group has recently developed a protein delivery platform based on an engineered Pos3Aa protein that forms sub-micrometer-sized crystals in Bacillus thuringiensis cells. In this report, we describe the further development of this platform (Pos3AaTM) via rationally designed site-directed mutagenesis, and its resultant potency for the delivery of cargo proteins to cells. Pos3AaTM-based fusion protein crystals are shown to exhibit improved release of their cargo proteins as demonstrated using a model mCherry protein. Importantly, this Pos3AaTM platform is able to mediate the efficient intracellular delivery of p16 protein with significant endosomal escape, resulting in p16-mediated inhibition of CDK4/6 kinase activity and Rb phosphorylation, and as a consequence, significant cell cycle arrest and cell growth inhibition. These results validate the ability of these improved Pos3AaTM crystals to mediate enhanced cytosolic protein delivery and highlight the potential of using protein therapeutics as selective CDK4/6 inhibitors for cancer therapy.
Statement of Significance
Cytosolic delivery of bioactive therapeutic proteins capable of eliciting therapeutic benefit remains a significant challenge. We have previously developed a protein delivery platform based on engineered Pos3Aa protein crystals with excellent cell-permeability and endosomal escape properties. In this report, we describe the rational design of an improved Pos3Aa triple mutant (Pos3AaTM) with enhanced cargo release. We demonstrate that Pos3AaTM-mCherry-p16 fusion crystals can efficiently deliver p16 protein, a CDK4/6 inhibitor frequently inactivated in human cancers, into p16-deficient UM-SCC-22A cells, where it promotes significant G1 cell cycle arrest and cell growth inhibition. These results highlight the ability of the Pos3AaTM platform to promote potent cytosolic delivery of protein therapeutics, and the efficacy of p16 protein delivery as an effective strategy for treating cancer.
Insect-specific toxins derived from Bacillus thuringiensis (Bt) provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the host range of Bt crystal (Cry) and cytolytic (Cyt) toxins. These strategies include toxin truncation, modification of protease cleavage sites, domain swapping, site-directed mutagenesis, peptide addition, and phage display screens for mutated toxins with enhanced activity. Toxin optimization provides a useful approach to extend the utility of these proteins for suppression of pests that exhibit low susceptibility to native Bt toxins, and to overcome field resistance.
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that
scattering from other cell components did not obscure diffraction fromthe crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 μs) between exiting the injector to
intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.
