Native Protein Research Articles

Traditional cooking methods decreased the allergenicity of egg proteins.

Egg allergy is one of the most common food allergies globally. This study aimed to assess the impact of four traditional cooking methods on the allergenicity of egg proteins using a comprehensive strategy, including simulated gastrointestinal digestion in vitro, serology experiments, a rat basophilic leukemia (RBL)-2H3 cell degranulation model, and a passive cutaneous anaphylaxis (PCA) mice model, and the structure changes were detected by circular dichroism (CD)spectra and ultraviolet (UV) spectra. The results showed that the processed egg proteins were more readily digested compared to raw egg proteins. The serological experiments revealed a significant reduction in immunoglobulin E binding of egg proteins after thermal treatments (p<0.05), particularly after frying. Subsequently, the RBL-2H3 cell degranulation experiment demonstrated a marked decrease in the level of egg allergens-induced β-hexosaminidase release after cooking (p<0.05). Moreover, the results from the PCA mice model indicated that the increase in vascular permeability was effectively relieved in the treated groups, especially in frying group (p<0.05). Additionally, the α-helix and β-turn contents of processed egg proteins were significantly decreased (p<0.05) compared with native egg proteins. The UV spectra findings showed that all cooking treatments caused significant alterations in the tertiary structure, and fluorescence analysis indicated that cooking decreased the surface hydrophobicity of egg proteins. In conclusion, four traditional cooking methods reduced the allergenicity of egg proteins, particularly frying, and this reduction was associated with structural changes that could contribute to the destruction or masking of epitopes of egg allergens. PRACTICAL APPLICATION: Egg allergy has a serious impact on public health, and there is no ideal treatment method at present. This study demonstrated that four traditional cooking methods (boiling, steaming, baking, and frying) reduced the allergenicity of egg proteins, especially frying, and the results will provide a basis for the development of hypoallergenic egg products.

TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL–CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL–CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

Enhancement of N-Methyl Amino Acid Incorporation into Proteins and Peptides Using Modified Bacterial Ribosomes and Elongation Factor P.

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

Mildly preheating induced conformational changes of soy protein isolates contributed to the binding interaction with blueberry anthocyanins for stabilization

Preheated proteins have been proven to stabilize anthocyanins better than natural proteins, but the binding mechanism remains to be further studied. This study investigated the interacting mechanism between soy protein isolate (SPI) that has been slightly heated and blueberry anthocyanins (ANs) using multi-spectroscopy and molecular dynamics approaches. It was found that preheated SPI (pre-SPI) increased the stability of the ANs and provided better protection than natural SPI. In particular, SPI preheated at 45°C provided the most effective protection against anthocyanin instability. Multi-spectroscopy demonstrates that SPI exhibits a robust affinity for and causes static quenching of malvidin-3-O-galactoside (M3G). The binding of SPI causes a considerable modification in its secondary structure, leading to a decrease in the β-sheet format and an increase in the α-helix, β-turn, and random coil structures. When heated to 45°C, the combination effect of SPI and M3G was the most significant, thus improving blueberry ANs' stability. The interaction between SPI and M3G predominantly occurs via hydrophobic interactions. Furthermore, molecular dynamics simulations utilized the Amber18 protein force field and demonstrated that hydrogen bonds played a role as a source of force. Notably, pre-SPI exhibited superior anthocyanin wrapping capabilities to natural SPI, mitigating external environmental damage resulting from pocket tightening. These findings offer a deeper understanding of how proteins and ANs interact.

Amyloid-like Aggregation of Wheat Gluten and Its Components during Cooking: Mechanisms and Structural Characterization.

Amyloid-like aggregation widely occurs during the processing and production of natural proteins, with evidence indicating its presence following the thermal processing of wheat gluten. However, significant gaps remain in understanding the underlying fibrillation mechanisms and structural polymorphisms. In this study, the amyloid-like aggregation behavior of wheat gluten and its components (glutenin and gliadin) during cooking was systematically analyzed through physicochemical assessment and structural characterization. The presence of amyloid-like fibrils (AFs) was confirmed using X-ray diffraction and Congo red staining, while Thioflavin T fluorescence revealed different patterns and rates of AFs growth among wheat gluten, glutenin, and gliadin. AFs in gliadin exhibited linear growth curves, while those in gluten and glutenin showed S-shaped curves, with the shortest lag phase and fastest growth rate (t1/2 = 2.11 min) observed in glutenin. Molecular weight analyses revealed AFs primarily in the 10-15 kDa range, shifting to higher weights over time. Glutenin-derived AFs had the smallest ζ-potential value (-19.5 mV) and the most significant size increase post cooking (approximately 400 nm). AFs in gluten involve interchain reorganization, hydrophobic interactions, and conformational transitions, leading to additional cross β-sheets. Atomic force microscopy depicted varying fibril structures during cooking, notably longer, taller, and stiffer AFs from glutenin.

Recent advances, challenges and functional applications of protein glycosylation modification in food industry

SummaryProtein glycosylation modification is an effective way to enhance the functional properties of natural proteins. This study aims to explore the preparation of glycosylation products (PPCs) through the Maillard reaction or enzymatic glycosylation using transglutaminase. The study discusses the advantages and disadvantages of preparing PPCs using dry heat, wet heat, and enzymatic glycosylation, as well as factors influencing the glycosylation process. Compared to natural proteins, PPCs demonstrate superior functional properties, including solubility, viscosity, emulsifying ability, gelling ability, thermal stability, antioxidant activity, and antibacterial activity. With improved functional performance, PPCs have broad application prospects as raw materials in food processing. For example, PPCs can be used as antioxidants, emulsifiers, gelling agents, and carriers for various bioactive molecules in the food industry. In the pharmaceutical field, PPCs are expected to serve as contrast agents and dressings. Furthermore, the application of PPCs in food processing should also consider specific processing requirements to achieve the intended enhancement of glycosylated protein functionality.

Effect of limited proteolysis and CaCl2 on the rheology, microstructure and in vitro digestibility of pea protein-carboxymethyl cellulose mixed gel

Limited proteolysis, CaCl2 and carboxymethyl cellulose (CMC) have individually demonstrated ability to increase the gel strength of laboratory-extracted plant proteins. However, the syneresis effects of their combination on the gelling capacity of commercial plant protein remains unclear. This was investigated by measuring the rheological property, microstructure and protein–protein interactions of gels formed from Alcalase hydrolyzed or intact pea proteins in the presence of 0.1 % CMC and 0–25 mM CaCl2. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed the molecular weight of pea protein in the mixture were < 15 kDa after hydrolysis. The hydrolysates showed higher intrinsic fluorescence intensity and lower surface hydrophobicity than the intact proteins. Rheology showed that the storage modulus (G') of hydrolyzed pea protein (PPH)-based gels sightly decreased compared to those of native proteins. 5–15 mM CaCl2 increased the G’ for both PP and PPH-based gels and decreased the strain in the creep-recovery test. Scanning electron microscopy (SEM) showed the presence of smaller protein aggregates in the PPH-based gels compared to PP gels and the gel network became denser, and more compact and heterogenous in the presence of 15 and 25 mM CaCl2. The gel dissociation assay revealed that hydrophobic interactions and hydrogen bonds were the dominant forces to maintain the gel structure. In vitro digestion showed that the soluble protein content in PPH-based gels was 10 ∼ 30 % higher compared to those of the PP counterpart. CaCl2 addition reduced protein digestibility with a concentration dependent behavior. The results obtained show contrasting effects of limited proteolysis and CaCl2 on the gelling capacity and digestibility of commercial pea proteins. These findings offer practical guidelines for developing pea protein-based food products with a balanced texture and protein nutrition through formulation and enzymatic pre-treatment.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6H-S-PaLPL) works optimally at 35°C and pH 6.5 with remarkable thermostability at 35-95°C. Thermo-inactivation kinetics of 6H-S-PaLPL were also studied at 35-95°C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0h at 95°C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Separation of ovomucin from duck egg white and its “acid-tight /alkali-loose” self-supporting gel properties under different pH

Ovomucin is a key component in maintaining the viscous nature of egg white and has vast potential in the utilization of food industry. The objectives of the study were to determine the effect of different pH on the extractability and gel properties of ovomucin. After removing lysozyme through ion exchange, the diluted duck egg white was acidified to a narrow pH range (4.50, 4.75, 5.00, 5.25, 5.50, 5.75 and 6.00) to precipitate ovomucin. By comprehensive study of purity, yield, and convenience of large-scale preparation, 4.75 was chosen as the optimal pH to extract ovomucin with the highest purity of 85.17 ± 2.31% and a compromised yield of 4.60 ± 0.06 mg/g duck egg white. A series of self-supporting gels were prepared with freeze-dried ovomucin powder at room temperature upon rehydration and centrifugation, and the gels properties were highly associated with pH. Gel formed around pH 5 was the aggregate of natural globular proteins with lowest hydrophobicity and electrostatic interaction, relatively dense structure and high water-holding capacity (WHC). Stronger acidic conditions disrupted the disulfide bonds and formed a rougher spherical aggregate. Under alkaline conditions, however, the protein gradually unfolded and broke into linear basic polypeptides, thus a porous three-dimensional network was formed. Overall, this study provided insights into the formation mechanism of the “acid-tight/alkali-loose” ovomucin gels, and introduced a potential candidate for the development of bioactive substance delivery materials.

Immunoinformatics design of a structural proteins driven multi-epitope candidate vaccine against different SARS-CoV-2 variants based on fynomer

The ideal vaccines for combating diseases that may emerge in the future require more than simply inactivating a few pathogenic strains. This study aims to provide a peptide-based multi-epitope vaccine effective against various severe acute respiratory syndrome coronavirus 2 strains. To design the vaccine, a library of peptides from the spike, nucleocapsid, membrane, and envelope structural proteins of various strains was prepared. Then, the final vaccine structure was optimized using the fully protected epitopes and the fynomer scaffold. Using bioinformatics tools, the antigenicity, allergenicity, toxicity, physicochemical properties, population coverage, and secondary and three-dimensional structures of the vaccine candidate were evaluated. The bioinformatic analyses confirmed the high quality of the vaccine. According to further investigations, this structure is similar to native protein and there is a stable and strong interaction between vaccine and receptors. Based on molecular dynamics simulation, structural compactness and stability in binding were also observed. In addition, the immune simulation showed that the vaccine can stimulate immune responses similar to real conditions. Finally, codon optimization and in silico cloning confirmed efficient expression in Escherichia coli. In conclusion, the fynomer-based vaccine can be considered as a new style in designing and updating vaccines to protect against coronavirus disease.

The study of relationship between taste and wheat bran protein isolate fortification during high-protein yogurt manufacturing

High-protein (HP) food is becoming a popular and nutritionally valuable commodity. The most common protein fortifiers are milk whey or soy protein isolates. However, one of the potentially promising sources of quality protein isolate is wheat bran, the secondary product of wheat milling. Wheat bran protein isolate (WBPI) can be obtained using a sustainable and environmentally friendly pH shift method. Considering its nutritional properties, incorporating WBPI into people's diet is convenient. This work focuses on protein fortification of plain yogurt using WBPI while maintaining sensory acceptability. Direct fortification of yogurts with WBPI to achieve 5 % and 10 % protein content revealed two main sensory defects – bitterness and sandiness. Several approaches, such as grinding WBPI, protein blending, and WBPI encapsulation, were applied to improve the overall impression score of HP yogurts. It was found that eliminating the sandiness of WBPI-fortified yogurt is not possible by reducing WBPI particle size. Blending WBPI with dried milk decreased bitterness, but the products lacked amino acid diversity. The most effective procedure to mask the sensory defects was WBPI encapsulation using alginate, revealing an appropriate way to incorporate natural plant proteins into HP food.

Localizing Protein Expression of Transporters in Epithelial Cell Membranes of Mosquitoes.

Identifying and localizing ion transport proteins in epithelia is important for understanding how these tissues can regulate salt and water balance in animals. Mosquitoes face distinct challenges regarding salt and water balance as larvae live in water of varying ionic composition while adult female mosquitoes must deal with periodic large blood meals. This protocol will explain how to localize ion transporters in epithelia of mosquitoes. Antibodies raised against specific proteins can be used to carry out immunohistochemistry to tag and visualize native proteins in tissues and cells. This technique may be used for whole tissues and organs or can also be used on histological thin sections of fixed tissue. This protocol will detail the use of immunohistochemistry to localize membrane proteins in mosquito organs.

Exploring Functionality Gain for (Recombinant) β-Lactoglobulin Through Production and Processing Interventions

Exploring Functionality Gain for (Recombinant) β-Lactoglobulin Through Production and Processing Interventions

Interfacial rheological properties of pepsin-hydrolyzed lentil protein isolate at oil-water interfaces

Lentil protein isolate (LPI) was investigated for its potential as a plant protein-based emulsifier. LPI consists mainly of globulins with high molecular weights which are relatively slow to adsorb and stabilize oil-water interfaces. Smaller proteins, such as whey protein, can stabilize the interface much faster, quickly forming a viscoelastic film that slows down the rate of droplet recoalescence, leading to smaller droplets. To increase the rate of adsorption, LPI was enzymatically hydrolyzed by pepsin at 1.5% and 4.5% degree of hydrolysis (DH), to obtain small peptides. The behavior of these peptides was studied at the oil-water interface in comparison to whey protein isolate (WPI) and native lentil protein isolate (LPI). Interfacial properties were investigated using dilatational and interfacial shear rheology in the linear viscoelastic regime (LVE) and nonlinear viscoelastic regime (NLVE) and these were related to protein characteristics and emulsion formation. The 1.5% and 4.5%DH LPI showed high surface hydrophobicity, and consisted mainly of low molecular weight peptides, which contributed to faster adsorption kinetics and consequently covered the interface faster than LPI. In dilatation, the native and hydrolyzed LPI-stabilized interfaces had comparable stiffness and were clearly weaker than WPI-stabilized interfaces, which contrasts with the results in shear deformations, where the former both had higher stiffness than WPI. Both hydrolyzed LPI and WPI formed more brittle and less stretchable interfaces compared to native LPI in both dilatational and shear rheology. In emulsification tests, native LPI produces the largest droplets, and the 4.5% DH LPI and WPI have the smallest droplet size. But, bridging flocculation was observed when emulsions were prepared with both hydrolyzed LPI, which may be attributed to inter-droplet hydrophobic interactions between small polypeptides. This can be linked to an increase in the surface hydrophobicity of hydrolyzed LPI. Thus, the use of native LPI as a stabilizer in emulsion preparation is preferable over the hydrolyzed samples, despite its larger droplet size.

Exploring the effect of high-pressure processing conditions on the deaggregation of natural major royal jelly proteins (MRJPs) fibrillar aggregates

High pressure processing is a safe and green novel non-thermal processing technique for modulating food protein aggregation behavior. However, the systematic relationship between high pressure processing conditions and protein deaggregation has not been sufficiently investigated. Major royal jelly proteins, which are naturally highly fibrillar aggregates, and it was found that the pressure level and exposure time could significantly promote protein deaggregation. The 100–200 MPa treatment favoured the deaggregation of proteins with a significant decrease in the sulfhydryl group content. Contrarily, at higher pressure levels (>400 MPa), the exposure time promoted the formation of disordered agglomerates. Notably, the inter-conversion of α-helix and β-strands in major royal jelly proteins after high pressure processing eliminates the solvent-free cavities inside the aggregates, which exerts a 'collapsing' effect on the fibrillar aggregates. Furthermore, the first machine learning model of the high pressure processing conditions and the protein deaggregation behaviour was developed, which provided digital guidance for protein aggregation regulation.

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as prototypes of enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it a potential player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. Therefore, it constitutes a prototype of a microreactor with potential use in bioremediation.

Mechanistic soft-sensor design for protein refolding processes based on intrinsic fluorescence measurements

In protein refolding processes the scarce availability of online measurements hampers effective process monitoring. In this work we developed a mechanistic soft-sensor for protein refolding based on online intrinsic fluorescence measurements of tryptophan and tyrosine. In validation experiments using two model proteins, lactate dehydrogenase (LDH) and galactose oxidase, the soft-sensor showed accurate estimates for the prediction of the total sum of folding products (NRMSE <6.1%) by calculating the changing rate of the average emission wavelength. For refolding of the enzyme LDH it was possible to obtain separate predictions of native protein and insoluble aggregates. The soft-sensor design was further extended by a model-based observer approach using particle filtering to incorporate kinetic formulations as well as physical constraints.The novel approach enabled the analysis of kinetic mechanisms during rapid reaction dynamics and can therefore be seen as an enabler to achieve a better understanding of kinetic refolding mechanisms.

Non-covalent delivery of native proteins and peptides by phenylboronic cell-penetrating poly(disulfide)s

Poly(disulfide)s have been proposed as delivery carriers, yet their design for native protein delivery without covalent conjugation remain elusive and challenging. Here, we present a type of poly(disulfide)s randomly copolymerized from cell-penetrating cyclic five-membered disulfide (CFMD) monomer (M1) and phenylboronic CFMD monomer (M2) by ring-opening polymerization. The resulted poly(disulfide)s can directly complex a broad range of native, unmodified proteins and peptides via multiple non-covalent forces, regardless of their chemical structure, molecular weight and isoelectric point. The complexation between poly(disulfide)s and proteins can be predominantly internalized by cells via strain-promoted, thiol-mediated translocation, bypassing the classical endocytic pathway. The degradation of the poly(disulfide) is induced by rich intracellular glutathione, thereby timely releasing protein or peptide cargoes in their active form and minimize the cytotoxicity of the carrier. Of note, the surface coating of poly(disulfide) complexes by hyaluronic acid enables the systemic delivery of functional proteins, demonstrating their therapeutic potentials in vivo.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1–155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Recombinant SpTransformer proteins are functionally diverse for binding and phagocytosis by three subtypes of sea urchin phagocytes.

The California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis. To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.