Biofunctional Proteins Research Articles

Intracellular Delivery of Therapeutic Protein via Ultrathin Layered Double Hydroxide Nanosheets.

The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable protein, gelonin, towards efficient and specific cancer treatment. The LDH nanosheet was synthesized via a facile method without using exfoliation agents and showed a high loading capacity of proteins (up to 182%). Using 2D and 3D 4T1 breast cancer cell models, LDH-gelonin demonstrated significantly higher cellular uptake efficiency, favorable endosome escape ability, and deep tumor penetration performance, leading to a higher anticancer efficiency, in comparison to free gelonin. This work provides a promising strategy and a generalized nanoplatform to efficiently deliver biofunctional proteins to unlock their therapeutic potential for cancer treatment.

Autotrophic production of bio-functional proteins from freshwater microalgae using natural water medium for an economical and ecofriendly approach

As natural repositories of a broad spectrum of bioactive compounds ranging from pigments and enzymes to unique fatty acids and proteins, microalgae have attracted growing interest for their potential to produce bio-functional proteins. This comprehensive study examines the autotrophic cultivation of freshwater microalgae, focusing on protein production using a photobioreactor and natural water as the growth medium. The cultivation strategy eliminated the need for supplemented nutrients, enhancing environmental sustainability and cost- effectiveness. The study achieved a biomass production rate of 0.262 mg/L/day and a notable protein yield of 27.69 mg/L/day. Our findings support the feasibility and efficacy of employing natural water as a cultivation medium for freshwater microalgae for protein production. This approach alleviates the environmental burden associated with synthetic growth media and contributes to reducing operational costs. The study thus demonstrates the potential for this methodology to pave the way for a new, eco-friendly, and economically sustainable paradigm in the production of algal bio-functional proteins. Moreover, the widespread availability of natural resources makes this approach highly adaptable and scalable for larger production systems.

Enrichment of Lactoferrin and Immunoglobulin G from Acid Whey by Cross-Flow Filtration.

The production of cream cheese, curd, high-protein yogurt, or caseinate results in large amounts of acid whey as a by-product. So far acid whey is often disposed as animal feed or organic fertilizer. However, these approaches ignore the valorization potential that arises from the unique composition of the whey protein fraction. Whey contains the biofunctional proteins lactoferrin and immunoglobulin G, which possess immune-supporting, antibacterial, antiviral, and numerous further health-promoting functions. However, the concentration of these proteins in bovine milk or whey is below a physiologically relevant level. Based on literature research we specified a daily intake of 200 mg lactoferrin as the minimal functional dose. By means of cross-flow ultrafiltration, an attempt was made to increase the concentration of biofunctional proteins. Therefore, a membrane for the selective retention of lactoferrin and immunoglobulin G was identified, and the process parameters were optimized. Finally, a concentration experiment was conducted, whereby the concentration of biofunctional proteins was increased up to factor 30. The biofunctionality was assessed in a microbiological assay. Surprisingly, the antimicrobial growth inhibition of the produced concentrate was even higher than in pure lactoferrin. The presented approach offers a strategy to convert an abundant but underutilized by-product into valuable products for human nutrition.

Dressing blood-contacting devices with platelet membrane enables large-scale multifunctional biointerfacing

•Platelet membrane were first used for coating macroscale blood-contacting devices •Platelet-membrane coating (PMC) endows material-improved biocompatibility •Biofunctional proteins confer favorable performance on PMC-modified vascular stents So far, cloaking materials with platelet membranes have been widely studied, mainly focused on covering nanosized materials but rarely forwarded to dressing large-scale materials. Herein, a platelet-membrane coating (PMC) constructed on the surface of a macroscopic device is reported, driven by the spontaneous fusion of platelet-membrane vesicles (PMVs) with a negatively charged superhydrophilic surface. The coating contains not only phospholipids but also a variety of functional proteins of the platelet membrane, which is a further leap compared with surface-modification strategies of imitating cell-membrane structure with inert synthetic amphiphilic molecules. The preservation of membrane proteins by dressing PMC endows the surface of macroscopic devices with bioactivity. The implantation of vascular stents covered with PMC demonstrates the potential to address the desired biointerface properties, such as thromboprotection and anti-inflammation, and promote re-endothelialization. Overall, dressing large-scale blood-contacting materials with PMC provides a multifunctional biointerface by naturally preserved membrane proteins. So far, cloaking materials with platelet membranes have been widely studied, mainly focused on covering nanosized materials but rarely forwarded to dressing large-scale materials. Herein, a platelet-membrane coating (PMC) constructed on the surface of a macroscopic device is reported, driven by the spontaneous fusion of platelet-membrane vesicles (PMVs) with a negatively charged superhydrophilic surface. The coating contains not only phospholipids but also a variety of functional proteins of the platelet membrane, which is a further leap compared with surface-modification strategies of imitating cell-membrane structure with inert synthetic amphiphilic molecules. The preservation of membrane proteins by dressing PMC endows the surface of macroscopic devices with bioactivity. The implantation of vascular stents covered with PMC demonstrates the potential to address the desired biointerface properties, such as thromboprotection and anti-inflammation, and promote re-endothelialization. Overall, dressing large-scale blood-contacting materials with PMC provides a multifunctional biointerface by naturally preserved membrane proteins.

Artificial Nanocage Formed via Self-Assembly of β-Annulus Peptide for Delivering Biofunctional Proteins into Cell Interiors.

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the β-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.

Protease-Triggered Release of Stabilized CXCL12 from Coated Scaffolds in an Ex Vivo Wound Model.

Biomaterials are designed to improve impaired healing of injured tissue. To accomplish better cell integration, we suggest to coat biomaterial surfaces with bio-functional proteins. Here, a mussel-derived surface-binding peptide is used and coupled to CXCL12 (stromal cell-derived factor 1α), a chemokine that activates CXCR4 and consequently recruits tissue-specific stem and progenitor cells. CXCL12 variants with either non-releasable or protease-mediated-release properties were designed and compared. Whereas CXCL12 was stabilized at the N-terminus for protease resistance, a C-terminal linker was designed that allowed for specific cleavage-mediated release by matrix metalloproteinase 9 and 2, since both enzymes are frequently found in wound fluid. These surface adhesive CXCL12 derivatives were produced by expressed protein ligation. Functionality of the modified chemokines was assessed by inositol phosphate accumulation and cell migration assays. Increased migration of keratinocytes and primary mesenchymal stem cells was demonstrated. Immobilization and release were studied for bioresorbable PCL-co-LC scaffolds, and accelerated wound closure was demonstrated in an ex vivo wound healing assay on porcine skin grafts. After 24 h, a significantly improved CXCL12-specific growth stimulation of the epithelial tips was already observed. The presented data display a successful application of protein-coated biomaterials for skin regeneration.

PiggyBac Transposition and the Expression of Human Cystatin C in Transgenic Chickens.

Simple SummaryThe genetic modification of livestock genomes showed the great potential for production of industrial biomaterials as well as improving animal production. Particularly, the transgenic hen’s eggs have been considered for a massive production system of the genetically engineered biomaterials as a bioreactor animal. Virus-mediated transgene transduction is the most powerful strategy to generate the transgenic animals. However, industrial applications were hampered by many obstacles such as relatively low germline transmission and transgene silencing effects, as well as viral safety issues. In this study, a piggyBac transposon which is a non-viral integration technical platform was introduced into chicken primordial germ cells. Finally, we developed transgenic chickens and assayed the bioactivity of human cystatin C in the transgenic chicken’s tissues.A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow’s milk and hen’s eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.

Magnetically Driven Nanotransporter-Assisted Intracellular Delivery and Autonomous Release of Proteins.

Biofunctional proteins such as active enzymes and therapeutic proteins show tremendous promise in disease treatment. However, intracellular delivery of proteins is facing substantial challenges owing to their vulnerability to degradation and denaturation and the presence of various biological barriers such as their low membrane transport efficiency. Herein, we report a magnetically driven and redox-responsive nanotransporter (MRNT) for highly efficient intracellular delivery of biofunctional proteins. The MRNT has remarkably high cargo capacity, compared with that without nanoscale cargo compartments. We have demonstrated the directional and dynamic motion of the MRNT using both nanoparticle tracking analysis and magnetic driving evaluation. Moreover, the active MRNT can translocate into the cytosol and sense the reducing cytosolic environment to discharge protein cargoes autonomously. The internalization mechanism of the MRNT has been studied using endocytosis inhibitors. Under the magnetic drive, the MRNT can promote a protein transduction efficiency of over 95%, and the intracellular protein delivery by the active MRNT shows significantly higher (∼4 times) enzymatic activity and therapeutic efficiency than those achieved by the static ones. Our proof-of-concept study provides a valuable tool for intracellular protein transduction and contributes to biotechnology and protein therapeutics.

Study on the antibacterial activities of emodin derivatives against clinical drug-resistant bacterial strains and their interaction with proteins.

Novel haloemodin (HEI2) synthesized by modifying emodin, a traditional Chinese medicine component, possesses remarkable antibacterial activity, being much more effective than its parent nucleus, emodin. Firstly, we discovered that HEI2 increases bacterial cell membrane permeability to potassium ions more drastically than emodin. We thus further investigated the interaction of haloemodin and protein using a fluorescence quenching and circular dichroism (CD) study based on bovine serum albumin (BSA). HEI2 spontaneously bound to BSA at Trp 212 residue (subdomain IIA) by hydrogen bonds and van der Waals interactions to forms HEI2-BSA complexes, and this binding decreased the α-helical content of BSA. We also confirmed that emodin bound to BSA by hydrophobic interaction alone. These results suggest that the main responses for the substantial antibacterial activities of HEI2 are a disruption of the bacterial plasma membrane function and the interaction with biological functional proteins. Furthermore, the study of the interaction of drugs with BSA, which has a fluorescent group tryptophan residue similar to many bio-functional proteins, will be a simple and inexpensive scope-reducing method in screening new drugs.

Supramolecular Nanofibers with Superior Bioactivity to Insulin-Like Growth Factor-I.

Bioactive peptides derived from proteins generally need to be folded into secondary structures to activate downstream signaling pathways. However, synthetic peptides typically form random-coils, thus losing their bioactivities. Here, we show that by introducing a self-assembling peptide motif and using different preparation pathways, a peptide from insulin-like growth factor-I (IGF-1) can be folded into an α-helix and β-sheet. The β-sheet one exhibits a low dissociation constant to the IGF-1 receptor (IGF-1R, 11.5 nM), which is only about 3 times higher than that of IGF-1 (4.3 nM). However, the α-helical one and the peptide without self-assembling motif show weak affinities to IGF-1R ( KD = 179.1 and 321.6 nM, respectively). At 10 nM, the β-sheet one efficiently activates the IGF-1 downstream pathway, significantly enhancing HUVEC proliferation and preventing cell apoptosis. The β-sheet peptide shows superior performance to IGF-1 in vivo, and it improves ischemic hind-limb salvage by significantly reducing muscle degradation and enhancing limb vascularization. Our study provides a useful strategy to constrain peptides into different conformations, which may lead to the development of supramolecular nanomaterials mimicking biofunctional proteins.

Self-Assembly of Extracellular Vesicle-like Metal-Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins.

The intracellular delivery of biofunctional enzymes or therapeutic proteins through systemic administration is of great importance in therapeutic intervention of various diseases. However, current strategies face substantial challenges owing to various biological barriers, including susceptibility to protein degradation and denaturation, poor cellular uptake, and low transduction efficiency into the cytosol. Here, we developed a biomimetic nanoparticle platform for systemic and intracellular delivery of proteins. Through a biocompatible strategy, guest proteins are caged in the matrix of metal-organic frameworks (MOFs) with high efficiency (up to ∼94%) and high loading content up to ∼50 times those achieved by surface conjunction, and the nanoparticles were further decorated with the extracellular vesicle (EV) membrane with an efficiency as high as ∼97%. In vitro and in vivo study manifests that the EV-like nanoparticles can not only protect proteins against protease digestion and evade the immune system clearance but also selectively target homotypic tumor sites and promote tumor cell uptake and autonomous release of the guest protein after internalization. Assisted by biomimetic nanoparticles, intracellular delivery of the bioactive therapeutic protein gelonin significantly inhibits the tumor growth in vivo and increased 14-fold the therapeutic efficacy. Together, our work not only proposes a new concept to construct a biomimetic nanoplatform but also provides a new solution for systemic and intracellular delivery of protein.

Mild cell disruption methods for bio-functional proteins recovery from microalgae—Recent developments and future perspectives

Bio-functional proteins from microalgae have numerous biological properties with health-promoting effects. However, efficient harnessing of bio-functional proteins from microalgae is still in its infancy. One of the major obstacles that hinder the mass production of bio-functional proteins is the presence of resistant cell wall that diminishes the liberation of cell contents. As the bio-functional proteins are very sensitive to denaturation, selecting a mild disruption method to rupture the cell wall, while preserving their bioactivity and functionality, is of vital importance in downstream processing. To ensure the future development of efficient mild disruption methods for maximum recovery of bio-functional proteins from microalgae, this review provides useful information on various mild disruption approaches, current status, potential technologies that are still under development, as well as their advantages and constraints. In particular, those potential technologies that require further attention in the future (namely, explosive decompression, microfluidization, pulsed arc technology and cationic polymer coated membranes) are also discussed in this review.

Phycobiliproteins: A Novel Green Tool from Marine Origin Blue-Green Algae and Red Algae.

Marine species are comprising about a half of the whole global biodiversity; the sea offers an enormous resource for novel bioactive compounds. Several of the marine origin species show multifunctional bioactivities and characteristics that are useful for a discovery and/or reinvention of biologically active compounds. For millennia, marine species that includes cyanobacteria (blue-green algae) and red algae have been targeted to explore their enormous potential candidature status along with a wider spectrum of novel applications in bio- and non-bio sectors of the modern world. Among them, cyanobacteria are photosynthetic prokaryotes, phylogenetically a primitive group of Gramnegative prokaryotes, ranging from Arctic to Antarctic regions, capable of carrying out photosynthesis and nitrogen fixation. In the recent decade, a great deal of research attention has been paid on the pronouncement of bio-functional proteins along with novel peptides, vitamins, fine chemicals, renewable fuel and bioactive compounds, e.g., phycobiliproteins from marine species, cyanobacteria and red algae. Interestingly, they are extensively commercialized for natural colorants in food and cosmetics, antimicrobial, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective agents and fluorescent neo-glycoproteins as probes for single particle fluorescence imaging fluorescent applications in clinical and immunological analysis. However, a comprehensive knowledge and technological base for augmenting their commercial utilities are lacking. Therefore, this paper will provide an overview of the phycobiliproteins-based research literature from marine cyanobacteria and red algae. This review is also focused towards analyzing global and commercial activities with application oriented-based research. Towards the end, the information is also given on the potential biotechnological and biomedical applications of phycobiliproteins.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Comparison of Different Mechanical Methods for the Modification of the Egg White Protein Ovomucin, Part A: Physical Effects

It is indispensable to modify the physical properties of egg white prior to a fractionation of the included bio-functional proteins. It was already demonstrated that this can be realized with mechanical devices. However, until now, it was not clear by which kind of molecular changes this is accompanied. Thus, this study reports on the molecular changes in egg white proteins induced by various mechanical treatments (high-pressure homogenizer, colloid mill, toothed disc dispersing machine). Evaluation criteria were the particle size of the long-chain protein ovomucin, the content of thiol groups, and disulfide bridges in egg white as well as the amount of free lysozyme. In general, it was shown that these treatments led to changes in the molecular structure and that the obtained modifications were more pronounced the higher the applied energy was. In detail, it was found that the applied mechanical forces in the experimental range of this study were able to disrupt strong covalent bonds in the fibrillar protein ovomucin. Additionally, the bio-functional protein lysozyme that is partly entrapped in the natural egg white structure was released by the applied forces. Summing up, this study generates comprehensive knowledge concerning the underlying mechanisms that enable the release of lysozyme as well as the use of egg white for fractionation processes.

Molecular cloning and characterization of prokineticin receptors

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.

ChemInform Abstract: Analysis of Protein Structure: Design and Application of Photoaffinity Reagents

The study of the structure of biofunctional proteins and receptors has become one of the most exciting and rapidly moving areas of life sciences. These fields are undergoing a rapid advancement with a tremendous growth of studies at the molecular level. The molecular resolution of the receptor protein reguires working with chemical methodologies and techniques. Photoaffinity labeling is a promising chemical approach for investigation of the structure of ligand binding sites within receptor proteins. The properties of various photogenerated reactive intermediates and its precursors are evaluated, while application of properly designed photolabeling reagents are discussed to highlight the potential of the method.

タンパク質の構造と機能解析のための有機試薬 光アフィニティラベル試薬の設計と応用

The study of the structure of biofunctional proteins and receptors has become one of the most exciting and rapidly moving areas of life sciences. These fields are undergoing a rapid advancement with a tremendous growth of studies at the molecular level. The molecular resolution of the receptor protein reguires working with chemical methodologies and techniques. Photoaffinity labeling is a promising chemical approach for investigation of the structure of ligand binding sites within receptor proteins. The properties of various photogenerated reactive intermediates and its precursors are evaluated, while application of properly designed photolabeling reagents are discussed to highlight the potential of the method.