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Biochemical and Biophysical Research... Jun 2024Human heavy-chain ferritin is a naturally occurring protein with high stability and multifunctionality in biological systems. This study aims to utilize a prokaryotic...
Human heavy-chain ferritin is a naturally occurring protein with high stability and multifunctionality in biological systems. This study aims to utilize a prokaryotic expression system to produce recombinant human heavy-chain ferritin nanoparticles and investigate their targeting ability in brain tissue. The human heavy-chain ferritin gene was cloned into the prokaryotic expression vector pET28a and transformed into Escherichia coli BL21 (DE3) competent cells to explore optimal expression conditions. The recombinant protein was then purified to evaluate its immunoreactivity and characteristics. Additionally, the distribution of the administered protein in normal mice and its permeability in an in vitro blood-brain barrier (BBB) model were measured. The results demonstrate that the purified protein can self-assemble extracellularly into nano-cage structures of approximately 10 nm and is recognized by corresponding antibodies. The protein effectively penetrates the blood-brain barrier and exhibits slow clearance in mouse brain tissue, showing excellent permeability in the in vitro BBB model. This study highlights the stable expression of recombinant human heavy-chain ferritin using the Escherichia coli prokaryotic expression system, characterized by favorable nano-cage structures and biological activity. Its exceptional brain tissue targeting and slow metabolism lay an experimental foundation for its application in neuropharmaceutical delivery and vaccine development fields.
Topics: Animals; Humans; Recombinant Proteins; Mice; Blood-Brain Barrier; Brain; Escherichia coli; Nanoparticles; Ferritins; Apoferritins; Tissue Distribution
PubMed: 38640729
DOI: 10.1016/j.bbrc.2024.149939 -
Langmuir : the ACS Journal of Surfaces... Apr 2024Recombinant protein production is an essential aspect of biopharmaceutical manufacturing, with serving as a primary host organism. Protein refolding is vital for...
Recombinant protein production is an essential aspect of biopharmaceutical manufacturing, with serving as a primary host organism. Protein refolding is vital for protein production; however, conventional refolding methods face challenges such as scale-up limitations and difficulties in controlling protein conformational changes on a millisecond scale. In this study, we demonstrate the novel application of flow microreactors (FMR) in controlling protein conformational changes on a millisecond scale, enabling efficient refolding processes and opening up new avenues in the science of FMR technology. FMR technology has been primarily employed for small-molecule synthesis, but our novel approach successfully expands its application to protein refolding, offering precise control of the buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed for gram-scale production. This FMR system allows flash changes in the reaction conditions, effectively circumventing protein aggregation during refolding. To the best of our knowledge, this is the first study to use FMR for protein refolding, which offers a more efficient and scalable method for protein production. The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The FMR technique offers a promising approach for enhancing protein refolding efficiency and has demonstrated its potential in streamlining the process from laboratory-scale research to industrial-scale production, making it a game-changing technology in the field.
PubMed: 38618876
DOI: 10.1021/acs.langmuir.4c00024 -
Nature Communications Apr 2024Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or...
Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.
Topics: DNA Damage; Heat-Shock Proteins, Small; Intranuclear Inclusion Bodies; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation; Vesicular Transport Proteins
PubMed: 38615096
DOI: 10.1038/s41467-024-47615-8 -
International Journal of Molecular... Apr 2024Refolding multi-disulfide bonded proteins expressed in into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and...
Refolding multi-disulfide bonded proteins expressed in into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the N-HSQC NMR spectrum exhibited several H-N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.
Topics: Humans; Influenza, Human; Escherichia coli; Influenza A Virus, H1N1 Subtype; Orthomyxoviridae Infections; Disulfides
PubMed: 38612753
DOI: 10.3390/ijms25073943 -
Frontiers in Molecular Biosciences 2024COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the...
COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the world. The viral positive-sense single-stranded RNA encodes 31 proteins among which the spike (S) is undoubtedly the best known. Recently, protein E has been reputed as a potential pharmacological target as well. It is essential for the assembly and release of the virions in the cell. Literature describes protein E as a voltage-dependent channel with preference towards monovalent cations whose intracellular expression, though, alters Ca homeostasis and promotes the activation of the proinflammatory cascades. Due to the extremely high sequence identity of SARS-CoV-2 protein E (E-2) with the previously characterized E-1 (i.e., protein E from SARS-CoV) many data obtained for E-1 were simply adapted to the other. Recent solid state NMR structure revealed that the transmembrane domain (TMD) of E-2 self-assembles into a homo-pentamer, albeit the oligomeric status has not been validated with the full-length protein. Prompted by the lack of a common agreement on the proper structural and functional features of E-2, we investigated the specific mechanism/s of pore-gating and the detailed molecular structure of the most cryptic protein of SARS-CoV-2 by means of MD simulations of the E-2 structure and by expressing, refolding and analyzing the electrophysiological activity of the transmembrane moiety of the protein E-2, in its full length. Our results show a clear agreement between experimental and predictive studies and foresee a mechanism of activity based on Ca affinity.
PubMed: 38606285
DOI: 10.3389/fmolb.2024.1334819 -
Journal of Animal Science Jan 2024Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation; a thermal imbalance that compromises female reproduction. This study...
Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation; a thermal imbalance that compromises female reproduction. This study investigated the hypothesis that HS alters the ovarian proteome and negatively impacts proteins engaged with insulin signaling, inflammation, and ovarian function. Prepubertal gilts (n = 19) were assigned to one of three environmental groups: thermal neutral with ad libitum feed intake (TN; n = 6), thermal neutral pair-fed (PF; n = 6), or HS (n = 7). For 7 d, HS gilts were exposed to 12-h cyclic temperatures of 35.0 ± 0.2 °C and 32.2 ± 0.1 °C, while TN and PF gilts were housed at 21.0 ± 0.1 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on ovarian protein homogenates. Relative to TN gilts, 178 proteins were altered (P ≤ 0.05, log2foldchange ≥ 1) by HS, with 76 increased and 102 decreased. STRING gene ontology classified and identified 45 biological processes including those associated with chaperone protein refolding, cytoplasmic translational initiation, and immune activation; with a protein-protein interaction web network of 158 nodes and 563 edges connected based on protein function (FDR ≤ 0.05). Relative to PF, HS altered 330 proteins (P ≤ 0.05, log2foldchange ≥ 1), with 151 increased and 179 decreased. Fifty-seven biological pathways associated with protein function and assembly, RNA processing, and metabolic processes were identified, with a protein-protein interaction network of 303 nodes and 1,606 edges. Comparing HS with both the TN and PF treatments, 72 ovarian proteins were consistently altered by HS with 68 nodes and 104 edges, with biological pathways associated with translation and gene expression. This indicates that HS alters the ovarian proteome and multiple biological pathways and systems in prepubertal gilts; changes that potentially contribute to female infertility.
Topics: Swine; Female; Animals; Proteome; Chromatography, Liquid; Tandem Mass Spectrometry; Sus scrofa; Heat-Shock Response; Hot Temperature
PubMed: 38605681
DOI: 10.1093/jas/skae053 -
Preparative Biochemistry & Biotechnology Apr 2024The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors: sweetness, saltiness, sourness, bitterness, and umami....
The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors: sweetness, saltiness, sourness, bitterness, and umami. Sweetness is primarily mediated through the sweet taste receptor, a membrane-bound heterodimeric structure comprising T1R2-T1R3. However, unraveling the structural and mechanistic intricacies of the sweet taste receptor has proven challenging. This study aimed to address this knowledge gap by expressing an extracellular N-terminal domain encompassing the cysteine-rich domain of human hT1R3 (hT1R3-TMD) in . The expressed protein was obtained as inclusion bodies, purified by metal affinity chromatography, and refolded using the dilution-refolding method. Through rigorous analysis, we confirmed the successful refolding of hT1R3-TMD and elucidated its structural characteristics using circular dichroism spectroscopy. Notably, the refolded protein was found to exist as either a monomer or a dimer, depending on its concentration. A tryptophan fluorescence quenching assay revealed that the dissociation constants for sucrose, sucralose, and brazzein were >9500 μM, 2380 μM and 14.3 μM, respectively. Our findings highlight the utility of this expression system for producing functional hT1R3-TMD for investigations and demonstrate the efficacy of the tryptophan fluorescence quenching assay in revealing complex interactions between sweet taste receptors and various sweeteners.
PubMed: 38578840
DOI: 10.1080/10826068.2024.2336985 -
Analytical and Bioanalytical Chemistry May 2024Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of...
Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.
Topics: Inclusion Bodies; Protein Refolding; Spectrometry, Fluorescence; Recombinant Proteins; Tryptophan; Escherichia coli; Tyrosine; Fluorescence; Protein Folding
PubMed: 38573344
DOI: 10.1007/s00216-024-05249-1 -
Journal of the Science of Food and... Apr 2024This study explored the denaturation of 11S globulin, a protein known for its diverse functional properties in soy protein applications, at pH 3.0 and pH 10.0,...
BACKGROUND
This study explored the denaturation of 11S globulin, a protein known for its diverse functional properties in soy protein applications, at pH 3.0 and pH 10.0, followed by a gradual return to pH 7.0 to facilitate renaturation. It investigated the structural and functional changes during renaturation induced by a change in pH, revealing the stabilization mechanism of 11S globulin.
RESULTS
The findings revealed that during pH adjustment to neutral, the denatured soybean 11S globulin - resulting from alkaline (pH 10.0) or acidic (pH 3.0) treatments - experienced a refolding of its extended tertiary structure to varying extents. The particle size and the proportions of α-helix and β-sheet in the secondary structure aligned progressively with those of the natural-state protein. However, for the alkali-denatured 11S, the β-sheet content decreased upon adjustment to neutral, whereas an increase was observed for the acid-denatured 11S. In terms of functional properties, after alkaline denaturation, the foaming capacity (FC) and emulsifying activity index (EAI) of 11S increased by 1.4 and 1.2 times, respectively, in comparison with its native state. The solubility, foamability, and emulsifiability of the alkali-denatured 11S gradually diminished during renaturation but remained superior to those of the native state. Conversely, these properties showed an initial decline, followed by an increase during renaturation triggered by pH neutralization.
CONCLUSIONS
This research contributes to the enhancement of protein functionality, offering a theoretical foundation for the development of functional soy protein products and expanding their potential applications. © 2024 Society of Chemical Industry.
PubMed: 38567792
DOI: 10.1002/jsfa.13505 -
Journal of Microbiology and... May 2024The production of disulfide bond-containing recombinant proteins in has traditionally been done by either refolding from inclusion bodies or by targeting the protein to... (Comparative Study)
Comparative Study
The production of disulfide bond-containing recombinant proteins in has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of : i) engineered strains with deletions in the disulfide reduction pathways, SHuffle, and ii) the co-expression of oxidative folding catalysts, CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.
Topics: Escherichia coli; Disulfides; Cytoplasm; Recombinant Proteins; Protein Folding; Escherichia coli Proteins; Oxidation-Reduction; Protein Disulfide-Isomerases; Periplasm; Culture Media
PubMed: 38563095
DOI: 10.4014/jmb.2311.11025