Protein Refolding Research Articles

The encapsulation and controlled release of proteins from “meltable” chemically cross‐linked hydrogels

AbstractChemically crosslinked hydrogels can be used for the controlled delivery of therapeutic proteins, however, cargo loading can be challenging. A common approach involves performing the hydrogel crosslinking step in the presence of the cargo proteins, however, this can require the utilization of selective chemistries to ensure the cargos do not chemically react with the forming hydrogel network. We demonstrate that chemically crosslinked hydrogels based upon the protein polymer Capsular antigen fragment 1 (Caf1) can be used to encapsulate proteins within a chemically crosslinked hydrogel network. This encapsulation exploits the “meltable” feature of Caf1 hydrogels, where upon heating the Caf1 protein unfolds with concomitant depolymerization of the hydrogel network. Protein cargos can be loaded into the “molten” form of the hydrogel which cools with protein refolding and concomitant reformation of the hydrogel network to entrap the cargos, ensuring a more homogenous dispersion of the cargo. Analysis of release profiles indicated lower burst release in comparison to release from identical hydrogels loaded by an absorption method. This work is significant because it suggest that the “meltable” feature of Caf1 hydrogels may present an improved method of loading protein cargos into crosslinked hydrogel networks whilst avoiding the need for crosslinking chemistries.

Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.

The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis.

Amino acids and glycine derivatives differently affect refolding of mesophilic and thermophilic like α-amylases: implications in protein refolding and aggregation

α-amylases are industrially important enzymes which are used in different starch-based industries. They are adapted to different environmental conditions like extremes of temperature, pH and salinity. Herein, α-amylases from Bacillus amyloliquifaciens (BAA) and Bacillus licheniformis (BLA), representing mesophilic and thermophilic-like proteins, respectively, have been used to investigate the effect of naturally occurring osmolytes like arginine, proline, glycine and its methyl derivatives, sarcosine and betaine on their refolding. In this study, we have shown that among amino acids and glycine derivatives, betaine is the most promising osmolyte, while arginine and glycine exhibit moderately positive effect at their lower concentrations on the refolding of BAA only. Except betaine, all other osmolytes above 0.25 M showed inhibitory effect on the native enzyme activity of BLA and BAA. However, aggregation kinetics monitored by static light scattering indicates suppression of aggregation by all of these osmolytes. Further investigation by tryptophan and ANS fluorescence spectroscopy indicates the formation of compact hydrophobic core in the presence of the osmolytes. The morphology of protein aggregates having different sizes was visualized by atomic force microscopy ,and it was observed that amorphous aggregates of variable heights were formed. Our study highlights the importance of differential effects of arginine, proline, glycine, sarcosine and betaine on the native state as well as on refolding of BLA and BAA which may be helpful in devising strategies for developing effective protein formulation and prevention of aggregation of industrially and therapeutically important proteins. Communicated by Ramaswamy H. Sarma

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

Do Ionic Liquids Exhibit the Required Characteristics to Dissolve, Extract, Stabilize, and Purify Proteins? Past-Present-Future Assessment.

Proteins are highly labile molecules, thus requiring the presence of appropriate solvents and excipients in their liquid milieu to keep their stability and biological activity. In this field, ionic liquids (ILs) have gained momentum in the past years, with a relevant number of works reporting their successful use to dissolve, stabilize, extract, and purify proteins. Different approaches in protein-IL systems have been reported, namely, proteins dissolved in (i) neat ILs, (ii) ILs as co-solvents, (iii) ILs as adjuvants, (iv) ILs as surfactants, (v) ILs as phase-forming components of aqueous biphasic systems, and (vi) IL-polymer-protein/peptide conjugates. Herein, we critically analyze the works published to date and provide a comprehensive understanding of the IL-protein interactions affecting the stability, conformational alteration, unfolding, misfolding, and refolding of proteins while providing directions for future studies in view of imminent applications. Overall, it has been found that the stability or purification of proteins by ILs is bispecific and depends on the structure of both the IL and the protein. The most promising IL-protein systems are identified, which is valuable when foreseeing market applications of ILs, e.g., in "protein packaging" and "detergent applications". Future directions and other possibilities of IL-protein systems in light-harvesting and biotechnology/biomedical applications are discussed.

Application of ensemble learning to augment fluorescence-based PAT and enable real-time monitoring of protein refolding

Recently, there has been a noticeable increase in the attention given to Process Analytical Technology (PAT) within biopharmaceutical manufacturing. Specifically, there has been a focus on using spectroscopic tools in conjunction with chemometrics analysis. The emergence of computational advancements, such as artificial intelligence and machine learning, has resulted in a shift away from traditional regression models towards more contemporary learning algorithms. Examples of these algorithms include artificial neural networks and ensemble learning. This transition has significantly enhanced the effectiveness of analytical tools within the field. This investigation aims to showcase the development and implementation of an ensemble-boosting regression model. This model relies on fluorescence spectroscopy to create a PAT tool based on XGBoost. This tool’s effectiveness is demonstrated by examining the refolding process of ranibizumab, a Fab fragment biotherapeutic. This case study highlights the model’s utility in real-time monitoring of protein refolding. The accuracy and efficacy of the proposed PAT tool are substantiated through an analysis of various statistical indicators. These include the coefficient of determination and its variants, which consistently fall within the range of 0.940–0.990. Additionally, the RMSE is 0.196, the NRMSE is 0.040, and the RMSE-CV is 0.637. These results provide evidence of the reliability and precision of the PAT tool. The overall framework has been implemented in real-time to validate the developed framework's robustness. The ability of the framework to accurately predict protein refolding has been examined, with the error percentage consistently below 10 percent. In conclusion, the proposed framework represents a comprehensive approach for integrating process understanding, advanced monitoring sensors, and control strategies to consistently achieve refolding targets over extended periods of continuous protein refolding.

An all-in-one state-observer for protein refolding reactions using particle filters and delayed measurements

Proper monitoring as basis for process optimization and control of protein refolding reactions in real-time is difficult and currently available techniques are either expensive, not applicable in real-time or give only limited information about the ongoing process. Model-based methods such as particle filters (PFs) have been used in different biological systems for state-estimation to overcome difficulties arising from states that are hard or impossible to measure, often low measurement frequencies and high measurement delay. Since recent approaches had difficulties to overcome all these problems, a novel approach via a PF including a mechanistic model is used. The PF is calibrated and tuned with experimental data and its applicability validated with two additional experiments. It is shown how augmentation of model parameters can be used for state-estimation in real-time to better adapt to model inaccuracies, poor model calibration or application of the calibrated model to a new process. Furthermore, it is shown that the PF can deal with low measurement frequencies and high measurement delay, resulting in reliable tracking of the process with normalized root mean squared errors (NRMSE) of the native protein and folding intermediates between 3.44 and 6.62%, values in the range of 18 to 93% less compared to simple feed-forward simulation.

Hemin competitively inhibits HSPA8 ATPase activity mitigating its foldase function

Hemolysis in red blood cells followed by hemoglobin degradation results in high hemin levels in the systemic circulation. Such a level of hemin is disastrous for cells and tissues and is considerably responsible for the pathologies of diseases like severe malaria. Hemin's hydrophobic chemical nature and structure allow it to bind several proteins leading to their functional modification. Such modifications in physiologically relevant proteins can have a high impact on various cellular processes. HSPA8 is a chaperone that has a protective role in oxidative stress by aiding protein refolding. Through ATPase activity assays we found that hemin can competitively inhibit ATP hydrolysis by the chaperone HSPA8. Hemin as such does not affect the structural integrity of the protein which is inferred from CD spectroscopy and Gel filtration but it hinders the ATP-dependent foldase function of the chaperone. HSPA8 was not able to cause the refolding of the model protein lysozyme in the presence of hemin. The loss in HSPA8 function was due to competition between hemin and ATP as the chaperone was able to regain the foldase function when the concentration of ATP was gradually increased with hemin present at the inhibitory concentration. In-silico studies to establish the competition for the specific binding site revealed that ATP was unable to replace hemin from the ATP binding pocket of HSPA8 and was forced to form a non-specific and unstable complex. In-vitro isothermal calorimetry revealed that the affinity of ATP for binding to HSPA8 was reduced 22 folds in the presence of hemin. The prevention of HSPA8's cytoprotective function by hemin can be a major factor contributing to the overall cellular damage during hemin accumulation in the case of severe malaria and other hemolytic diseases.

Sodium Dodecyl Sulfate Analogs as a Potential Molecular Biology Reagent.

In this study, we review the properties of three anionic detergents, sodium dodecyl sulfate (SDS), Sarkosyl, and sodium lauroylglutamate (SLG), as they play a critical role in molecular biology research. SDS is widely used in electrophoresis and cell lysis for proteomics. Sarkosyl and, more frequently, SDS are used for the characterization of neuropathological protein fibrils and the solubilization of proteins. Many amyloid fibrils are resistant to SDS or Sarkosyl to different degrees and, thus, can be readily isolated from detergent-sensitive proteins. SLG is milder than the above two detergents and has been used in the solubilization and refolding of proteins isolated from inclusion bodies. Here, we show that both Sarkosyl and SLG have been used for protein refolding, that the effects of SLG on the native protein structure are weaker for SLG, and that SLG readily dissociates from the native proteins. We propose that SLG may be effective in cell lysis for functional proteomics due to no or weaker binding of SLG to the native proteins.

Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response

The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

Heat stress alters the ovarian proteome in prepubertal gilts.

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.

Glycation-induced Amyloid Formation in Proteins: An Emerging Perspective to Explore Diabetes Associated Onset of Neurodegenerative Symptoms

Abstract: Non-enzymatic protein glycation occurs spontaneously via the formation of sugarprotein Schiff adducts. The end products of this pathway are terminally misfolded proteins popularly known as Advanced Glycation End (AGE) Products. Glycated proteins account for a diverse spectrum of physiological maladies including arteriosclerosis, renal failure, diabetic complications, obesity, and neurological disorders. AGEs not only jeopardise the functionality of modified proteins but also induce the formation of Covalent protein cross-links. Glycation has the potential to induce the unfolding and refolding of globular proteins into cross-β structures thus resembling many amyloid deposits like amyloid beta, tau protein, and prions. However, glycation-induced amyloid formation is not a generic property of proteins; instead, it is guided by the nature and conformation of the protein, the type of glycation agent as well as the solution conditions governing the glycation reaction. The half-lives of AGE adducts are prolonged by their impaired proteasomal clearance since glycation modifies the lysine residues and renders them unavailable for ubiquitination. AGEs are cleared via sequestration with specific cell surface receptors (RAGE); subsequently, downstream signalling events involving MAPK and NF-κB result in the activation of inflammatory response or the apoptotic pathway. This perspective article discusses the current developments in understanding the various facets of glycationassociated protein aggregation and the corresponding development of amyloid-like entities.

Exploiting cyclodextrins as artificial chaperones to enhance enzyme protection through supramolecular engineering.

We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of β-cyclodextrin (β-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied in silico using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the β-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.

Retraction: Stacking of nanorings to generate nanotubes for acceleration of protein refolding.

Retraction of 'Stacking of nanorings to generate nanotubes for acceleration of protein refolding' by N. Kameta and D. Wing, Nanoscale, 2021, 13, 1629-1638, https://doi.org/10.1039/D0NR07660K.

Inhibitors of amyloid fibril formation.

Many diseases are caused by misfolded and denatured proteins, leading to neurodegenerative diseases. In recent decades researchers have developed a variety of compounds, including polymeric inhibitors and natural compounds, antibodies, and chaperones, to inhibit protein aggregation, decrease the toxic effects of amyloid fibrils, and facilitate refolding proteins. The causes and mechanisms of amyloid formation are still unclear, and there are no effective treatments for Amyloid diseases. This section describes research and achievements in the field of inhibiting amyloid accumulation and also discusses the importance of various strategies in facilitating the removal of aggregates species (refolding) in the treatment of neurological diseases such as chemical methods like as, small molecules, metal chelators, polymeric inhibitors, and nanomaterials, as well as the use of biomolecules (peptide and, protein, nucleic acid, and saccharide) as amyloid inhibitors, are also highlighted.

The effect of HSP70 overexpression on the cerebral cortex development

Heat shock proteins (HSPs) make up a large family of molecular chaperones, recognized for their role in protein maturation, refolding, and degradation. HSP70 was shown to promote cell survival during several pathological processes in the brain, such as stroke, neurodegenerative diseases, epilepsy, and trauma [1]. In addition, HSPs serve to promote the proper embryonic and postnatal development of many organ systems, such as the central nervous system [2]. Heat shock proteins demonstrate specific expression patterns throughout the development of the nervous system, notably during crucial embryonic and postnatal moments [1]. During embryonic development, neural and glial progenitors must survive within a hypoxic microenvironment while performing energetically demanding actions, such as cell migration and neurite outgrowth. HSPs can activate or inhibit development pathways in the nervous system that modulate cell differentiation, neurite outgrowth, cell migration, or angiogenesis [1]. Indeed, recent studies demonstrated that HSP70 directly regulates the development of the nervous system by modulating signaling cascades involved in cell growth and migration [3]. Additionally, research demonstrated [4] that introducing HSP70 from an external source significantly augments the populations of proliferating cells and differentiated neuroblasts within the mouse hippocampus. Nevertheless, some researchers [1] contend that overexpression of HSPs may negatively impact cell survival. Therefore, the precise role of these chaperones remains largely unexplored. In our study, we used in utero electroporation to introduce plasmids that controlled HSP70 overexpression into neuron progenitors of mouse embryos on the 14th day of gestation. Additionally, plasmid DNA encoding GFP was used to facilitate subsequent visualization of transformed cells. Brain samples were collected on the 18th day of gestation for immunohistochemical analysis of the sections. Confocal microscopy was used to compare the characteristics of neuronal migration in both control and HSP70 overexpression conditions. Cells that received plasmids inducing HSP70 were discovered to migrate at a lower pace in comparison to the control. Additionally, it is hypothesized that the induction of HSP70 expression could lead to neuronal malformations and impact the development of neurites. In the future, the study of cytoarchitectonics in the cortex will continue, examining the identification of electroporated cells in individual neuron populations using Satb2 and Ctip2 markers. Additionally, the differentiation of these cells will be assessed by counting those that have not exited the mitotic cycle, and the hypothesis of apoptosis induction in cells electroporated with HSP70 will be tested.

Elucidation of Site-Specific Ubiquitination on Chaperones in Response to Mutant Huntingtin.

Huntington's disease (HD) is one of the prominent neurodegenerative diseases, characterized by the progressive decline of neuronal function, due to the accumulation and aggregation of misfolded proteins. Pathological progression of HD is hallmarked by the aberrant aggregation of the huntingtin protein (HTT) and subsequent neurotoxicity. Molecular chaperones (heat shock proteins, HSPs) play a pivotal role in maintaining proteostasis by facilitating protein refolding, degradation, or sequestration to limit the accumulation of misfolded proteins during neurotoxicity. However, the role of post-translational modifications such as ubiquitination among HSPs during HD is less known. In this study, we aimed to elucidate HSPs ubiquitin code in the context of HD pathogenesis. In a comprehensive proteomic analysis, we identified site-specific ubiquitination events in HSPs associated with HTT in HD-affected brain regions. To assess the impact of ubiquitination on HSPs during HD, we quantified the abundance of ubiquitinated lysine sites in both the rat cortex/striatum and in the mouse primary cortical neurons. Strikingly, we observed highly tissue-specific alterations in the relative ubiquitination levels of HSPs under HD conditions, emphasizing the importance of spatial perturbed post-translational modifications (PTMs) in shaping disease pathology. These ubiquitination events, combined with other PTMs on HSPs, are likely to influence the phase transitions of HTT. In conclusion, our study uncovered differential site-specific ubiquitination of molecular chaperones and offers a comprehensive view of the intricate relationship between protein aggregation, and PTMs in the context of Huntington's disease.

Quantitative analytics for protein refolding states

Development and optimization of inclusion body (IB) refolding processes is often based on empirical strategies. Thus, generation of in-depth process understanding is crucial to shift towards more knowledge-driven approaches. Within this contribution the goal was to establish a systematic approach for the reliable quantification of process dynamics in protein refolding processes. Established offline- analytical tools like HPLC-based methods or photometric assays were analyzed regarding their specifications and consequent suitability to quantify protein states. An in-silico study was conducted to define requirements for state quantification by analyzing the influences of error, discrete sampling intervals and the pace of the reaction on signal quality. Refolding and aggregation reaction rates were calculated by finite difference approximation with errors propagated from the corresponding folding state. Application of the defined principles on two real Lactate dehydrogenase fed-batch refolding processes resulted in two individual sampling strategies. At reaction rates of up to 0.58 g h−1 description of the ongoing dynamics could be done accurately with high sampling intervals during the first few hours of processing as shown by comparison to a noise free simulation. The proposed method has the potential to be transferred to other proteins and to facilitate the development of model-based monitoring & control strategies.

Excessive free radical grafting interferes with the macromolecular association and crystallization of brined porcine myofibrils during heat-set gelatinization

Free radical grafting and oxidative modification show superiority in myofibrillar protein (MP) aggregation patterns during salting process, but their consequent formation mechanisms of protein hydration network require further evaluation. Herein, we explored the effect of salt-curing (0, 1, 3 and 5 %) on MP protein polymer substrate, water-protein interaction, crystallization events and thermal stability under H2O2/ascorbate-based hydroxyl radical (•OH)-generating system (HRGS) (1, 10, 20 mM H2O2). Results showed that moderate salting (≤3%) favored the water binding of MP gels during the oxidation course. Accordingly, the maximum thermal stability (Tm) of MP gels was obtained at 3 % salting could be greatly attributed to the protein chain solubilization and refolding process. However, 5 % salt synergized with •OH oxidation intensified diffraction peak 2 (the most striking diffraction feature). Microstructural analysis validated a maximum compactness of MP gel following brining with 5 % salt at potent oxidation strength (20 mM H2O2). This study maybe promises efficient strategy to the myogenetic fibril products and biomaterials.

Harnessing Liquid Crystal Sensors for High-Throughput Real-Time Detection of Structural Changes in Lysozyme during Refolding Processes.

Despite the rapid advances in process analytical technology, the assessment of protein refolding efficiency has largely relied on off-line protein-specific assays and/or chromatographic procedures such as reversed-phase high-performance liquid chromatography and size exclusion chromatography. Due to the inherent time gap pertaining to traditional methods, exploring optimum refolding conditions for many recombinant proteins, often expressed as insoluble inclusion bodies, has proven challenging. The present study describes a novel protein refolding sensor that utilizes liquid crystals (LCs) to discriminate varying protein structures during unfolding and refolding. An LC layer containing 4-cyano-4'-pentylbiphenyl (5CB) intercalated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used as a sensing platform, and its proof-of-concept performance is demonstrated using lysozyme as a model protein. As proteins unfold or refold, a local charge fluctuation at their surfaces modulates their interaction with zwitterionic phospholipid DOPE. This alters the alignment of DOPE molecules at the aqueous/LC interface, affecting the orientational ordering of bulk LC (i.e., homeotropic to planar for refolding and planar to homeotropic for unfolding). Differential polarized optical microscope images of the LC layer are subsequently generated, whose brightness directly linked to conformational changes of lysozyme molecules is quantified by gray scale analysis. Importantly, our LC-based refolding sensor is compatible with diverse refolding milieus for real-time analysis of lysozyme refolding and thus likely to facilitate the refolding studies of many proteins, especially those lacking a method to determine structure-dependent biological activity.