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Principle of isoelectric focusing. Two proteins with varying isoelectric points will migrate in the presence of a pH gradient and electric field until the net charge of a protein is zero, in which migration will cease.
Source publication
The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative met...
Contexts in source publication
Context 1
... IEF, ampholytes travel according to their charge under the influence of an electric field, in the presence of a pH gradient, until the net charge of the molecule is zero (e.g., isoelectric point, pI). When considering peptides and proteins, the separation is deemed according to the composition of amino acids and exposed charged residues, which behave as weak acids and bases ( Figure 1). The migration of the ampholytic species will follow basic principles of electrophoresis; however, the mobility will change in the presence of the pH gradient by slowing down migration at values close to the pI value. ...
Context 2
... this separation method is advantageous in its ability to concentrate large quantities of samples while simultaneously removing common interfering agents or unwanted analytes. Additionally, IEF can be carried out in capillaries, microfluidic channels and multi-compartment electrolyzers (MCE) as Figure 1. Principle of isoelectric focusing. ...
Citations
... The physicochemical properties in Table 3 and Fig. 3 include the pI as the pH at which no electrical charge is present on the molecule or the total number of negative and positive charges are equal [30]. The isoelectric focusing technique is performed based on the pI values for separating the molecule from the complex [48]. These values help to isolate the respective protein of interest in the wet lab experiments upon digestion. ...
... AI is another parameter that describes protein stability at temperatures. AI is defined as the relative volume occupied by aliphatic side chains like alanine (Ala), valine (Val), leucine (Leu), and isoleucine (Ile) [30,48]. The high value of AI indicates the increase in the thermostability nature of the protein which is an additive factor for wet lab studies. ...
Background
The Junín virus (JUNV) is well known for causing argentine haemorrhagic fever (AHF), a severe endemic disease in farming premises. The glycoprotein of JUNV is an important therapeutic target in vaccine design. Despite using drugs and neutralizing weakened antibodies being used in the medication, neither the severity reduced nor eradicated the infection. However, this constraint can be resolved by immunoinformatic approaches.
Results
The glycoprotein fasta sequence was retrieved from NCBI to anticipate the B cell and T cell epitopes through the Immune Epitope Database. Furthermore, each epitope underwent validation in Vaxijen 2.0, Aller Top, and Toxin Pred to find antigenic, nonallergic, and non-toxic peptides. Moreover, the vaccine is designed with appropriate adjuvants and linkers. Subsequently, physicochemical properties were determined in ProtParam including solubility and disulphide bonds in the SCRATCH server. The vaccine 3D structure was built using I-TASSER and refined in ModRefine. Docking between JUNV glycoprotein (PDB ID:5NUZ) with a built vaccine revealed a balanced docked complex visualized in the Drug Discovery studio, identified 280 hydrogen bonds between them. The docking score of − 15.5 kcal/mol was determined in the MM/GBSA analysis in HawkDock. MD simulations employed using the GROMACS at 20 ns resulted in minimal deviation and fewer fluctuations, particularly with high hydrogen bond-forming residues.
Conclusion
However, these findings present a potential vaccine for developing against JUNV glycoprotein after validating the epitopes and 3D vaccine construct through in silico methods. Therefore, further investigation in the wet laboratory is necessary to confirm the potentiality of the predicted vaccine.
... Using discrete pH gates allows targeted separation of proteins and it offers some advantages such as pH inherent consistency [22]. IET has been performed using different types of membranes such as PVA, agarose, and poly(acryloylaminoethoxyethanol) [23]. Microscale IET devices for the isolation of molecular species have been introduced [22,24]. ...
Histones are basic proteins with an isoelectric point around 11. It has been shown that the level of plasma circulating histones increases significantly during sepsis, and circulating free histones are associated with sepsis severity and mortality. It was found that the median plasma total free histone concentration of sepsis ICU non-survivors is higher compared to survivors. Therefore, histone concentration can serve as a prognostic indicator and there is a need for a simple, low-cost, and rapid method for measuring histone levels. In this work, we have developed a microfluidic device containing an isoelectric membrane made of dehydrated agarose gel of a specific pH embedded in a porous membrane for isoelectric trapping of histones rapidly. Although isoelectric gates have been used for trapping proteins before, they have to be introduced at the time of the experiment. Here, we show that isoelectric gates formed by gels loaded in a scaffold can be integrated directly into the fabrication process flow, dehydrated for storage, and rehydrated during the experiment and still function effectively to achieve isoelectric trapping. A low-cost and rapid microfabrication technique, xurography, was used for agarose integration and device fabrication. The integrated device was tested with samples containing buffered histone, histone in the presence of high-concentration bovine serum albumin (BSA), and histone spiked in blood plasma. The results show that the device can be used to distinguish between survivors and non-survivors of sepsis in less than 10 min, making it suitable as a point-of-care device for sepsis prognosis.
Graphical abstract
... The isoelectric focusing technique is performed on the basis of the pI values to separate molecules from complexes. 29 These values aid in the isolation of proteins of interest from the WNV polyprotein in wet laboratory experiments after digestion. ...
Objectives
West Nile virus (WNV) belongs to the Flaviviridae family and causes West Nile fever. The mechanism of transmission involves the culex mosquito species. Infected individuals are primarily asymptomatic, and few exhibit common symptoms. Moreover, 10 % of neuronal infection caused by this virus cause death. The proteins encoded by these genes had been uncharacterized, although understanding their function and structure is important for formulating antiviral drugs.
Methods
Herein, we used in silico approaches, including various bioinformatic tools and databases, to analyse the proteins from the WNV polyprotein individually. The characterization included GC content, physicochemical properties, conserved domains, soluble and transmembrane regions, signal localization, protein disorder, and secondary structure features and their respective 3D protein structures.
Results
Among 11 proteins, eight had >50 % GC content, eight proteins had basic pI values, three proteins were unstable under in vitro conditions, four were thermostable according to >100 AI values and some had negative GRAVY values in physicochemical analyses. All protein-conserved domains were shared among Flaviviridae family members. Five proteins were soluble and lacked transmembrane regions. Two proteins had signals for localization in the host endoplasmic reticulum. Non-structural (NS) 2A showed low protein disorder. The secondary structural features and tertiary structure models provide a valuable biochemical resource for designing selective substrates and synthetic inhibitors.
Conclusions
WNV proteins NS2A, NS2B, PM, NS3 and NS5 can be used as drug targets for the pharmacological design of lead antiviral compounds.
... When it comes to the performance of a biosensor, it is important to consider the pI associated with the charge of the protein and how changes in the solution pH may affect this charge. As previously reported [85][86][87], when the pH of the solution deviates from its pI, proteins can carry a net positive charge at a lower pH or a net negative charge at a higher pH; this pH-dependent charge state significantly affects the binding and interaction of proteins with the MIP electrode surface. For example, the charge state of interfering proteins, such as Myo and IgG, affect the binding affinity on the sensing electrodes by changing the molecular interaction in the specific environments designed to detect specific biomarkers of IL-1β. Figure 4i displays the EIS responses obtained by separately measuring the solution samples of IL-1β, Myo, IgG, IL-1β/Myo, and IL-1β/IgG. ...
Molecularly imprinted polymers (MIPs) have garnered significant attention as a promising material for engineering specific biological receptors with superior chemical complementarity to target molecules. In this study, we present an electrochemical biosensing platform incorporating MIP films for the selective detection of the interleukin-1β (IL-1β) biomarker, particularly suitable for mobile point-of-care testing (POCT) applications. The IL-1β-imprinted biosensors were composed of poly(eriochrome black T (EBT)), including an interlayer of poly(3,4-ethylene dioxythiophene) and a 4-aminothiophenol monolayer, which were electrochemically polymerized simultaneously with template proteins (i.e., IL-1β) on custom flexible screen-printed carbon electrodes (SPCEs). The architecture of the MIP films was designed to enhance the sensor sensitivity and signal stability. This approach involved a straightforward sequential-electropolymerization process and extraction for leaving behind cavities (i.e., rebinding sites), resulting in the efficient production of MIP-based biosensors capable of molecular recognition for selective IL-1β detection. The electrochemical behaviors were comprehensively investigated using cyclic voltammograms and electrochemical impedance spectroscopy responses to assess the imprinting effect on the MIP films formed on the SPCEs. In line with the current trend in in vitro diagnostic medical devices, our simple and effective MIP-based analytical system integrated with mobile POCT devices offers a promising route to the rapid detection of biomarkers, with particular potential for periodontitis screening.
... The pI of the L1 protein of HPV 52 is 8.2, and the pH of the buffer used in the purification protocols was 6.2. The L1 HPV protein was successfully separated and purified using cation exchange chromatography, which is considered the most suitable method for protein or peptide separation [43]. Size-exclusion chromatography, which is commonly used in protein aggregation analysis for peptide and protein biotherapeutics [44], is the next purification step after ion exchange. ...
Background
Cervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein.
Result
The recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 μg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP.
Conclusion
The recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.
... In isoelectric focusing, proteins or peptides are separated according to their isoelectric points (pI) in a pH gradient formed by carrier ampholytes (amphoteric electrolytes) upon the application of an electric potential [48,49]. The process includes the following steps: (1) the sample, mixed with carrier ampholytes, is filled into the capillary with the inlet and outlet ends immersed into a high-pH and low-pH solutions, respectively; (2) the electric field is applied to establish a pH gradient and the protein molecules with different pI values migrate until their net charge becomes zero [50]; ...
This review summarizes the fundamental principles, basic methodologies, strength and weaknesses of capillary gel electrophoresis of proteins by providing both a short historical overview and highlighting new developments and applications in biopharmaceutical, biomedical as well as food and agriculture fields. The subsets of the method including native capillary gel electrophoresis, SDS capillary gel electrophoresis, capillary gel isoelectric focusing, capillary gel isotachophoresis and capillary affinity gel electrophoresis of proteins are all critically reviewed. Relevant protein labeling techniques are also addressed.
... Every protein has a characteristic isoelectric point. In this technique, pH gradient is applied and proteins movement is based on charge in the applied electric field region, until reaches isoelectric point (Pergande and Cologna, 2017). When the pH is increased, loss of proton by the protein happens, so carrying negative charge and this decisively provokes their movement toward anode. ...
... A holder was designed to create a uniform well Materials for these microchips are typically glass, quartz, silicon, polymers (such as PDMS and PMMA), graphene and paper; carrier ampholytes or immobilised gels are used to form the pH gradients needed for these techniques (Colyer et al., 1997;Cui et al., 2005;Mohamadi et al., 2007;Peng et al., 2008;Dutta, 2019;Yu et al., 2019;Niu et al., 2021;Naghdi et al., 2022). (Herbert et al., 1998;Musante et al., 1998;Sommer and Hatch, 2009;Walker, 2009;Lengqvist et al., 2011;Pergande and Cologna, 2017) Free Flow Isoelectric Focusing (ff-IEF) ...
During the recent pandemic outbreak, Lab-on-Chip devices did not manage to fully reach their potential in rapid diagnosis of pathogens, mainly due to the lack of cost-effective LoC solutions integrated with all required sample preparation modules. This paper presents such a critical step, aiming to translate electrochemical pH control into practical protein preconcentration modules, easy to integrate with subsequent quantification modules seamlessly via Lab-on-PCB technology. In this work we present a device capable of electrochemically controlling the pH of a solution local to an individually addressed electrode in a PCB array. The electrodes were functionalised with an electropolymerised self-assembled monolayer of 4-Aminothiophenol and were subjected to voltages of 0.2–0.4 V, evaluating for the first time the bias effect both over time and over space. This study enables for the first time the implementation of this technique for electrochemical pH control into practical Lab-on-PCB devices such as isoelectric focusing, via the informed design of such electrode arrays of appropriate size and spacing.
... It is only until recently that this method is employed in the recovery of algal proteins Veide Vilg and Undeland 2017;Harrysson et al. 2018) for the purpose of generating seaweed protein products for food, feed and potential therapeutic application (Angell et al. 2016;Harrysson et al. 2019;Cermeño et al. 2020;Trigo et al. 2021). This protein recovery technique is considered to be old since isoelectric point-based protein and peptide separation technologies has gone through major developmental milestones (Pergande and Cologna 2017). However, its increasing use this decade proves the greater appreciation of the technology for sustainable recovery of functional proteins both from marine and terrestrial sources (Jiang et al. 2018;Tang et al. 2021). ...
Previous work on Pacific dulse (Devaleraea mollis), a fast-growing protein-rich red algae, revealed that protein recovery can be significantly improved with cellulase pretreatment and sequential extraction approach. Since solubilized protein fractions need to be precipitated out of solution for enrichment and pellet recovery for downstream applications, this study aimed to evaluate the effects of precipitation strategies on macroalgal protein yield. Extracted protein fractions were precipitated using trichloroacetic acid (TCA) or pH-shift method (HCl/NaOH), and protein concentrations were assessed using three quantification methods, namely modified Lowry, Dumas, and total amino acid analysis (TAA). Specific to each Osborne fraction (albumin, globulin, glutelin and prolamin), the pH-shift precipitation approach was optimized in consideration to the amount of protein pellet recovered, protein retained in supernatant, and volume of acid required for pH adjustment. This work shows that the optimized pH-shift method has competitive yield compared to that of TCA precipitation. Ethanol wash post-pellet collection improved purity of the freeze-dried powders in both precipitation approaches but had more pronounced effects to TCA pellets. This then suggests that a single step precipitation using the optimized pH-shift method can be employed as a food-grade method in the recovery of extracted Pacific dulse proteins. Overall, this work provides a pioneering insight on the recovery of Pacific dulse protein using a pH-shift approach, and how three protein quantification methods were streamlined for protein recovery assessment. As a promising complementary food protein and potential bioactive peptide (BAP) source, this work offers an upscalable and ecologically sustainable recovery approach for seaweed protein from an abundant natural resource on the Pacific coast.
... including theoretical isoelectric point (pI), in vitro and in vivo half-life, instability index, and aliphatic index according to a previous study (Wilkins et al., 1999). The theoretical pI of a protein can facilitate the selection of methods for protein purification, such as ion exchange chromatography or isofocusing electrophoresis (Pergande and Cologna, 2017). Half-life refers to the time it takes for half of the synthesized protein to disappear (Ciechanover and Schwartz, 1989). ...
Background
Our previous study developed a novel peptide-based vaccine, MP3RT, to fight against tuberculosis (TB) infection in a mouse model. However, the consistency between the immunoinformatics predictions and the results of real-world animal experiments on the MP3RT vaccine remains unclear.
Method
In this study, we predicted the antigenicity, immunogenicity, physicochemical parameters, secondary structure, and tertiary structure of MP3RT using bioinformatics technologies. The immune response properties of the MP3RT vaccine were then predicted using the C-ImmSim server. Finally, humanized mice were used to verify the characteristics of the humoral and cellular immune responses induced by the MP3RT vaccine.
Results
MP3RT is a non-toxic and non-allergenic vaccine with an antigenicity index of 0.88 and an immunogenicity index of 0.61, respectively. Our results showed that the MP3RT vaccine contained 53.36% α-helix in the secondary structure, and the favored region accounted for 98.22% in the optimized tertiary structure. The binding affinities of the MP3RT vaccine to the human leukocyte antigen (HLA)-DRB1*01:01 allele, toll-like receptor-2 (TLR-2), and TLR-4 receptors were -1234.1 kcal/mol, -1066.4 kcal/mol, and -1250.4 kcal/mol, respectively. The results of the C-ImmSim server showed that the MP3RT vaccine could stimulate T and B cells to produce immune responses, such as high levels of IgM and IgG antibodies, IFN-γ, TNF-α, and IL-2 cytokines. Results from real-world animal experiments showed that the MP3RT vaccine could stimulate the humanized mice to produce high levels of IgG and IgG2a antibodies and IFN-γ⁺ T lymphocytes. Furthermore, the levels of IFN-γ, IL-2, and IL-6 cytokines in mice immunized with the MP3RT vaccine were significantly higher than those in the control group.
Conclusion
MP3RT is a highly antigenic and immunogenic potential vaccine that can effectively induce Th1-type immune responses in silico analysis and animal experiments. This study lays the foundation for evaluating the value of computational tools and immunoinformatic techniques in reverse vaccinology research.
