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Processing of edible insects typically involves fractionating into high-value food ingredients, which results in by-products containing chitin and insoluble proteins. This study examined the effectiveness of lactic acid bacteria (LAB) in removing proteins from chitin in insect processing residues. Lesser mealworm processing residues were biologically treated for 48 and 120 h using LAB strains without added carbon sources. Results showed partial deproteinization, up to 29 % with Levilactobacillus brevis after 120 h. Most LAB grew up to 2 log 10 colony-forming units/mL in the first 48 h. Confocal microscopy and Fourier-transform infrared spectra indicated that some protein remained attached to chitin. The molecular weight of solubilized proteins was affected by strain and time of incubation, with antioxidant activity increasing significantly after 120 h with Lacticaseibacillus paracasei. The biological treatment of insect processing streams can be a sustainable approach to producing high amounts of LAB biomass with subsequent protein solubilization and chitin release., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

This study aimed to valorize brewer's spent grain (BSG) from a side-stream into protein ingredients suitable for human consumption. The impact of protease treatments was studied for solubilizing BSG proteins. Treatment with Protamex or simultaneous co-incubation of Protamex and Flavourzyme solubilized up to 60% of the total protein in BSG, whereas co-incubation with Flavourzyme increased the availability of hydrophobic amino acids (Val, Phe, Ala, Leu, Ile) in extracts. The scale-up of protease treatments demonstrated comparable solubilized protein fractions in 0.1 L and 10 L reaction volumes. Thorough sequence-based peptide analysis by liquid chromatography-ion mobility-mass spectrometry resulted in the identification of 479 and 451 water-soluble peptides in the hydrolysates obtained with Protamex or co-incubation of Protamex and Flavourzyme, respectively. Main cutting sites on BSG proteins were identified between Leu-Gln, Tyr-Phe, Pro-X (Protamex), complementing with a variety of cutting sites mainly next to Gln, Pro, Ile, and Phe when combined with Flavourzyme. Uniform protease activity throughout the entire B-hordein sequence and the formation of peptides with varying sequence lengths did not increase the bitterness of the hydrolysates compared to the BSG sample with water extraction. These results support the characterization of enzymatic treatments in plant-based materials and the production of hydrolysates with desired composition.

Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Results: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of the anti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive. [ABSTRACT FROM AUTHOR]

Introduction: Hepatitis E virus (HEV) causes emerging diseases in poor regions of the world. The ORF2 is the only protein encoded by the virus to make the viral capsid. The aggregation of proteins into inclusion bodies (IBs) while expressing ORF2 is a major challenge in bioengineering. Methods: The ORF2 conserved sequence was expressed in Escherichia coli BL21 and assessed by a modified SDS-PAGE containing a weak denaturing environment to solubilize the recombinant ORF2 protein and Western blotting. The protein of interest was evaluated by secondary structure prediction using SOPMA, homology modeling by I-TASSER and Circular Dichroism analyses. The function of the recombinant protein was investigated by an in-house ELISA using serum specimens of HEV infected patients. Results: The solubilized form of ORF2 protein was successfully expressed in E. coli BL21 and was confirmed by SDS-PAGE and Western blotting. Secondary structure prediction, homology modeling and CD analysis of the protein of interest demonstrated that the native structure of ORF2 was almost intact. The specific anti-HEV antibody was detected using this recombinant protein and an in-house ELISA test. Conclusion: We achieved new combinations of chemical agents, consisted of low concentrations of urea and detergents to overcome the aggregation of ORF2 protein in IBs inside E. coli BL21.

In conventional cell lysate protocols, cell debris is typically discarded to obtain a cleaner lysate. However, this approach has limitations, as it may overlook vital cellular components. By discarding cell debris, researchers may inadvertently exclude crucial elements. Retaining all cellular components offers several advantages for studying molecular biology within various cellular compartments. Firstly, it provides a more accurate representation of the cellular environment. Secondly, it enables the study of complex cellular interactions, including those involving cellular structures and signaling pathways associated with debris. This shift in perspective highlights the importance of a holistic approach to lysate preparation. By obtaining lysates that include all cellular components, researchers can gain deeper insights into cellular processes, leading to more accurate data and a better understanding of cellular function and dysfunction. This study aimed to develop a protocol for the preparation of total cell lysates that retain all cellular components, including debris. Our method involves:•A three-step solubilization process using a combination of detergents, saccharides, and chelators, coupled with sonication, in contrast to the classical one-step approach using an all-detergent cocktail.•A comprehensive strategy ensuring the solubilization of all cellular components, providing a more complete lysate for analysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Results: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MWproteins (<15 kDa). Conclusions: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well. [ABSTRACT FROM AUTHOR]

The current interest in using olive biophenols to promote functional ingredients and antioxidant additives is increasing. These compounds can be obtained from olive fruit and olive-derived biomasses using different technologies. However, other components can be co-extracted. Therefore, the main objective of this study was to evaluate the effect on protein solubilization of several extraction technologies, which were applied to obtain olive biophenols from olive-derived biomasses. For this purpose, conventional (Soxhlet and water bath) and non-conventional technologies (ultrasound and microwave) were evaluated. The total phenolic content was measured using the Folin and Ciocalteu method and the protein content was measured using the Dumas combustion method. The phenolic profile and the hydroxytyrosol content were also determined. Overall, the highest total phenolic content was obtained using the Soxhlet method, while the microwave-assisted extraction at 100 °C led to the highest protein solubilization (closer to 60%) using water.

The protein extraction method based on the phenol solution and combined with protein precipitation with ammonium acetate in methanol and purification in the same solution, and additionally in acetone and ethanol, is recommended for proteomic studies of plant tissues. The obtained protein samples do not require additional nucleic acid digestion and removal of interfering contaminations. The presented protocol was used to analyze the proteome of common buckwheat flowers and leaves., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein function is generally dependent on its subcellular localization. In gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different compartments: the cytoplasm, the inner membrane, the periplasm, the outer membrane, and the extracellular medium. Different approaches can be used to determine the protein localization within cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labeling, optical fluorescence microscopy, and biochemical technics. In this chapter, we describe a simple and efficient method to isolate the different compartments of Escherichia coli by a fractionation method and to determine the presence of the protein of interest. For inner membrane proteins, we propose a method to discriminate between integral and peripheral membrane proteins., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii , and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas , Synechocystis , and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis , respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 2 (BMP-2) has a high tendency to aggregate at physiological pH and physiological ionic strength, which can complicate the development of growth factor delivery systems. The aggregation behavior in differently concentrated BMP-2 solutions was investigated using dynamic and static light scattering. It was found that at higher concentrations larger aggregates are formed, whose size decreases again with increasing dilution. A solubilizing effect and therefore less aggregation was observed upon the addition of albumin. Imaged capillary isoelectric focusing and the simulation of the surface charges of BMP-2 were used to find a possible explanation for the unusually low solubility of BMP-2 at physiological pH. In addition to hydrophobic interactions, attractive electrostatic interactions might be decisive in the aggregation of BMP-2 due to the particular distribution of surface charges. These results help to better understand the solubility behavior of BMP-2 and thus support future pharmaceutical research and the development of new strategies for the augmentation of bone healing.

Insects have the ability to convert biowaste into valuable functional compounds, such as proteins, fat and chitin. Currently, unlike with conventional commodities, there are few fractionation methods to isolate these compounds for diversification of use. Enzymatic hydrolysis is a possible method to fractionate Hermetia illucens larvae, into protein, fat and chitin. This method is a particularly attractive due to the milder process conditions required compared to chemical methods, the relative ease to control the reaction and minimal formation of unpalatable and toxic by-products. A central composite design was used to help identify the optimum hydrolysis conditions for fractionation. At these conditions the fat recovery was ?~81%; substantially more than previous attempts and the fatty acid profile stayed unchanged from the original larvae material. The protein solubility was ~57%, with a degree of hydrolysis of ~22%, and was on par with previous studies. The amino acid profile of the soluble proteins differed from the original larvae material with a slight decrease in the ratio of essential to total amino acids. The insoluble proteins were accounted for with the chitin. A washing-and-sieving step, as means to recover the insoluble proteins from the chitin is envisaged. Also, the spray drying of the protein hydrolysate produced and its functional properties allows for future investigation. This technology allows for higher margins to be made, both environmentally as well as financially, compared to the use of the ‘intact’ biomass.

Background: Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins. Methods: Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously. Results: After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots. Conclusion: By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.

Protein aggregation is a major hindrance in many in vivo and in vitro studies of proteins. It results in the formation of inclusion bodies and non-functional aggregates. Chemical chaperones also known as osmolytes which are accumulated during the stress conditions in the cells can influence the protein stability through various mechanisms. They act as osmoprotectants and contribute to the protein folding by enabling the protein to bury the backbone into the core of protein fold. In the current study, we observed the effect of chemical chaperones from four different classes on the stability and functionality of aggregation prone protein zebrafish dihydrofolate reductase (zDHFR). We also used UV–visible and circular dichroism (CD) spectroscopy to explore the protecting action of chemical chaperones on the structure and activity of zDHFR in vitro and in vivo conditions. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Response surface methodology (RSM) was used in a sequential manner to optimize solubilization and precipitation conditions in the recovery of protein from bovine lung using pH shift. RESULTS: Separate D‐optimal designs were employed for protein solubilization and precipitation. Independent variables investigated for protein solubilization were time (10–120 min), temperature (4–20 °C), pH (8.0–11.0) and solvent/sample ratio (2.5–10). Variables for protein precipitation were time (0–60 min) and pH (4.25–6.00). Soluble protein yields ranged from 323 to 649 g kg−1 and the quadratic model for protein solubilization revealed a coefficient of determination R2 of 0.9958. Optimal conditions for maximum protein solubility were extraction time 140 min, temperature 19 °C, pH 10.8 and solvent/sample ratio 13.02. Protein precipitation yields varied from 407 to 667 g kg−1, giving a coefficient of determination R2 of 0.9335. Optimal conditions for maximum protein precipitation were pH 5.03 and 60 min. Based on the RSM model, solubilization conditions were manipulated to maximize protein solubilization under reduced water and alkaline usage. These conditions were also validated. CONCLUSION: Models for solubilization and precipitation using bovine and porcine lung were validated; predicted and actual yields were in good agreement, showing cross‐species applicability of the results. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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[Display omitted] • A simplified whey protein-catechins system was prepared in the same ratio as in milk chocolate. • Interaction between whey proteins and catechins determined by (chiro-)optical spectroscopy. • Protein hydrolysis/solubilisation of whey proteins during digestion unaffected by catechins. • Milk chocolate proteins completely digested. • Interactions of catechins with milk proteins does not impair in vitro protein digestibility. Flavonoids are largely present in plant food such as cocoa and derived products. These compounds can interact with proteins inherently contained in the food matrix and/or the proteolytic enzymes involved in gastrointestinal digestion. The flavonoid/protein interaction might hamper protein bioaccessibility and digestibility, affecting the nutritional quality. However, information on the digestion fate of proteins in food matrices containing both proteins and flavonoids is limited. The aim of this work was to evaluate the interaction between proteins and flavonoids and verify the potential effects of this interaction on protein digestibility. Taking milk chocolate as model, first a simple whey proteins/catechins mixed system was evaluated, and then the effects on digestibility were also verified in a real sample of commercial milk chocolate. The effects of the catechins/whey proteins interaction in the model system were evaluated by optical and chiro-optical spectroscopy, outlining a slight protein structure modification upon interaction with catechins. The digestibility of the protein fraction both in the model system, with and without catechins, and also in milk chocolate, was then determined by the application of INFOGEST in vitro digestion method: the bioaccessibility was evaluated in terms of protein hydrolysis and protein solubilisation, and major peptides generated by the digestion were also determined by LC/HR-MS. Despite the slight interaction with proteins, flavonoids were found to not hinder nor modify protein solubilization, protein hydrolysis and peptide profile by digestive enzymes. Also protein digestibility in milk chocolate, evaluated by SDS-PAGE, was found to be complete. The present data clearly indicate that the interaction of the proteins with the flavonoids present in the cocoa matrix does not to affect protein bioaccessibility during digestion. [ABSTRACT FROM AUTHOR]

Stable and soluble proteins are ideal candidates for functional and structural studies. Unfortunately, some proteins or enzymes can be difficult to isolate, being sometimes poorly expressed in heterologous systems, insoluble and/or unstable. Numerous methods have been developed to address these issues, from the screening of various expression systems to the modification of the target protein itself. Here we use a hydrophobic, aggregation-prone, phosphate-binding protein (HPBP) as a case study. We describe a simple and fast method that selectively uses ancestral mutations to generate a soluble, stable and functional variant of the target protein, here named sHPBP. This variant is highly expressed in Escherichia coli, is easily purified and its structure was solved at much higher resolution than its wild-type progenitor (1.3 versus 1.9 Å, respectively).

The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3ʹ untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5ʹ-TAATG-3ʹ between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes.

Background Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets. New method We optimized a buffer for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer. Results Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples. Comparison with existing method(s) Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation. Conclusions This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects. [ABSTRACT FROM AUTHOR]

The aqueous two-phase extraction (ATPE) of therapeutic proteins is a promising separation alternative to cost-intensive chromatography, still being the workhorse of nowadays downstream processing. As shown in many publications, using NaCl as displacement agent in salt-polymer ATPE allows for a selective purification of the target protein immunoglobulin G ( IgG ) from human serum albumin ( HSA , represents the impurity). However a high yield of the target protein is only achievable as long as the protein is stabilized in solution and not precipitated. In this work the combined influence of NaCl and polyethylene glycol (Mw = 2000 g/mol) on the IgG - IgG interactions was determined using composition gradient multi-angle light scattering (CG-MALS) demonstrating that NaCl induces a solubilization of IgG in polyethylene glycol 2000 solution. Moreover it is shown that the displacement agent NaCl has a significant and beneficial influence on the IgG solubility in polyethylene glycol 2000-citrate aqueous two-phase system (ATPS) which can also be accessed by these advanced B 22 measurements. By simultaneous consideration of IgG solubility data with results of the ATPS phase behavior (especially volume fraction of the respective phases) allows for the selection of process tailored ATPS including identification of the maximum protein feed concentration. Through this approach an ATPS optimization is accessible providing high yields and selectivity of the target protein ( IgG ). [ABSTRACT FROM AUTHOR]

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for different diseases, which may help to identify the proteins that may serve as markers for diagnostics as well as targets for development of new therapeutic potential. [ABSTRACT FROM AUTHOR]

The effects of high pressure (to 600 MPa) at different temperatures (20 to 60 °C) for 20 min on protein solubilization and electrophoretic pattern in beef post-rigor longissimus dorsi muscle were studied. The results showed that protein solubilization increased with increasing temperature, especially from 40 °C to 60 °C. A regular trend of protein solubilization was found when isolated myofibrils were subjected to high pressure at different temperatures, an increase was observed with increasing pressure up to about 400 MPa, solubility then decreasing to 600 MPa. Electrophoretic profiles showed that myosin light chains and actin thin filaments were sensitive to pressure, and were released from myofibrils subjected to 100 MPa and higher pressures at the different temperatures.

We describe a rapid, simple, and efficient method for recovering glutathione S-transferase (GST)- and His6-tagged maltose binding protein (MBP) fusion proteins from inclusion bodies. Incubation of inclusion bodies with 10% sarkosyl effectively solubilized >95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ratio of Triton X-100 and CHAPS. We demonstrate for the first time that this combination of three detergents significantly improves binding efficiency of GST and GST fusion proteins to gluthathione (GSH) Sepharose.

During the W1/O phase (in the W1/O/W2 process) of protein microencapsulation within poly-lactide-co-glycolide (PLGA), hydrophobic interfaces are expanded where interfacial adsorption occurs followed by protein unfolding and aggregation. Spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on Diphtheria toxoid (Dtxd) stability during the W1/O phase. A correlation was established between salts used in aqueous solutions and the changes in Dtxd solubility and conformation. The Dtxd α-helical content was quite stable thus leading to the conclusion that encapsulation was followed by protein aggregation, with minor exposition of hydrophobic residues and a small change at the S-S dihedral angle. Dtxd aggregation is 95% avoided by the chaotropic SCN-. This was used to prepare a stable Dtxd and immunologically recognized/PLGA formulation in the presence of 30 mM SNC-. The recovery increased by 10.42% or 23.2% when microencapsulation was within the -COOMe or -COOH (12kDa) PLGA, respectively. In conclusion, the aim of this work was achieved, which was to obtain the maximum of Dtxd stability after contact with CH2Cl2 to begin its PLGA microencapsulation within ideal conditions. This was a technological breakthrough because a simple solution like salt addition avoided heterologous proteins usage.

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M-1) than ASB-16 (Kb = 15610 M-1). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

• NP-40 and DM are highly effective in rhSTEAP1-His 6 solubilization. • Phenyl-Sepharose recovers pre-purified rhSTEAP1-His 6 at 10 mM Tris step. • Nickel resin recuperates pre-purified rhSTEAP1-His 6 at 175 mM Imidazole step. • Q-Sepharose is an effective polishing step to purify rhSTEAP1-His 6 at 500 mM NaCl. • First-time strategies for rhSTEAP1-His 6 purification from K. pastoris lysates. The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His 6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-β-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His 6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His 6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His 6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His 6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment. [ABSTRACT FROM AUTHOR]

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications. [ABSTRACT FROM AUTHOR]

Membrane proteins account for approximately 30% of the coding regions of all sequenced genomes, and they play crucial roles in many fundamental cell processes. However, there are relatively few membrane proteins with known three-dimensional structures. This is likely due to technical challenges associated with membrane protein extraction, solubilization, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, with the latter's extraction requiring phospholipid bilayer disruption using detergents or organic solvents. In this chapter, important considerations for membrane protein purification are addressed, with a focus on the initial stages of membrane protein solubilization, where problems are most frequently encountered. Protocols are outlined for the extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and sample clean-up and concentration., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra-molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly-folded recombinant proteins. Methods: Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously. Results: After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots. Conclusion: By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5 M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins. [ABSTRACT FROM AUTHOR]

A simple and efficient method for preparing Tricine–SDS–PAGE protein sample of extracted oil bodies (OBs) was supplied: OB suspension was vortexed with SDS buffer (pH 6.8) for 2 min at room temperature with SDS/protein of 1.52/1 (w/w), which could be analyzed by Tricine–SDS–PAGE after simple treatments (dilution and 2-mercaptoethanol). At SDS/protein of 1.52/1, about 95% of proteins in soybean OB suspension were solubilized, whereas residual 5% of proteins were weakly bound to SDS-destroyed OBs; proteins in destroyed OBs might be further solubilized by SDS in the gel and cathode buffer of Tricine–SDS–PAGE, causing about 99% of proteins in soybean OB suspension recover on Tricine–SDS–PAGE gel, which was better than acetone (89%) and diethyl ether (96%) harvested protein samples. Higher or lower SDS/protein was unbeneficial for protein solubilization from OBs. Additionally, the above method was also better than organic solvent method for peanut, sesame, and rapeseed OB suspensions. [ABSTRACT FROM AUTHOR]

In this study, we attempted to solubilize protein in slaughter blood (SB) using ultrasonic technology. The application of ultrasonic technology can make enzymatic degradation of SB more effective, which has no comparable alternative for treatment. The SB was homogenized by grinding it for 10 minutes at 10,000 rpm as a pretreatment for preventing its clotting, and then ultrasonic treatment was attempted to solubilize protein in SB. To maximize the efficiency of ultrasonic treatment for SB, the optimum condition of ultrasonic frequency (UF) was determined to be 20 kHz. To optimize the operation conditions of ultrasonification with 20 kHz of frequency, we used response surface methodology (RSM) based on ultrasonic density (UD) and ultrasonification time (UT). The solubilization rate (SR) of protein (%) was calculated to be 101.304 - 19.4205 X1 + 0.0398 X2 + 7.9411 X1² + 0.0001 X2² + 0.0455 X1X². From the results of the RSM study, the optimum conditions of UD and UT were determined at 0.5 W/mL and 22 minutes, respectively, and SB treated under these conditions was estimated to have a 95% SR. Also, experimentally, a 95.53% SR was observed under same conditions, accurately reflecting the theoretical prediction of 95%. [ABSTRACT FROM AUTHOR]

For the first time, the full length recombinant HER-2[neu] receptor has been produced in a yeast ( Arxula adeninivorans ). It is one of the most studied membrane receptors in oncology and is involved in aggressive tumor formation. A yeast integration rDNA cassette containing the human gene coding for the HER-2[neu] protein was constructed and a screening procedure was performed to select the most productive transformant. Different detergents were tested for efficient solubilization of the membrane bound protein, with CHAPS giving the best results. To increase the yield of the recombinant protein from HER-2[neu] producing A. adeninivorans , optimal culture parameters were established for cultivation in bioreactor. The recombinant protein was subsequently assayed using ELISA and SPR immunoassays systems with antibodies raised against two different epitopes of the human receptor. In both cases, elution fractions containing the recombinant HER-2[neu] receptor successfully reacted with the immunoassays with limits of quantification below 100 ng ml −1 . These results demonstrate that the full length recombinant HER-2[neu] reported here has the potential to be a new standard for the detection of HER-2 type cancer. [ABSTRACT FROM AUTHOR]

Stable and soluble proteins are ideal candidates for functional and structural studies. Unfortunately, some proteins or enzymes can be difficult to isolate, being sometimes poorly expressed in heterologous systems, insoluble and/or unstable. Numerous methods have been developed to address these issues, from the screening of various expression systems to the modification of the target protein itself. Here we use a hydrophobic, aggregation-prone, phosphate-binding protein (HPBP) as a case study. We describe a simple and fast method that selectively uses ancestral mutations to generate a soluble, stable and functional variant of the target protein, here named sHPBP. This variant is highly expressed in Escherichia coli , is easily purified and its structure was solved at much higher resolution than its wild-type progenitor (1.3 versus 1.9 Å, respectively). [ABSTRACT FROM AUTHOR]

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Membrane proteins are inherently amphipathic and undergo dynamic conformational changes for proper function within native membranes. Maintaining the functional structures of these biomacromolecules in aqueous media is necessary for structural studies but difficult to achieve with currently available tools, thus necessitating the development of novel agents with favorable properties. This study introduces several new glucose-neopentyl glycol (GNG) amphiphiles and reveals some agents that display favorable behaviors for the solubilization and stabilization of a large, multi-subunit membrane protein assembly. Furthermore, a detergent structure-property relationship that could serve as a useful guideline for the design of novel amphiphiles is discussed. [ABSTRACT FROM AUTHOR]

Abstract: The impact of high pressure/temperature treatment on structure modification and functional sensory properties of frankfurter batter was investigated. The degree of solubilization of meat proteins, particularly of myosin, was identified as a key process with significant effect on the batter's structural properties. The maximal solubilization level was at 200MPa/40°C IT for all formulations which was found to be treatment time dependent. The impact of the pressurizing gradient — PG=40MPa/s and PG=2.5MPa/s was investigated and estimated to have a significant effect on the protein network and functional properties, respectively. These were improved at low PG (2.5MPa/s) as a phenomenon of secondary network formation parallel to the main matrix. Batter secondary-structure characteristics were found to be ionic-strength dependent. According to SDS-PAGE analysis, the major role in the solubilization, aggregation and gelation processes occurring in the aqueous phase was due to the myosin S-1 and S-2, N-terminal, C-terminals, the MLC and actin during the high pressure/temperature treatment. [Copyright &y& Elsevier]

It is important to solubilize acetone-precipitated proteins before isoelectric focusing (IEF) to achieve high resolution 2-DE gels. To resolve the maximum possible number of plant protein spots, we developed an improved solubilization buffer for plant proteins. We demonstrated that the resolution of 2-DE gels increased dramatically as the concentration of Tris-base increased, with maximum solubilization obtained at 200 mM Tris-base (Ly200T). The Ly200T buffer was more effective than the commonly used solubilization buffer containing 40 mM Tris at solubilizing acetone-precipitated plant proteins. Use of the Ly200T buffer to solubilize proteins resulted in an increase in intensity of approximately 30% of plant protein spots in the larger-than-40 kDa region of the gel. The Ly200T buffer also improved the resolution of abundant and basic proteins. Thus, the Ly200T buffer can be used to achieve greater resolution of protein spots in plant proteomics research. [ABSTRACT FROM AUTHOR]