The contrasting roles of co-solvents in protein formulations and food products.

Protein aggregation is a major hurdle in developing biopharmaceuticals, in particular protein formulation area, but plays a pivotal role in food products. Co-solvents are used to suppress protein aggregation in pharmaceutical proteins. On the contrary, aggregation is encouraged in the process of food product making. Thus, it is expected that co-solvents play a contrasting role in biopharmaceutical formulation and food products. Here, we show several examples that utilize co-solvents, e.g., salting-out salts, sugars, polyols and divalent cations in promoting protein-protein interactions. The mechanisms of co-solvent effects on protein aggregation and solubility have been studied on aqueous protein solution and applied to develop pharmaceutical formulation based on the acquired scientific knowledge. On the contrary, co-solvents have been used in food industries based on empirical basis. Here, we will review the mechanisms of co-solvent effects on protein-protein interactions that can be applied to both pharmaceutical and food industries and hope to convey knowledge acquired through research on co-solvent interactions in aqueous protein solution and formulation to those involved in food science and provide those involved in protein solution research with the observations on aggregation behavior of food proteins.

Different behavior of Ferguson plot between agarose and polyacrylamide gels.

In this study, we conducted Ferguson plot analyses using both agarose and polyacrylamide gels in native electrophoresis and SDS-PAGE. The results revealed intriguing differences in the behavior of bovine serum albumin (BSA) and other model proteins. Specifically, BSA exhibited Ferguson plot slopes that were dependent on the oligomer size in agarose native gel electrophoresis, while such size-dependent behavior was not observed in native-PAGE or SDS-PAGE. These findings suggest that Ferguson plot analysis is a suitable approach when using agarose gel under the electrophoretic conditions employed in this study. Furthermore, our investigation extended to model proteins with acidic isoelectric points and larger molecular weights, namely Ferritin and caseinolytic peptidase B (ClpB). Notably, these proteins displayed distinct Ferguson plot slopes when subjected to agarose gel electrophoresis. Intriguingly, when polyacrylamide gel was employed, ClpB exhibited multiple bands, each with its unique Ferguson plot slope, deviating from the expected behavior based on molecular size. This divergence in Ferguson plot characteristics between agarose and polyacrylamide gels points to an interesting and complex interplay between protein properties and gel electrophoresis conditions.

Electrophoresis, a transport technology that transitioned from moving boundary method to zone method.

Gel electrophoresis, a transport technology, is one of the most widely used experimental methods in biochemical and pharmaceutical research and development. Transport technologies are used to determine hydrodynamic or electrophoretic properties of macromolecules. Gel electrophoresis is a zone technology, where a small volume of sample is applied to a large separation gel matrix. In contrast, a seldom-used electrophoresis technology is moving boundary electrophoresis, where the sample is present throughout the separation phase or gel matrix. While the zone method gives peaks of separating macromolecular solutes, the moving boundary method gives a boundary between solute-free and solute-containing phases. We will review electrophoresis as a transport technology of zone and moving boundary methods and describe its principles and applications.

Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin.

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.

Elucidating the mechanisms of additive effects at high concentrations on hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is a commonly used chromatography technique for purifying proteins. It utilizes salting-out salts to facilitate the binding of native proteins to weakly hydrophobic ligands. There have been three proposed mechanisms for the promoting effects of salting-out salts, which include the dehydration of proteins by salts, cavity theory, and salt exclusion. To evaluate the above three mechanisms, an HIC study was conducted on Phenyl Sepharose using four different additives. These additives included a salting-out salt (NH4)2SO4, sodium phosphate that increases the surface tension of water, a salting-in salt MgCl2, and an amphiphilic protein-precipitant polyethylene glycol (PEG). Results indicated that the first two salts resulted in protein binding, while MgCl2 and PEG led to flow-through. These findings were then used to interpret the three proposed mechanisms, which showed that MgCl2 and PEG deviated from the dehydration mechanism, and MgCl2 also deviated from the cavity theory. The observed effects of these additives on HIC were reasonably well explained for the first time by their interactions with proteins.

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and ß-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Agarose native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series.

The effects of salting-in and salting-out salts defined by Hofmeister series on the solution state of bovine serum albumin (BSA) in 50 mM Tris-HCl buffer at pH 7.4 before and after thermal unfolding at 80 °C for 5 min were examined using agarose native gel electrophoresis and mass photometry. Gel electrophoresis showed that salting-in MgCl2, CaCl2 and NaSCN resulted in formation of intermediate structures of BSA upon heating on native gel, while heating in buffer alone resulted in aggregated bands. Mass photometry showed large loss of monomer and oligomers when heated in this buffer, but retaining these structures in the presence of 1 M MgCl2 and NaSCN. To our surprise, salting-out MgSO4 also showed a similar effect on gel electrophoresis and mass photometry. Salting-out NaCl and (NH4)2SO4 resulted in smearing and aggregated bands, which were supported by mass photometry. Aggregation-suppressive ArgHCl also showed oligomer aggregates upon gel electrophoresis and mass photometry.

Non-Affinity Purification of Antibodies.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis.

The nucleoprotein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is abundantly expressed during infection, making it a diagnostic target protein. We analyzed the structure of the NP in solution using a recombinant protein produced in E. coli. A codon-optimized Profinity eXact™-tagged NP cDNA was cloned into pET-3d vector and transformed into E. coli T7 Express. The recombinant protein was first purified via chromatographic step using an affinity tag-based system that was followed by tag cleavage with sodium fluoride, resulting in proteolytic removal of the N-terminal tag sequence. The digested sample was then loaded directly onto a size exclusion chromatography run in the presence of L-Arg-HCl, resulting in removal of host nucleic acids and endotoxin. The molecular mass of the main NP fraction was determined by mass photometry as a dimeric form of NP, consistent with the blue native PAGE results. Interestingly, analysis of the purified NP by our newly developed agarose native gel electrophoresis revealed that it behaved like an acidic protein at low concentration despite its alkaline isoelectric point (theoretical pI = 10) and displayed a unique character of concentration-dependent charge and shape changes. This study should shed light into the behavior of NP in the viral life cycle.

Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis.

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.

A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting.

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.

Ladder observation of bovine serum albumin by high resolution agarose native gel electrophoresis.

A commercially available bovine serum albumin (BSA) was examined by agarose native gel electrophoresis using two different agarose sources, UltraPure and MetaPhor agarose. While UltraPure agarose up to 5 % showed no clear separation of BSA oligomers, MetaPhor agarose clearly demonstrated oligomer bands above 4 %, indicating that the latter agarose has greater molecular sieving effects and is hence characterized to have high resolution for size differences, as probed by a greater slope of Ferguson plot. Physical properties are different between two agaroses. In general, UltraPure agarose has physical strength, while MetaPhor agarose is considerably fragile, but MetaPhor agarose solution is less viscous so that even 10 % gel can be made. Cause of oligomers was shown to be not associated with inter-chain disulfide bonds, but is due to association of native or native-like molecules.

Gel-electrophoresis based method for biomolecular interaction.

Electrophoresis is one of the most important analytical technologies for characterization of macromolecules and their interactions. Among them, native gel electrophoresis is used to analyze the macromolecules in the native structure. It differs in principle and information from those obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or blue native polyacrylamide gel electrophoresis (BN-PAGE). SDS-PAGE is carried out in the presence of strong denaturant, SDS, while BN-PAGE is done in the presence of negatively charged dye, e.g., Coomassie brilliant blue, G-250. Here, we describe native gel electrophoresis using agarose gel and a buffer at pH 6.1 composed of histidine and 2-(N-morpholino) ethanesulfonic acid. First, a protocol for vertical and horizontal formats of agarose native gel electrophoresis is described followed by different staining procedures. Then, various examples obtained using the developed procedure will be shown to demonstrate how the technology can be applied to specific cases and the advantages or caveats of the present technology.

Western blotting of native proteins from agarose gels.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.

Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives.

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.

Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis.

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis.

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

TORC1 inactivation stimulates autophagy of nucleoporin and nuclear pore complexes.

The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor-binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope-derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term "NPC-phagy" and "nucleoporinophagy."