The pH-responsive precipitation-redissolution of the CspB fusion protein, CspB50TEV-Teriparatide, triggered by changes in secondary structure.

Cell surface protein B (CspB) fusion proteins can undergo reversible pH-responsive precipitation-redissolution. A pH-responsive precipitation-redissolution of CspB tag purification (pPRCP) method was established for protein purification using this property. However, the mechanism of the pH-responsive precipitation of CspB fusion proteins is unknown, which has made it difficult to set process parameters for pPRCP. In this study, we investigated the mechanism of the pH-responsive precipitation of CspB fusion proteins using CspB50TEV-Teriparatide (CspB-teri) as a model. As expected, CspB-Teri was reversibly precipitated at acidic pH. By contrast, CspB-Teri was not precipitated under unfolding conditions induced by trifluoroethanol, urea, or guanidine hydrochloride, even at acidic pH. The conformation of CspB-Teri changed to a ß-sheet-rich structure as the pH decreased, followed by the formation of intermolecular interactions, which caused precipitation. The particle size of the CspB-Teri precipitate increased in a protein concentration-dependent manner. These results indicated that the pH-responsive precipitation of CspB-Teri is triggered by the formation of a ß-sheet structure in response to decreasing pH, and the growth of the precipitate particles occurred through intermolecular interactions.

Lipocalin 15 in the olfactory mucus is a biomarker for Bowman's gland activity.

Olfactory mucus contributes to the specific functions of the olfactory mucosa, but the composition and source of mucus proteins have not been fully elucidated. In this study, we used comprehensive proteome analysis and identified lipocalin 15 (LCN15), a human-specific lipocalin family protein, as an abundant component of the olfactory mucus. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using a newly generated anti-LCN15 antibody showed that LCN15 was concentrated in olfactory mucus samples, but not in respiratory mucus samples. Immunohistochemical staining using anti-LCN15 antibody revealed that LCN15 localized to the cytokeratin 18-positive Bowman's glands of the olfactory cleft mucosa. Quantitative image analysis revealed that the area of LCN15 immunoreactivity along the olfactory cleft mucosa significantly correlated with the area of neuron-specific Protein-Gene Product 9.5 (PGP9.5) immunoreactivity, suggesting that LCN15 is produced in non-degenerated areas of the olfactory neuroepithelium. ELISA demonstrated that the concentration of LCN15 in the mucus was lower in participants with normal olfaction (≥ 50 years) and also tended to be lower in patients with idiopathic olfactory loss (≥ 50 years) than in participants with normal olfaction (< 50 years). Thus, LCN15 may serve as a biomarker for the activity of the Bowman's glands.

Solution design to extend the pH range of the pH-responsive precipitation of a CspB fusion protein.

Cell surface protein B (CspB) from Corynebacterium glutamicum has been developed as a reversible pH-responsive tag for protein purification. CspB fusion proteins precipitate at acidic pH, after that they completely dissolve at neutral pH. This property has been used in a non-chromatographic protein purification method named pH-responsive Precipitation-Redissolution of CspB tag Purification (pPRCP). However, it is difficult to apply pPRCP to proteins that are unstable under acidic conditions. In an effort to shift the precipitation pH to a milder range, we investigated the solution conditions of CspB-fused Teriparatide (CspB50TEV-Teriparatide) during the process of pH-responsive precipitation using pPRCP. The purified CspB50TEV-Teriparatide in buffer without additives precipitated at pH 5.3. By contrast, CspB50TEV-Teriparatide in buffer with 0.5 M Na2SO4 precipitated at pH 6.6 because of the kosmotropic effect. Interestingly, the pH at which precipitation occurred was independent of the protein concentration. The precipitated CspB50TEV-Teriparatide was fully redissolved at above pH 8.0 in the presence or absence of salt. The discovery that proteins can be precipitated at a mild pH will allow pPRCP to be applied to acid-sensitive proteins.

Non-chromatographic purification of Teriparatide with a pH-responsive CspB tag.

Cell surface protein B (CspB) from Corynebacterium glutamicum is used as a pH-responsive peptide tag to enable a simple solid-liquid separation method for isolating a CspB fusion protein. Here we demonstrate the first application of a CspB tag for the purification of Teriparatide, which is a biologic drug that is prescribed for osteoporosis. The Teriparatide was constructed as CspB50TEV-Teriparatide, comprising 50 amino acid residues of CspB, the cleavage site of TEV protease, and Teriparatide. CspB50TEV-Teriparatide was expressed in a culture supernatant by C. glutamicum secretion system at 3.0 g/L (equivalent to approximately 1.2 g/L Teriparatide). The CspB50TEV-Teriparatide was precipitated by reducing the pH of the culture supernatant, and the precipitate was then dissolved in a neutral buffer. A TEV protease treatment was applied to cleave the Teriparatide from the CspB50TEV-Teriparatide. Then, the remaining digested CspB50TEV, undigested CspB50TEV-Teriparatide, and TEV protease were precipitated in an acidic pH, whereas the soluble Teriparatide remained in the supernatant. The process had a yield of 96.5% and resulted in Teriparatide with a purity of 98.0% and productivity of 1.1 g/L of C. glutamicum culture. Thus, tag-free Teriparatide was successfully purified from the CspB fusion protein using only pH changes, centrifugation, and protease digestion without the need for chromatography. This versatile purification protocol is expected to be applicable to various proteins from laboratory to industrial scales.

A new pH-responsive peptide tag for protein purification.

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH.

Protein refolding using chemical refolding additives.

In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-based methods that contribute to the emergence of a universal refolding method.

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor.

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.

Solid-phase artificial chaperone-assisted refolding using insoluble beta-cyclodextrin-acrylamide copolymer beads.

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins.

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.