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.

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.

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).

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.

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.

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.

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.

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.

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.