This disclosure relates to methods for the prevention of the reduction of disulfide bonds in a polypeptide expressed in a recombinant host cell, comprising, following fermentation, adding selenite and/or its salts or derivatives to a harvest solution of the recombinant host cell, wherein the disulfide bond in the polypeptide remains non-reduced.

The present invention relates to an in vitro method for production of heterodimeric proteins.

The present invention provides a method for sequencing a protein/polypeptide based on Aerolysin nanopores to achieve specific discrimination of natural amino acids and post-translational modifications thereof and accurate acquisition of a sequence of a single-molecule protein, the method including the following steps: (1) unfolding of the protein; (2) terminus labeling of protein sequencing; (3) protein charge screening; (4) unfolding of a tertiary structure of the polypeptides; (5) orthogonal identification of amino acids; (6) confined perturbation-assisted identification of amino acids; and (7) single-molecule protein sequencing. The present invention aims at sensitive detection of sequence information about 20 amino acids and establishes an innovative method for accurately determining sequences of the amino acids and post-translational modifications of a single protein molecule.

Dithioamine reducing agents useful for the reduction of disulfide bonds. The reducing agents of this invention are useful, for example, to reduce disulfide bonds, particularly in proteins, or to prevent the formation of disulfide bonds, particularly in proteins and other biological molecules. Reducing agents of this invention can be employed to regulate protein function in proteins in which a sulfhydryl group is associated with biological activity. Reducing agents of this invention can prevent inactivation of a given protein or enhance activation of a given protein or other biological molecule in vitro and/or in vivo. Reducing agents of this invention can prevent or reduce oxidation of cysteine residues in proteins and prevent the formation of reduced activity protein dimers (or other oligomers). Reducing agents of this invention are useful and suitable for application in a variety of biological applications, particularly as research and synthetic reagents. The invention provides S-acylated dithioamines which can be selectively activated reducing agents by removal of the S-acyl groups enzymatically or chemically. The invention further provides dithiane precursors of thioamino reducing agents. The invention provides dithioamine reducing agents, S-acylated dithioamines and dithianes which are immobilized on surfaces, including among others, glass, quartz, microparticles, nanoparticles and resins.

The present invention relates to a method for modifying and purifying peptides comprising an immobilizing step, a modification step and a releasing step. In the immobilizing step, a crude linker-tagged peptide L-P is coupled to a solid support yielding an immobilized linker-tagged peptide S-L-P. Subsequently, the immobilized linker-tagged peptide S-L-P is modified with one or more organic molecules yielding an immobilized linker-tagged peptide S-L-mP. Finally, the modified peptide is released via a reduced intermediate RI. The linker molecule is a compound of formula 1, X—Tb—Va-U—Y—Z (1), which can be coupled to a purification resin via the moiety X and to a peptide via the moiety Y under the release of the leaving group Z. T is an optional spacer moiety and V is an optional electron withdrawing moiety. U is an aryl or 5- or 6-membered heteroaryl moiety bound to at least one electron withdrawing moiety V, W or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.

The present invention relates to an in vitro method for production of heterodimeric proteins.

A composition of renaturation buffer solution for dimeric proteins and method for using it. The renaturation buffer solution comprises buffer, oxido shuffling system, primary additives, chelating agent and polysorbate compound. The method for renaturation of dimeric proteins includes solubilization of target proteins using a solubilization buffer, solution dilution of the renatured target protein using a renaturation buffer containing polysorbate compound(s), concentrating of the target protein solution to a concentration approaching saturation. A method of applying renature denatured or misfolded proteins to become correctly folded and bioactive. A method of proper renaturation of recombinant proteins that have been expressed using translation vehicles (e.g., bacteria, insects, etc.) and proteins damaged by mechanical shearing, chemical stresses, and other stresses.

A method for cleaving a disulfide bond in a protein includes: cleaving a disulfide bond in a protein present in a reaction system by reduction via a reduced redox protein; and reducing an oxidized redox protein produced by oxidation of the reduced redox protein in the cleaving to the reduced redox protein by donating an electron from an electrode connected to an external power supply outside the reaction system to the oxidized redox protein.

An allergen inactivation method includes an inactivation step of inactivating an allergen present in a reaction system by reduction via a reduced redox protein, and a reduction process of reducing an oxidized redox protein produced by oxidation of the reduced redox protein in the inactivating to the reduced redox protein by donating an electron from an electrode connected to an external power supply outside the reaction system to the oxidized redox protein.

The present disclosure relates to methods of re-oxidizing an antigen-binding protein.