Dimeric disulfide-linked peptides are formed by the regioselective oxidative folding of thiol precursors containing the CX3CX2CX3C tetracysteine motif. Here, we investigate the general applicability of this peptide as a dimerization motif for different proteins. By recombinant DNA technology, the peptide CHWECRGCRLVC was loaded with proteins, and functional homodimers were obtained upon oxidative folding. Attached to the N-terminus of the dodecapeptide, the prokaryotic enzyme limonene epoxide hydrolase (LEH) completely forms a covalent antiparallel dimer. In a diatom expression system, the monoclonal antibody CL4 mAb is released in its functional form when its natural CPPC central parallel hinge is exchanged for the designed tetra-Cys hinge motif. To improve our understanding of the regioselectivity of tetra-disulfide formation, we provoked the formation of heterodimeric hinge peptides by mixing two different tetra-Cys peptides and characterizing the heterodimer by mass spectrometry and nuclear magnetic resonance spectroscopy.