Monovalent streptavidin is a heterotetramer consisting of 3 dead subunits which do not bind biotin and one alive subunit which binds biotin with high affinity. Dead (D) and Alive (A) subunits are expressed separately in E. coli, where they form inclusion bodies. D and A inclusion bodies are dissolved in guanidinium hydrochloride, mixed in an appropriate ratio, and refolded by rapid dilution into PBS. The refold creates a statistical mixture of tetramers, and monovalent streptavidin is purified from the other tetramers by a nickel affinity column, since its single His6 tag gives it different elution properties than the tetramers with 0, 2, 3 or 4 His6 tags.