Purification of his-tagged proteins with [desthiobiotin-BSA-EDTA] conjugates exhibiting resistance to EDTA

Two His-tagged proteins (His6-P38 and His6-Protein A) were purified by specific precipitation utilizing nonsoluble macrocomplexes composed of: BSA conjugates (modified with desthiobiotin-NHS and EDTA-dianhydride), tetrameric avidin, and Cu²⁺ ions. The generated pellets containing bound His-tagged proteins are washed with EDTA (25-100 mM) and then eluted in relatively high purity (>90%) devoid the macrocomplexes. Three different BSA conjugates were synthesized (DB-BSA-EDTA, DB-BSA-EDTA-A, DB-BSA-EDTA-B) and their adsorption capacities (3.8-6.4 μmol/g of BSA conjugate) as well as the recovery yields of His-tagged proteins obtained with them (44-84%) determined. The data demonstrate that capacity is dependent on the stochiometric ratio of modifying reagents (i.e., desthiobiotin-NHS and EDTA-dianhydride) used during the synthesis of the BSA conjugates. Copper ions were found to be significantly superior to Zn²⁺, Co²⁺, and Ni²⁺. BSA conjugates could be regenerated in moderate yields (74-83%) by incubating them at 88 0C in the presence of biotin (10 mM) at pH 7. The absence of resins leads to formation of small pellets (1-5 mg) and utilization of minute volumes of elution buffer (50-100 μL). Hence, concentrated preparations can be obtained, and a reconcentration step may be circumvented.