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 °C 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.