| Abstract
| - 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.
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