Purification of His-tagged proteins with [desthiobiotin-BSA-EDTA] conjugates exhibiting resistance to EDTA.
Two His-tagged proteins (His 6-P38 and His 6-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 (> or =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 micromol/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 degrees 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 microL). Hence, concentrated preparations can be obtained, and a reconcentration step may be circumvented.
Patchornik
Guy
G
Affisink Biotechnology Ltd, 11 Hamaccabee St. Kiryat-Ono 55572, Israel. guy@affisink.com
D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002498:Centrifugation; D002847:Chromatography, Agarose; D003300:Copper / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000302:isolation & purification; D014508:Urea / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000302:isolation & purification
D008670:Metals; D011506:Proteins; D013205:Staphylococcal Protein A; D001360:Avidin; D012710:Serum Albumin, Bovine; D006639:Histidine; D001710:Biotin; C004749:desthiobiotin; D003300:Copper; D014508:Urea; D004492:Edetic Acid; D048051:p38 Mitogen-Activated Protein Kinases