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ELSEVIER
DOI: 10.1016/j.jchromb.2023.123759
关键词
Capillary monolith; Immobilized metal affinity chromatography; Nano-liquid chromatography; His tagged protein; Green fluorescent protein
A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (mu-IMAC). The monolith was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and mercaptosuccinic acid (MSA) as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. His-GFP was successfully isolated with high yield and purity using the Ni(II)@MSA@poly(POSS-MA) capillary monolith. The monolith showed stable performance over consecutive His-GFP purifications.
A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (mu-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 mu m in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. mu-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly (POSS-MA)] capillary monolith. His-GFP was succesfully isolated by mu-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.
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