Ni(II) functionalized polyhedral oligomeric silsesquioxane based capillary monolith for purification of histidine-tagged proteins by immobilized metal affinity micro-chromatography

Çambay Kuban F., Koçer İ., Kip Ç., Çelik E., Tuncel A.

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, vol.1225, 2023 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 1225
  • Publication Date: 2023
  • Doi Number: 10.1016/j.jchromb.2023.123759
  • Journal Name: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Aerospace Database, BIOSIS, CAB Abstracts, Chemical Abstracts Core, Chimica, Communication Abstracts, Compendex, EMBASE, Food Science & Technology Abstracts, MEDLINE, Metadex, Veterinary Science Database, Civil Engineering Abstracts
  • Keywords: Capillary monolith, Green fluorescent protein, His tagged protein, Immobilized metal affinity chromatography, Nano-liquid chromatography
  • Hacettepe University Affiliated: Yes


A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (μ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 μm 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. μ-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 μ-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.