Tiyol-Metakrilat Polimerizasyonu ile Affinite Monolitlerinin Sentezi ve Histidin İşaretli Protein İzolasyonunda Kullanımı

Abstract

There are many methods currently available for the purification of proteins. It is important that the purification process is carried out in as little steps as possible, with high purity and in a short time. Therefore, it is necessary to choose the most efficient and effective separation method by taking into consideration the properties of the protein to be purified by isolations (such as amino acid residue - histidine, tryptophan or cysteine). Within the scope of this thesis, metal coordination interaction between histidine and transition metal ions is used to selectively and reliably separate histidine-labeled proteins using immobilized metal affinity chromatography (IMAC). Histidine groups form coordination complex with divalent metal ion Ni2+. Column packing material was synthesized in a capillary column with internal diameter of 300 μm with thiol-methacrylate polymerization using MSA as hydrophilic ligand, POSS-MA as main monomer and crosslinker and AIBN as thermal initiator. NiCl2 solution was passed through the column so that Ni2+ ions were immobilized on the monolith surface. Ni2+ ion-immobilized MSA- (POSS-MA) capillary monolithic column (IMAC sorbent) was used for the selective separation of green fluorescent protein (GFP), a histidine-labeled protein, from E. coli bacteria lysate. SEM images showed that the monolithic surface was porous and it successfully adhered to the inner wall of the column. Due to the use of micro and macro porosity agent (n-butanol and ethylene glycol), the monolith packing material has a very large surface area. According to the results of BET analysis, the surface area of MSA-(POSS-MA) monolith was 143 m2/g and the surface area of Ni2+-MSA-(POSS-MA) monolith was 116 m2/g. Effect of change in imidazole concentration on protein isolation was investigated in the synthesized Ni2+-MSA-(POSS-MA) cappilary column at 4 µL/min flow rate and 3.52 mg/mL initial protein concentration at different desorption conditions. According to SDSPAGE analysis, 25.55 µg GFP was adsorbed and 18.74 µg GFP was desorbed under suitable experiment conditions. Adsorption efficiency was determined as 61.9% and desorption efficiency as 100%, while the flow rate was 2 µL/min. In order to determine the effect of the initial protein concentration change on protein isolation, lysates containing different protein concentrations were studied in synthesized standard monolithic capillary columns at 0.4 µL/min flow rate. When the initial protein concentration was 1.2 mg/mL; adsorption efficiency was found 87.6%, desorption efficiency was found 100% and isolation efficiency was found 89.3%. When the initial protein concentration was 2.1 mg/mL; adsorption efficiency was found 77.6%, desorption efficiency was found 73.9% and isolation efficiency was found 57.3%. In order to determine the effect of Ni2+ ion on protein isolation, both nickel-free and nickel-MSA-free columns were synthesized. Adsorpsion efficiency was found 9% for nickel-MSA-free column, 45.8% for nickel-free column and 77.6% for Ni2+-MSA-(POSS-MA) column. For nickel- free column, desorption efficiency was found 19.8% and the isolation efficiency was found 9%. For standard Ni2+-MSA-(POSS-MA) column, desorption efficiency was found 73.9% and the isolation efficiency was found 57.3%.