Characterisation of metal-chelate methacrylate monoliths.
Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.
Peterka
M
M
BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia.
Jarc
M
M
Banjac
M
M
Frankovic
V
V
Bencina
K
K
Merhar
M
M
Gaberc-Porekar
V
V
Menart
V
V
Strancar
A
A
Podgornik
A
A
D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003300:Copper / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000302:isolation & purification; D008670:Metals / Q000737:chemistry*; D008689:Methacrylates / Q000737:chemistry*; D014409:Tumor Necrosis Factor-alpha / Q000302:isolation & purification
D002614:Chelating Agents; D008670:Metals; D008689:Methacrylates; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D003300:Copper