Regulation of Protein Binding toward a Ligand on Chromatographic Matrixes by Masking and Forced-Releasing Effects Using Thermoresponsive Polymer

Abstract

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

“…However, with very few exceptions [47], all reported investigations on thermoresponsive liquid chromatography use conventional fixed-bed columns filled with thermoresponsive adsorbents, and the whole column is tempered by a water bath, a water jacket connected to a water bath, or a column oven [16][17][18][19]24,26].…”

“…Most of this has concentrated on exploiting the principle of thermally induced extension and collapse of polymer chains for modulating the fractionation range in size exclusion chromatography [11,12,14], hydrophobicity in HPLC [13,[15][16][17][18], and balancing electrostatic and hydrophobic interactions in ion exchange chromatography, through the use of copolymers containing both thermoresponsive and ion exchange components [19][20][21][22][23][24][25], rather than mitigating ligand masking and 'forced elution' in pseudoaffinity chromatography [3,26]. To this day, the vast body of work on thermoresponsive chromatography pertains to the modification and subsequent use of small pored inorganic (glass, silica) or hydrophobic (polystyrene based) chromatography supports in analytical HPLC separations of small biomolecules (especially steroids).…”

Novel temperature-responsive polymer brushes with carbohydrate residues facilitate selective adhesion and collection of hepatocytes

“…Yoshizako et al reported reversible capture/release of bovine serum albumin (BSA) using PNIPAAm-grafted silica beads co-immobilized with Cibacron Blue F3G-A (CB), and the association constant of BSA against CB on the surface was 10 2 -10 4 M −1 [40]. The affinity constant value, K A , for RCA 120 binding to the PLAMA brush has been measured as 1.86 × 10 5 M −1 by surface plasmon resonance spectroscopy [41].…”

Heparin-functionalized thermoresponsive surface

“…Yoshizako et al have investigated the possibility of affinity regulation by both PIPAAm and spacer length for forced-releasing effects. 8 In case of Cibacron Blue ligand immobilized with shorter spacer-length than the mean size of the extended PIPAAm, captured albumin could be released when temperature is lowered below LCST. Hoffman et al have immobilized PIPAAm molecule in the vicinity of genetically engineered binding sites of biotin in streptavidin and demonstrated that the binding ability of biotin decreases with increasing temperature higher than LCST.…”