Affinity Chromatography with Collapsibly Tethered Ligands

Abstract

We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA120). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA120 and lactose. At 5 degrees C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 degrees C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA120. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA120 ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA120 lectin.

Temperature-responsive intelligent interfaces for biomolecular separation and cell sheet engineering

“…Ricinus communis agglutinin (RCA 120 ), galactose-specific lectin and lactose were introduced into the PIPAAm chain, and the copolymer was attached to sepharose beads ( Yamanaka et al 2003). The glycoprotein target, asialotransferrin, was loaded onto a column packed with copolymerimmobilized beads.…”

Tuning Specific Biomolecular Interactions Using Electro‐Switchable Oligopeptide Surfaces

“…[2][3][4] Biological surfaces in which the biomolecules "statically" interact with the outside environment have been explored over the past few years [ 2 ] and have provided, for instance, important new insights into how cells probe their surroundings. [ 5 ] Whereas important progress on static biological surfaces has been made in the past, much research is now focusing on the development of switchable biological surfaces [ 3 , 6 ] as it can be tremendously useful in diverse biological and medical applications, including biofouling, [ 7 ] chromatography, [ 8 ] drug delivery, [ 9 ] cell culture [ 10 ] and tissue engineering. [ 11 ] This fi eld is in its infancy and early examples of switchable biological surfaces includes surfaces that switch between bio-inert and bio-active states, under an external thermal-, photo-, chemical/biochemical-or electrical-induced stimulus, to trigger capture or release of biological entities such as DNA, [ 12 ] proteins, [ 13 ] antibodies, [ 14 ] enzymes, [ 15 ] and cells.…”

Thermoresponsive Poly(N-vinylcaprolactam) as Stationary Phase for Aqueous and Green Liquid Chromatography

“…Columns containing copolymers of NIPAAm with hydrophobic [5][6][7]9], ionic [8,10], and affinity ligands [11] have extended the applicability range of TR-LC.…”