Applications of hydrodynamic forces for membrane chromatography
Licentiate thesis, 2013
Membrane proteins are proteins that reside in the cell membrane. Due to their position in the cell membrane, the barrier between the cell interior and exterior, they are vital to cell signaling. Moreover, the presence of membrane proteins in the cell's signaling pathways make membrane proteins important drug targets. In fact, one of the most important drug target classes are the membrane residing G protein coupled receptors. Despite this apparent importance there is currently no good technique for purifying membrane proteins that does not also have a high risk of causing denaturation of the protein. Taking into account that the cell membrane and its constituents can be described as a two-dimensional fluid, combined with the fact that many chromatography techniques are two-dimensional systems, it may seem peculiar that membrane chromatography is not yet solved in a satisfactory way. The aim of this thesis is to contribute to improved membrane protein chromatography.
Hydrodynamic forces have recently been used to induce lateral movement of membrane associated molecules, a principle which has been utilized to improve the efficiency of the accumulation and separation of membrane associated molecules situated in supported lipid bilayers (SLB). This was done by forming SLBs in microfluidic channels, and by further introducing a gold barrier situated on the floor of the microfluidic channel. The barrier restricts the SLB to the center of the microfluidic channel where the spatial variation of the hydrodynamic force is small. Thanks to the so obtained homogeneous force, it was possible to show that separation of cholera toxin B subunit based on the number of attachment points to the SLB was complete, with no material in between the different populations.
Also, a method for label-free diffusivity measurements on a substrate that is sensitive to changes in the effective refractive index in a small volume above the substrate is described. The technique is based on local application of hydrodynamic forces, in a so called hydrodynamic trap, to locally accumulate proteins at a surface coverage significantly higher than the equilibrium of the system. The trap is subsequently turned off, allowing the accumulated proteins to diffuse out of the trap, allowing the diffusivity of the accumulated proteins can be studied. The latter method was shown to provide a unique possibility to compare the diffusivity of labeled and non-labeled proteins in otherwise identical systems. It was shown that the inclusion of fluorescent labels decreases the diffusivity by approximately 12%.
Supported lipid bilayer