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Abstract
Structural determination of integral membrane proteins has been exceedingly difficult to study. In fact, only seven membrane proteins have had their structures solved to atomic resolution and only two of these (prostaglandin H2 synthase, bovine cytochrome oxidase) are mammalian. The primary impediment to structural analysis is the lack of suitable methods for high-level expression and large-scale purification of intact, functional membrane protein. The focus of this thesis was to develop methods for the overexpression and purification of a mammalian voltage-gated potassium channel protein, Kv1.3, in an intact, functional state for use in structural studies.
Kv1.3, a well-characterized K$\sp+$ channel which is critical for lymphocyte function, was overexpressed as a fusion-protein in mammalian cells using a recombinant vaccinia virus (VV). CV-1 cells infected with the recombinant VV displayed large K$\sp+$ currents ($\sim$60,000-500,000 channels/cell) which were identical to the native Kv1.3 channel found in lymphocytes. Using a two-step purification method involving nickel-chelate and ion exchange chromatography, CHAPS- or cholate-solubilized Kv1.3-fusion protein was purified to homogeneity, yielding $\sim$300-400 $\mu$g protein/10$\sp8$ CV-1 cells. The purified protein is appropriately N-glycosylated with a core-sugar moiety like its native counterpart, and has an estimated mass of 270 kDa in sedimentation experiments, consistent with it being a homotetramer of $\sim$64 kDa subunits.
Functional reconstitution of the protein into lipid bilayers produced a K$\sp+$-selective channel that was blocked by the high affinity scorpion-derived peptide, margatoxin (MgTX). The pure protein bound specifically to ($\sp{125}$I) MgTX with an affinity similar to that of native Kv1.3 (40pM), indicating that the pure protein is intact. Finally, reconstitution of Kv1.3 protein into lipid bilayers at high protein:lipid ratios, and visualization by negative-staining electron microscopy, reveals a mosaic consisting of tetramers with a unit cell size of 65 x 65 A; on several occasions 2D crystals were obtained which yielded an optical diffraction pattern extending out to $\sim$3.5A. This methodology may also be adapted to the purification and structural characterization of related ion channels, as well as other mammalian integral membrane proteins.