Section 4.2.5. Membrane-protein crystallization with the help of antibody Fv fragments

The number of detergents that can be tried is limited for rather unstable membrane proteins. For instance, the four-subunit cytochrome c oxidase from P. denitrificans is sufficiently stable only with dodecyl-β-D-maltoside as detergent. When other detergents are employed, subunits III and IV and some lipids are removed from subunits I and II. These lipids might contribute to the binding of the protein subunits III and IV to the central subunits I and II. Therefore, the size of the detergent micelles can not be varied by using different detergents. In order to obtain crystals, the size of the extramembraneous part of this important enzyme was enlarged by the binding of Fv fragments of monoclonal antibodies. For this purpose, monoclonal antibodies against the four-subunit cytochrome c oxidase were generated using the classical hybridoma technique. Then hybridoma cell lines producing conformation-specific antibodies were selected (such antibodies react positively in enzyme-linked immunosorbent assays, but negatively in Western blot assays). The cDNA strands coding for the respective VL and VH genes were cloned and expressed in Escherichia coli. Binding of conformation-specific antibody fragments can be expected to lead to a more homogeneous protein preparation. The first Fv fragment worked, and well ordered crystals of the four-subunit and two-subunit cytochrome c oxidases were obtained (Ostermeier et al., 1995, 1997). As an important additional advantage of this approach, an affinity tag can be fused to the recombinant antibody Fv fragment and used for efficient isolation. The affinity tag can then be used to purify the complex of antibody fragment and membrane protein rapidly and with a high yield (Kleymann et al., 1995). Because Fv fragment cocrystallization also worked with the yeast cytochrome complex, again with the first and only conformation-specific antibody Fv fragment tried (Hunte et al., 2000), we are rather confident about this method for the future. The usefulness of single-chain Fv fragments, which can be obtained by the phage-display technique, has not been investigated, because the linker region joining the VL and VH chains must be expected to be flexible. Such flexibility would induce inhomogeneity and reduce the chance of obtaining crystals.