PROTEIN MICROHETEROGENEITY AND CRYSTAL HABITS - THE CASE OF EPIDERMALGROWTH-FACTOR RECEPTOR ISOFORMS AS ISOLATED IN A MULTICOMPARTMENT ELECTROLYZER WITH ISOELECTRIC MEMBRANES

A purified, soluble form of the epidermal growth factor receptor (sEGF R) was found, by isoelectric focusing in immobilized pH gradients, to consist of three major isoforms (with pi values 6.45, 6.71 and 6.96, r espectively) and ca. a dozen minor components. This wild-type sEGFR, w hile producing crystals, has so far defied any attempt at decoding the structure, due to the very poor diffraction pattern. When the wild-ty pe sEGFR was purified in a multicompartment electrolyzer with isoelect ric Immobiline membranes, it yielded the three major isoforms as singl e-pi components, collected in three separate chambers of the recycling electrolyzer. The pi 6.71 and the pi 6.96 isoforms produced large cry stals of apparent good quality. However, while the former produced a h igh-quality diffraction pattern, which may lead to decoding of the thr ee-dimensional structure, the pi 6.96 produced crystals which did not diffract at all. It is concluded that, in the case of ''tough'' protei ns (large size, heterogeneous glycosylation, high water content of cry stals), purification to single-charge components might be an essential step for growing proper crystals. The unique advantage of purificatio n via isoelectric membranes is that the protein is collected both isoe lectric and isoionic, i.e. uncontaminated by soluble buffers (such as the carrier ampholytes used in conventional focusing).