A purified, soluble form of the epidermal growth factor receptor (sEGFR) 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, respectively) and ca. a dozen minor components. This wild-type sEGFR, while producing crystals, has so far defied any attempt at decoding the structure, due to the very poor diffraction pattern. When the wild-type sEGFR was purified in a multicompartment electrolyzer with isoelectric Immobiline membranes, it yielded the three major isoforms as single-pi components, collected in three separate chambers of the recycling electrolyzer. The pi 6.71 and the pi 6.96 isoforms produced large crystals of apparent good quality. However, while the former produced a high-quality diffraction pattern, which may lead to decoding of the three-dimensional structure, the pi 6.96 produced crystals which did not diffract at all. It is concluded that, in the case of ''tough'' proteins (large size, heterogeneous glycosylation, high water content of crystals), purification to single-charge components might be an essential step for growing proper crystals. The unique advantage of purification via isoelectric membranes is that the protein is collected both isoelectric and isoionic, i.e. uncontaminated by soluble buffers (such as the carrier ampholytes used in conventional focusing).

Leukemia inhibitory factor (LIF) is a polyfunctional molecule with significant and diverse biological activities. LIF is a glycoprotein secreted by a number of different cell types in vitro. It is induced in fibroblasts, lymphocytes, monocytes and astrocytes by various inducers such as serum, TNF, interleukin-IP and EGF. Due to extensive and variable glycosylation the molecular weight can range from 38 to 67 kDA. The biological functions of LIF are mediated through a receptor and a signal transducer, gp130, which is also used by factors like interleukin-6 (IL-6), cilliary neurotropic factor (CNTF), and oncostatin M (OSM). Here, we report the crystallization of the non-glycosylated human-like LIF expressed in E. coli. The present crystals diffract to 2.0 A using synchrotron radiation. They belong to the monoclinic space group C2, and the cell dimensions are a = 61.5 A, b = 45.3 A, c = 77.7 A and beta = 112.3 degrees.