Protein microheterogeneity and crystal habits: the case of epidermal growth factor receptor isoforms as isolated in a multicompartment electrolyzer with isoelectric membranes.
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Protein microheterogeneity and crystal habits: the case of epidermal growth factor receptor isoforms as isolated in a multicompartment electrolyzer with isoelectric membranes. / Weber, Wolfgang; Wenisch, E; Günther, N; Marnitz, U; Betzel, C; Righetti, P G.
in: J CHROMATOGR A, Jahrgang 679, Nr. 1, 1, 1994, S. 181-189.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Protein microheterogeneity and crystal habits: the case of epidermal growth factor receptor isoforms as isolated in a multicompartment electrolyzer with isoelectric membranes.
AU - Weber, Wolfgang
AU - Wenisch, E
AU - Günther, N
AU - Marnitz, U
AU - Betzel, C
AU - Righetti, P G
PY - 1994
Y1 - 1994
N2 - 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 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).
AB - 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 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).
KW - Animals
KW - Mice
KW - Crystallography, X-Ray
KW - Isoelectric Focusing/methods
KW - Receptor, Epidermal Growth Factor/chemistry/isolation & purification
KW - Animals
KW - Mice
KW - Crystallography, X-Ray
KW - Isoelectric Focusing/methods
KW - Receptor, Epidermal Growth Factor/chemistry/isolation & purification
M3 - SCORING: Journal article
VL - 679
SP - 181
EP - 189
JO - J CHROMATOGR A
JF - J CHROMATOGR A
SN - 0021-9673
IS - 1
M1 - 1
ER -