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After SDS-PAGE analysis on a 15% gel under reducing conditions, the fraction containing the majority of the recombinant proteins was subjected to size-exclusion chromatography by loading the protein extract on an Ultrogel ACA44 (5 75 cm, Biosepra) column equilibrated with a 30 mM Tris-HCl pH 8.0, 1 mM EDTA, and 200 mM NaCl buffer

203,204 Delivery of substrates to the 26S proteasome for degradation To avoid the deleterious accumulation of unfolded intermediates, the protein extracted from the ER needs to enter the 26S proteasome to enable effective degradation

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Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor