PROTEIN-PURIFICATION IN MULTICOMPARTMENT ELECTROLYZERS WITH ISOELECTRIC MEMBRANES

Preparative purification of proteins under isoelectric conditions is r eviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desire d protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the nove l generation of non-amphoteric buffers, based on the chemistry of acry lamides, which can be insolubilized onto polyacrylamide supports. Afte r a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogen eous (by the most stringent criterion of surface charge) protein fract ions. Such a high charge purity has been found to be often a fundament al prerequisite for the growth of protein crystals. Interfacing the el ectrolyzer with mass spectrometry has permitted the decoding of the st ructure of minor components generated from a parental molecule, especi ally ones having a higher pi. It was found that these species were oft en generated either by proteolytic cleavage or by the formation of a t risulphide bridge between two Cys residues. A unique application of th e electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is o utstanding, in that the kinetic parameters of the immobilized enzyme a re identical to those of a free enzyme form. (C) 1997 Elsevier Science B.V.