Purification of the c-erbB2/neu membrane-spanning segment: a hydrophobic challenge

High quality purification of membrane-spanning peptides and proteins remain s a challenging problem. In this work we describe a tailored chromatographi c purification of a synthetic 35-residue peptide corresponding to the trans membrane region of the tyrosine kinase receptor c-erb2/neu. Composed to ove r 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and va line, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resi n matrix used during the solid-phase synthesis, represents a difficult task . We propose a three step strategy based on gel permeation and reversed-pha se high-performance liquid chromatography, using aprotic polar solvent mixt ures. The challenge consisted in obtaining a sufficient amount of an extrem ely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purificati on steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography-ESI TOF mass spectromet ry. (C) 2000 Elsevier Science BN. All rights reserved.