Purification of the c-erbB2/neu membrane-spanning segment: a hydrophobic challenge.
High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography-ESI TOF mass spectrometry.
Goetz
M
M
Institut Européen de Chimie et de Biologie, Ecole Polytechnique, Universités de Bordeaux I et II, B.P. 108, Talence, France. michael.goetz@iecb-polytechnique.u-bordeaux.fr
Rusconi
F
F
Belghazi
M
M
Schmitter
J M
JM
Dufourc
E J
EJ
D000595:Amino Acid Sequence; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases