Expression and membrane assembly of a transmembrane region from Neu

Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways. The present work addresses problems surrounding expression, isolation, secondary structure recovery, a nd assembly into membranes, of the relatively large quantities of transmemb rane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PACE behavior of such pe ptides and their H-2 NMR spectra in lipid bilayer membranes. A 50-residue p eptide, Neu(exp), containing the transmembrane portion of the receptor tyro sine kinase, Neu, was designed for expression in Escherichia coli. The sequ ence also contained 11-12 amino acids from each side of the transmembrane d omain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene p roduct. This difficulty was overcome by expressing the peptide as a TrpE fu sion protein in a pATH vector to target expression products to inclusion bo dies, and subsequently removing the TrpE portion by cyanogen bromide cleava ge. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag al lowed excellent recovery of the final peptide, while permitting use of dena turing solvents and avoiding the need for HPLC with its attendant adsorptio n losses. Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior. Dissolution in certain acidic organic solvents led to ma terial with increased alpha-helix content, which behaved in detergent as mi xtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes. For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression. The same acidic organic solvents used to dissolve and dissociate expressed transmembrane pe ptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, dem onstrated related phenomena.