Expression and Membrane Assembly of a Transmembrane Region from Neu

Abstract

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, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes. A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli. The sequence also contained 11-12 amino acids from each side of the transmembrane domain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product. This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption 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 material with increased alpha-helix content, which behaved in detergent as mixtures 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 peptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.

Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbReceptor Family of Proteins

“…42 Furthermore, it has been reported that a peptide corresponding to the TM of the EGF receptor (erbB1) migrated as a monomer in SDS-PAGE, 54 and isolated erbB2 TM peptides also migrate predominantly as monomers. 55 These results with isolated TM peptides are consistent with the SDS-PAGE analysis of our SN-fusion constructs. Some reports do indicate, however, peptides corresponding to the TM of the proto-oncogenic neu receptor from rats, a homolog of erbB2, migrate as something larger than monomers on SDS-PAGE.…”

“…Some reports do indicate, however, peptides corresponding to the TM of the proto-oncogenic neu receptor from rats, a homolog of erbB2, migrate as something larger than monomers on SDS-PAGE. 36,55,56 While specific protein-protein interactions determined by the amino acid sequence are important for TM helix interactions, the environment can also be a critical factor in the investigation of membrane proteins. Successful solution studies can depend on the choice of an appropriate detergent.…”

Cloning, Expression, and Assembly of Sericin-like Protein

“…5a). Reduced electrophoretic mobility has been observed previously for various proteins (27)(28)(29)(30)(31)(32), and this can stem from unusual amino acid composition, intrinsic net charge or protein oligomerization. The pI values of SRC proteins are 5.98, 5.02, and 4.61 for SRC4, 8, and 12, respectively.…”

Oligomerization of the integrin αIIbβ3: Roles of the transmembrane and cytoplasmic domains

“…The cleaved sample was dialyzed extensively at 4°C against water, and the turbid solution was lyophilized. The lyophilized proteins were solubilized by using a mixture of a formic acid-acetic acid-chloroform-trifluoroethanol (FACT) mix (21), acetonitrile, and water in a ratio of 1:3:3 and purified by preparative RP-HPLC as described (22). The purified TM-CYTO protein was lyophilized and stored at Ϫ80°C.…”