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, 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.
Jones
D H
DH
Department of Biochemistry, University of Western Ontario, London, Canada, N6A 5C1.
Ball
E H
EH
Sharpe
S
S
Barber
K R
KR
Grant
C W
CW
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D008565:Membrane Proteins; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins