High-yield synthesis and purification of an alpha-helical transmembrane domain.
Polypeptides corresponding to hydrophobic transmembrane alpha-helices, such as residues 69-101 of glycophorin A, are notoriously difficult to prepare in quantities sufficient for biophysical experiments. Simple synthetic and purification approaches reported here have been developed by combining a few modifications to standard procedures, without resorting to elevated temperatures, expensive activation strategies, or complex hydrophobic solvent mixtures. The cost of screening projects, preparing labeled peptides, and examining sequence variations is thereby significantly reduced. The quality of the peptide synthesized by this small-scale 9-fluorenylmethoxycarbonyl (Fmoc) strategy is comparable to that of the peptide synthesized by an experienced resource facility using a large-scale tert-butyloxycarbonyl strategy. Using reverse-phase HPLC, the desired peptide was separated from the primary side product (a Leu or Ile deletion) and quantitatively recovered at greater than 98% purity. Baseline resolution was achieved using a water:acetonitrile gradient to elute the peptides from a cyanopropyl column at ambient temperature. Combining these approaches readily yields 10 to 20 mg of pure transmembrane peptide from a small-scale Fmoc synthesis. The approaches are readily transferable to transmembrane sequences not previously synthesized and do not require setting up a specialized facility. The time and start-up expense required to launch new studies are thereby reduced expanding the range and detail with which questions in membrane protein biophysics can be explored.
Fisher
L E
LE
Department of Chemistry, Yale University, New Haven, CT 06520, USA.
Engelman
D M
DM
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006021:Glycophorins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; D006021:Glycophorins; D010455:Peptides