High-Yield Synthesis and Purification of an a-Helical ransmembrane Domain

Examination of genome sequences for stretches of Polypeptides corresponding to hydrophobic transmembrane a-helices, such as residues 69–101 of glycophrin A, are notoriously difficult to prepare in quantities ufficient for biophysical experiments. Simple synthetic nd purification approaches reported here have been eveloped by combining a few modifications to standard rocedures, without resorting to elevated temperatures, xpensive activation strategies, or complex hydrophobic olvent mixtures. The cost of screening projects, preparng labeled peptides, and examining sequence variaions is thereby significantly reduced. The quality of the eptide synthesized by this small-scale 9-fluorenylmehoxycarbonyl (Fmoc) strategy is comparable to that of he peptide synthesized by an experienced resource faility using a large-scale tert-butyloxycarbonyl strategy. sing reverse-phase HPLC, the desired peptide was seprated from the primary side product (a Leu or Ile deleion) and quantitatively recovered at greater than 98% urity. Baseline resolution was achieved using a water: cetonitrile gradient to elute the peptides from a cyanoropyl column at ambient temperature. Combining hese approaches readily yields 10 to 20 mg of pure ransmembrane peptide from a small-scale Fmoc synhesis. The approaches are readily transferable to transembrane sequences not previously synthesized and do ot require setting up a specialized facility. The time nd start-up expense required to launch new studies are hereby reduced expanding the range and detail with hich questions in membrane protein biophysics can be xplored. © 2001 Academic Press