Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.
Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.
Khemtémourian
Lucie
L
UMR 5144 MOBIOS, CNRS-University Bordeaux 1, IECB, 2 rue Robert Escarpit, 33607 Bordeaux-Pessac, France.
Lavielle
Sébastien
S
Bathany
Katell
K
Schmitter
Jean-Marie
JM
Dufourc
Erick J
EJ
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D011138:Polytetrafluoroethylene / Q000737:chemistry; D017433:Protein Structure, Secondary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D010446:Peptide Fragments; D011138:Polytetrafluoroethylene; D018719:Erb-b2 Receptor Tyrosine Kinases; D012997:Solvents