Synthesis of a membrane protein with two transmembrane regions.
A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.
Sato
Takeshi
T
Institute for Protein Research, Osaka University, Suita, Japan.
Kawakami
Toru
T
Akaji
Kenichi
K
Konishi
Hiroki
H
Mochizuki
Koji
K
Fujiwara
Toshimichi
T
Akutsu
Hideo
H
Aimoto
Saburo
S
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000138:chemical synthesis* / Q000737:chemistry*; D012995:Solubility; D012997:Solvents; D013696:Temperature
D008565:Membrane Proteins; D021122:Protein Subunits; D012997:Solvents; D006180:Proton-Translocating ATPases