Synthesis of the C-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency.
In the process of developing a method for the synthesis of membrane proteins, the conditions for native chemical ligation, namely, detergent concentration and the chemical characteristics of the thiol additive were investigated in detail. The C-terminal region of the opioid receptor like 1, ORL1(288-370), which contains C-terminal intracellular and transmembrane domains, was chosen as a model. The building blocks, ORL1(329-370) and ORL1(288-328)-SR-Gly-Arg(5)-Leu (-SR- : -SCH(2)CH(2)CO-) were most effectively condensed slightly below the critical micelle concentration of SDS and in the presence of mercaptoethanesulfonic acid as a thiol additive. The results showed that the concentration of SDS and the charge on the thiol additive are crucial factors for the effective synthesis of a membrane protein by native chemical ligation.
Sato
Takeshi
T
Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
Saito
Yasuhiro
Y
Aimoto
Saburo
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes / Q000737:chemistry; D008623:Mercaptoethanol / Q000031:analogs & derivatives* / Q000737:chemistry*; D008823:Micelles; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis*; D011957:Receptors, Opioid / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D000094942:Nociceptin Receptor
D000596:Amino Acids; D005449:Fluorenes; D008823:Micelles; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010446:Peptide Fragments; D011957:Receptors, Opioid; D013438:Sulfhydryl Compounds; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D001120:Arginine; D000094942:Nociceptin Receptor