Chemical synthesis of proteins using peptide hydrazides as thioester surrogates.
This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ NaNO2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ~30 working days.
Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China.
Ji-Shen
JS
Zheng
Shan
S
Tang
Yun-Kun
YK
Qi
Zhi-Peng
ZP
Wang
Lei
L
Liu
D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004952:Esters / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D051844:alpha-Synuclein / Q000138:chemical synthesis
D000596:Amino Acids; D004952:Esters; D005609:Free Radicals; D010455:Peptides; D051844:alpha-Synuclein