Chemical synthesis of proteins using peptide hydrazides as thioester surrogates

• Published in: Nature protocols Vol. 8; no. 12; pp. 2483 - 2495
• Main Authors: Zheng, Ji-Shen, Tang, Shan, Qi, Yun-Kun, Wang, Zhi-Peng, Liu, Lei
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2013; Nature Publishing Group
• Subjects: 631/1647/666; 631/92/469; 631/92/95; 639/638/549/978; alpha-Synuclein - chemical synthesis; Amino Acids - chemistry; Analytical Chemistry; Biological Techniques; Chemical synthesis; Chemistry; Chromatography, High Pressure Liquid; Computational Biology/Bioinformatics; Esters - chemistry; Free Radicals - chemistry; Life Sciences; Microarrays; Organic Chemistry; Parkinson's disease; Peptides; Peptides - chemistry; Properties; Protein biosynthesis; Protein Engineering - methods; Protein research; Proteins; Protocol; Spectrometry, Mass, Electrospray Ionization
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2013.152

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 NaNO 2 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.