Total chemical synthesis of proteins

Abstract

This tutorial review outlines the modern ligation methods that enable the efficient total chemical synthesis of enzymes and other protein molecules. Key to this success is the chemoselective reaction of unprotected synthetic peptides ('chemical ligation'). Notably, native chemical ligation enables the reaction of two unprotected peptides in aqueous solution at neutral pH to form a single product in near quantitative yield. Full-length synthetic polypeptides are folded to form the defined tertiary structure of the target protein molecule, which is characterized by mass spectrometry, NMR, and X-ray crystallography, in addition to biochemical and/or biological activity.

Selective chemical protein modification

“…The intermolecular formation of an intermediate thioester is followed by a rapid S-N-acyl shift 46 , resulting in the formation of native peptide bond. Applications in fully synthetic proteins have been reviewed elsewhere 45,47 and many amino acids can now be generated at the ligation site in place of cysteine 48 . NCL may also be utilized in the 'semi-synthesis' or 'expressed protein ligation' of proteins through the site-selective ligation of recombinantly derived thioesters and synthetic peptides 49 .…”

“…Since its introduction by Kent 8,44 , NCL has been used extensively for the generation of synthetic proteins 45 . The intermolecular formation of an intermediate thioester is followed by a rapid S-N-acyl shift 46 , resulting in the formation of native peptide bond.…”

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

“…Evidence is accumulating that racemic protein mixtures can be much easier to crystallize in cases in which the natural L-protein is refractory to crystallization (9). D-proteins can only be made by total chemical synthesis (11,12), but as Rv1738 is a small protein, we considered it a good candidate for total chemical synthesis using modern ligation methods.…”

Soluble factors from T cells inhibiting X4 strains of HIV are a mixture of β chemokines and RNases

“…Human RNase 4 was chemically synthesized via a threesegment native chemical ligation reaction (35,36). See SI Materials and Methods for detailed methods.…”