Expressed Enzymatic Ligation for the Semisynthesis of Chemically Modified Proteins

Abstract

Chemical synthesis is the most powerful way to assemble selectively modified proteins and is a tool for engineering proteins that are inaccessible by standard site-directed mutagenesis. A number of methods for the chemical synthesis of proteins have been developed, of which native chemical ligation (NCL) [1] is currently the most widely applied. This ligation approach is based on the chemoselective reaction of a C-terminal peptide thioester with a peptide that has a cysteine residue at its N terminus and results in the formation of a native peptide bond. Various attempts have been made to overcome the requirement for the cysteine residue at the ligation site. However, although some of these were successful, for example, with glycine [2] or selenocysteine [3] in place of cysteine, this feature still limits the flexibility of NCL. Expressed protein ligation (EPL) [4] has opened up a whole new dimension in terms of accessible protein targets, because it applies recombinant techniques. However, this method suffers from restrictions similar to those of native ligation. Proteases that are used as ligation catalysts tolerate greater structural diversity at the coupling site. However, the limited primary substrate specificity of these enzymes reduces the scope of their use in synthesis. In the subtiligase-catalyzed fragment-condensation strategy (SCFC), which is one of the most successful enzymatic ligation methods to date, a genetically optimized enzyme is used for the synthesis of native and modified ribonuclease A.[5] Although this method increased the size of synthetic targets to entire proteins, it still suffers from the typical requirement for the careful selection of suitable coupling sites, because of limited enzyme specificity. Thus, despite significant progress in protein synthesis, alternative ligation strategies with broader applicability are still needed.Herein we report a further step toward this ambitious aim-expressed enzymatic ligation (EEL), which combines the advantages of EPL with those of the substrate-mimeticmediated ligation strategy. The latter eliminates the specificity problem of classical protease-catalyzed synthesis through the attachment of an active-site-specific ester leaving group at the C-terminus of a nonspecific peptide moiety.[6] This combined approach also takes advantage of the recombinant expression of long-chain peptide thioesters, which can then act as substrate mimetics. Through this technique, peptide coupling can be carried out by an ordinary protease, for example, the Glu/Asp-specific serine protease V8 from Staphylococcus aureus, at nonspecific ligation sites, regardless of the specificity of the enzyme.The feasibility of EEL has been demonstrated through the synthesis of a cysteine-free carboxyfluorescein (CF)-labeled analogue of the 69-mer prohormone neuropeptide Y (proNPY; Scheme 1). ProNPY processing leads to one of the most abundant neuropeptides in the brain, neuropeptide Y (NPY), which is involved in numerous biological events, such as the regulation of food intake and...

Diels–Alder Ligation of Peptides and Proteins

“…Noteworthy is also the development of enzymatic ligation methods for the preparation of proteins with enzymes specifically engineered to perform reverse proteolysis and to act as "ligases". [17] Nevertheless, despite some notable successes, [18][19] such methods have not found widespread use (yet) after the development of the more simple and versatile chemical ligation methods. [1,[20][21][22][23][24] The size of proteins which can be chemically synthesized has been increased Because the mutually reactive groups (or at least one of them) are functionalities not normally found in peptides, the price to be paid for applying such chemoselective ligation is the formation of an unnatural structure at the ligation site.…”

Switch-Peptides as Folding Precursors in Self-Assembling Peptides and Amyloid Fibrillogenesis

“…Furthermore, the access of polypeptides and even proteins exhibiting high tendency for self-association and misfolding, notably in the context of neurodegenerative diseases, will become accessible by incorporating the elaborated methodologies into modern strategies of convergent syntheses and peptide ligation techniques. 64,65 In perspective, switch-peptides will become a powerful chemical tool for addressing fundamental biological questions, notably in the field of peptide and protein self-assembly, misfolding, and fibrillogenesis. In addition, by decipher-…”

Diels–Alder Cycloaddition in Protein Chemistry

“…maleimide cysteine conjugation, [7] aldehyde assisted ligations, [8] aromatic amino acid side chains of tryptophan [9] and tyrosine, [10] native chemical ligation, [11] expressed enzymatic ligation, [12] expressed protein ligation, [13] Staudinger ligation [14] or 1,3-dipolar cycloaddition reaction [15] have been developed as powerful new methods makes significant advances in this field.…”