Peptide ligation and its application to protein engineering

Abstract

The ability to assemble a target protein from a series of peptide fragments, either synthetic or biosynthetic in origin, enables the covalent structure of a protein to be modified in an unprecedented fashion. The present technologies available for performing such peptide ligations are discussed, with an emphasis on how these methodologies have been utilized in protein engineering to investigate biological processes.

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

“…Single-point energy ab initio calculations (HartreeFock model, 6-31G* basis set) (22,23) were performed for methionine and for analogs 2 and 3 with fully extended side chains. Electron density maps are shown as surfaces of electron density 0.08 electrons͞au (3). Isopotential plots are represented as surfaces where the energy of interaction between the amino acid and a point positive charge is equal to Ϫ10 kcal͞mole.…”

“…Protein engineering by means of the introduction of non-natural amino acids is an important approach to the investigation of protein folding, structure, and function as well as the design of novel protein reactivity (1). Indeed, incorporation of nonnatural amino acids into proteins by means of chemical methods such as solid-phase synthesis (2), native chemical ligation (3), and in vitro translation protocols (4,5) has permitted characterization of protein folding pathways, enzymatic mechanisms, and ligand-receptor interactions. Recent advances in the adaptation of ''unnatural amino acid mutagenesis'' to cellular systems suggests a rich future for heterologous expression of novel proteins (6)(7)(8)(9).…”

Synthesis of Native Proteins by Chemical Ligation

“…This "expressed protein ligation" 3 can be expected to lead to widespread use of the native chemical ligation method in biological research laboratories (109,110).…”

“…For proteins with no suitable Cys ligation sites in the natural sequence, it is possible to simply put a Cys wherever one is needed for ligation, usually without deleterious effects on function (63, 67; see Figure 9). The work of Muir and coworkers is illustrative of this expedient but effective approach (66,104,110). Their chemical ligation of recombinantly expressed polypeptide-α thioesters to synthetic peptides has typically made use of an arbitrarily introduced Cys residue at the desired ligation site, with no deleterious effects.…”

Injectable hydrogels for cartilage tissue engineering

“…However, some caution has to be taken in the use of an excess of thiol functional groups for unreacted thiols may cause cell death [53]. Chemical peptide ligation is a particularly appealing approach for the synthesis of proteins and enzymes [54]. The reaction is based on the chemoselective reaction and ligation of two unprotected peptide segments.…”