Regioselektive Cysteinbiokonjugation durch Anhängen eines modifizierten Cystein‐tags an ein Protein mithilfe von trans‐Proteinspleißen

Abstract

Orthogonale Cysteine: Die chemische Modifikation von Cysteinresten ist eine der wichtigsten Methoden zur chemoselektiven Biokonjugation von Proteinen, sie verläuft allerdings in der Regel nicht regioselektiv. Durch das Zusammensetzen eines Proteins mithilfe von trans‐Proteinspleißen kann ein einzelner Cysteinrest gezielt markiert werden. Dieser Ansatz ist sogar in komplexen Gemischen wie Zellextrakten durchführbar.

Bioorthogonal Ligation in the Spotlight

“…Suitably modified precursors for lipid and glycan biosynthesis could be processed in a similar way. Finally, one of the two functional groups could be attached to a protein fused with a tag amenable to chemical modification 30–32…”

Enzymatic “Click” Ligation: Selective Cysteine Modification in Polypeptides Enabled by Promiscuous Glutathione S‐Transferase

“…This result indicates that the GST-catalyzed arylation could greatly expand the scope of cysteine modification beyond that of previous methods, which necessitate the use of protecting groups or multiple steps for the differential functionalization of two or more cysteine residues. [19] Cyclic peptides constitute a very important class of medicinally relevant macrocycles. [20] Although various methods have been developed, [21] the synthesis of macrocyclic peptide fragments remains challenging.…”

Split Inteins as Versatile Tools for Protein Semisynthesis

“…As outlined in Scheme , a single cysteine in the appended protein/peptide corresponding to the extein can thus be selectively and quantitatively conjugated, excess bioconjugation reagent be separated or quenched, and subsequently the full‐length protein be reassembled by PTS 52. 73 Specifically, the Int C fragments of the artificially split Ssp DnaB and Mxe GyrA inteins are free of cysteines and operate with a serine and threonine, respectively, at the (+1) position. These intein fragments were expressed in fusion with a short peptide sequence with a single cysteine, termed the CysTag.…”