Chemical-Tag Labeling of Proteins Using Fully Recombinant Split Inteins

Abstract

Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.

Synthetic Biology: A New Tool for the Trade

“…Furthermore, covalent attachment is not seamless, and a peptide–protein complex will remain between assembled polypeptides. Other covalent protein assembly techniques, such as inteins, sortase‐based tagging, the disulfide‐based dock‐and‐lock method, or native chemical ligation may also be employed. However, these techniques tend to be more dependent on the experimental conditions for activity, and a discussion of these techniques is beyond the scope of this article.…”

“…In vitro protein assembly : Many options exist for the in vitro covalent assembly of proteins, including disulfide bonds, inteins, chemical ligation, or enzymatic assembly . However, these methods are either dependent on the redox status of the environment, lead to undesired side reactions or require accessory factors and restrictive experimental conditions for assembly.…”

Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications

“…Unnatural amino acids facilitate bioorthogonal reactions and expand the capabilities of protein chemistry. Among others, these comprise the creation of cyclic peptides by an incorporation of an unnatural amino acid followed by an oxime ligation [ 17 ] as well as site-specific chemical-tag labeling of proteins by recombinant split inteins [ 18 ].…”