Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid

Abstract

Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at a selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine.

New developments in RiPP discovery, enzymology and engineering

“…As an example, the remarkable promiscuity of butelase-1 as a macrocyclase (Section 3.19) and its tolerance to ncAAs (see Section 7.4.1) has been exploited on dihydrofolate reductase (DHFR). 551 Expression in E. coli of DHFR containing ncAA that display alkyne groups and a C-terminal extension with the Asn-His-Val recognition sequence for butelase-1 allowed the production of a cyclic DHFR variant. Next, CuAAC was used to conjugate either biotin or a cell-penetrating peptide to the cyclic DHFR for enzyme inmobilization or intracellular delivery, respectively.…”

Peptide asparaginyl ligases—renegade peptide bond makers

“…Butelase-mediated cyclization of peptides and proteins generally resulted in high yield and completed within minutes. Table 3 shows selected examples of macrocyclization by butelase-1, which ranges from 14 to a few hundred amino acids [35,54,55,[75][76][77].…”

“…However, advances in genetic code expansion allow the installation of unusual amino acids with desirable features to a protein for site-specific ligation for a diverse range of applications, including protein probing, imaging, and function controlling [85]. Liu and coworker [77] incorporated un-natural amino acids in the protein sequence by amber codon suppression technology to enable site-specific modification at the side chain of murine dihydrofolate reductase (mDHFR). Butelase-mediated ligation was then applied to cyclize the biotinylated linear mDHFR within 30 min.…”

“…However, the ability of the drug to penetrate the cell membrane and to remain functional in the cell has been hampering therapeutics targeting specific sites in vivo. By conjugating the cyclic biotinylated mDHFR with cellpenetrating peptide Liu and coworker [77] successfully delivered mDHFR into the cell, paving the way toward refinement and optimization of approaches for the enzymereplacement therapy.…”

Advances in Site-specific and Linkage-specific Ligation

“…It belongs to the asparaginyl endoprotease (AEP) family, but it is virtually devoid of protease activity. Butelase 1 recognizes a C-terminal Asx-containing tripeptide motif, Asn/Asp-HisVal, to form an Asx-Xaa peptide bond where Xaa is any amino acid, either intramolecularly or intermolecularly, yielding, respectively, head-to-tail cyclic peptides or site-specific modified proteins [5][6][7][8][9][10][11][12][13][14]. Figure 2).…”