No abstract

Butelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

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We describe the total synthesis of three active HIV-1 protease analogs by an orthogonal ligation through thiaproline formation of two large, unprotected peptide segments. The central element of this strategy in achieving orthogonality of peptide bond formation is through proximity effect, and the key reaction is a side chain thiol initiated aldehyde capture to overcome the entropic problem of coupling between two large molecular weight peptide segments. The capture step also leads to specific entropic activation of the acyl segment because the respective termini are brought to close proximity, resulting in a spontaneous acyl rearrangement to form the amide bond. A general method using a thioester for introducing an aldehyde moiety to the C-terminus of an unprotected peptide segment was also developed. Three active analogs of HIV-1 protease were obtained in excellent yield by ligating two segments of 38 and 61 residues. Two analogs contained a thioproline residue at position 39, and the third contained a 38−39 non-peptide backbone. Efficient ligation at pH 4 was attained at peptide segment concentrations as low as 50 μM, a concentration which is not feasible with conventional convergent methods using protected peptide segments.

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.