title: Butelase-mediated cyclization and ligation of peptides and proteins.
abstract: Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Giang K T
authors.initials: GK
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Yibo
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cao
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Xinya
authors.initials: X
authors.lastname: Hemu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Chuan-Fa
authors.initials: CF
authors.lastname: Liu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: James P
authors.initials: JP
authors.lastname: Tam