Sortase A as a tool for high‐yield histatin cyclization

Abstract

Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in ≤ 3% yields, whereas sortase-mediated cyclization provided a yield of >90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 μM. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.

Identification of Peptides Derived from the Human Antimicrobial Peptide LL-37 Active against Biofilms Formed by Pseudomonas aeruginosa Using a Library of Truncated Fragments

“…Peptides were purified by preparative reversed-phase high-pressure liquid chromatography. The purity of the peptides was at least 95%, and the authenticity of the peptides was confirmed by a Microflex LRF matrix-assisted laser desorption ionization-time of flight mass spectrometer equipped with an additional gridless reflectron (Bruker Daltonik, Bremen, Germany) as described previously (2).…”

Killing of Mycobacterium avium by Lactoferricin Peptides: Improved Activity of Arginine- and d -Amino-Acid-Containing Molecules

“…Peptides were purified to a purity of at least 95% by semipreparative RP-HPLC (Jasco Corporation Tokyo, Japan) on a Vydac C 18 column (218MS510; Vydac, Hesperia, CA, USA), and the authenticity of the peptides was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry on a Microflex LRF mass spectrometer equipped with an additional gridless reflectron (Bruker Daltonik, Bremen, Germany) as described previously (25).…”

Butelase 1: A Versatile and Multi-Purpose Ligase

“…Cyclization of histatin has been shown to improve the activity by a 1000 fold [3]. Figure 2 shows the cyclization of histatin-1 was highly efficient with >95% yield in 15 min in the presence of 0.002 molar equivalent of butelase 1.…”