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Current Topics in Peptide & Protein Research   Volumes    Volume 17 
Facile synthesis of intramolecular cyclic peptides using sortase A
Jinsheng Huang, Lasanthi Jayathilaka, Shalini Gupta, Baoshiang Lee
Pages: 57 - 69
Number of pages: 13
Current Topics in Peptide & Protein Research
Volume 17 

Copyright © 2016 Research Trends. All rights reserved

Sortase A (SrtA)-mediated peptide intramolecular transpeptidation is an effective method for peptide cyclization. We report here the recombinant protein production of SrtA and the optimization of its peptide intramolecular cyclization reaction. The effects of pH, temperature, concentration, time duration and organic solvent on this reaction are analyzed by HPLC and MS. In addition, methods of recycling SrtA for cost effectiveness have been investigated. Results show that SrtA purified from a Ni2+-nitrilotriacetic acid (NTA) affinity chromatography in the elution buffer containing 0.5 M imidazole can be used for peptide intramolecular cyclization directly without desalting. Although completion time is inversely proportional to the temperature, reaction at 4 °C is as effective as at 37 °C and is completed in 24 h with lesser degradation of SrtA. Higher SrtA-to-peptide mole ratio and higher concentration of both SrtA and peptide decrease the completion time of the reaction. Neutral pH is optimum whereas extreme pH is detrimental to the peptide intramolecular cyclization reaction. Dimethyl sulfoxide aqueous solution up to a concentration of 50% can be used to facilitate cyclization of a peptide with solubility issues. Active SrtA can be easily recovered using Ni+2-NTA, PD-10 desalting or RP-HPLC column after reaction. Cyclic peptides can be purified from the reaction directly, from flow-through of Ni+2 -NTA column, or from the fraction (small molecule) of PD-10 desalting column of the reaction by RP-HPLC. In certain cases, cyclic peptides can be recovered as a pellet when it is insoluble in reaction solution. The above-mentioned optimizations will facilitate the inclusion of this intramolecular cyclization method in a peptide research laboratory.
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