0597139 - ÚFCH JH 2025 RIV NL eng J - Článek v odborném periodiku
Riegerová, Petra - Horváth, Matej - Šebesta, Filip - Sýkora, Jan - Šulc, M. - Vlček, Antonín
Single-step purification and characterization of Pseudomonas aeruginosa azurin.
Protein Expression and Purification. Roč. 224, AUG 2024 (2024), č. článku 106566. ISSN 1046-5928. E-ISSN 1096-0279
Grant CEP: GA MŠMT(CZ) EH22_008/0004617; GA ČR(CZ) GA21-05180S
Výzkumná infrastruktura: e-INFRA CZ II - 90254
Institucionální podpora: RVO:61388955
Klíčová slova: Azurin * Glutathione S-Transferase * Protein purification
Obor OECD: Physical chemistry
Impakt faktor: 1.4, rok: 2023
Způsob publikování: Open access

Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.
Trvalý link: https://hdl.handle.net/11104/0355432