Single-step purification and characterization of Pseudomonas aeruginosa azurin

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 communit...

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Bibliographic Details
Published in:Protein expression and purification Vol. 224; p. 106566
Main Authors: Riegerová, Petra, Horváth, Matej, Šebesta, Filip, Sýkora, Jan, Šulc, Miroslav, Vlček, Antonín
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01.12.2024
Subjects:
Azurin
Azurin - chemistry
Azurin - genetics
Azurin - isolation & purification
Azurin - metabolism
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Bacterial Proteins - metabolism
Escherichia coli - genetics
Escherichia coli - metabolism
Glutathione S-Transferase
HeLa Cells
HRV 3C protease
Humans
Molecular dynamics
Protein purification
Pseudomonas aeruginosa - enzymology
Pseudomonas aeruginosa - genetics
Redox reaction
Molecular dynamics
Azurin
HRV 3C protease
Protein purification
Glutathione S-Transferase
Redox reaction
ISSN:1046-5928, 1096-0279, 1096-0279
Online Access:Get full text
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Summary: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. •Azurin is a scaffold protein with essential role in bacterial respiration and de-nitrification/nitrogen cycle.•Azurin is a model protein to study long-range electron transfer mechanism.•Azurin has significant antiviral, antimicrobial and anticancer properties.•Single step purification procedure yields high quantities of properly folded and metalated protein.•Purification procedure does not affect solubility, metalation or function of the protein.
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ISSN:1046-5928
1096-0279
1096-0279
DOI:10.1016/j.pep.2024.106566