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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Vydáno v:	Protein expression and purification Ročník 224; s. 106566
Hlavní autoři:	Riegerová, Petra, Horváth, Matej, Šebesta, Filip, Sýkora, Jan, Šulc, Miroslav, Vlček, Antonín
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	United States Elsevier Inc 01.12.2024
Témata:	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
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Popis
Shrnutí:	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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1046-5928 1096-0279 1096-0279
DOI:	10.1016/j.pep.2024.106566