Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388955%3A_____%2F24%3A00597139" target="_blank" >RIV/61388955:_____/24:00597139 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/24:10487642 RIV/00216208:11320/24:10487642

Výsledek na webu

<a href="https://hdl.handle.net/11104/0355432" target="_blank" >https://hdl.handle.net/11104/0355432</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.pep.2024.106566" target="_blank" >10.1016/j.pep.2024.106566</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10403 - Physical chemistry

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Protein Expression and Purification

ISSN

1046-5928

e-ISSN

1096-0279

Svazek periodika

224

Číslo periodika v rámci svazku

AUG 2024

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

9

Strana od-do

106566

Kód UT WoS článku

001297093200001

EID výsledku v databázi Scopus

2-s2.0-85201293335