relation: http://publicatio.bibl.u-szeged.hu/28465/
title: Temoneira-1 β-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography
creator:  Nafaee Zeyad Hasan Abdullah
creator:  Hunyadi-Gulyás Éva Csilla
creator:  Gyurcsik Béla
subject: 01.04. Kémiai tudományok
description: β-lactamases protect bacteria from β-lactam antibiotics. Temoneira (TEM) is a class A serine β-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 β-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 β-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15–200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 β-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of β-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated β-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair. © 2022 Elsevier Inc.
date: 2023
type: Folyóiratcikk
type: PeerReviewed
format: text
identifier: http://publicatio.bibl.u-szeged.hu/28465/3/33241868_megjelent.pdf
format: text
identifier: http://publicatio.bibl.u-szeged.hu/28465/1/ProteinExprPurif2023201106169Pre-proof_Gyurcsik.pdf
identifier:     Nafaee Zeyad Hasan Abdullah;  Hunyadi-Gulyás Éva Csilla;  Gyurcsik Béla: Temoneira-1 β-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography.   PROTEIN EXPRESSION AND PURIFICATION, 201.   ISSN 1046-5928 (2023)     
identifier: doi:10.1016/j.pep.2022.106169
relation: https://doi.org/10.1016/j.pep.2022.106169
relation: 33241868
language: eng