%0 Journal Article
%@ 1046-5928
%A  Nafaee Zeyad Hasan Abdullah
%A  Hunyadi-Gulyás Éva Csilla
%A  Gyurcsik Béla
%A Molekuláris és Analitikai Kémiai Tanszék SZTE / TTIK / KI MAKT [2023-],
%A Biokémiai Intézet SZBK BKI [1971-],
%D 2023
%F publicatio:28465
%J PROTEIN EXPRESSION AND PURIFICATION
%T Temoneira-1 β-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography
%U http://publicatio.bibl.u-szeged.hu/28465/
%V 201
%X 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.
%Z Export Date: 15 November 2022                         CODEN: PEXPE                         Correspondence Address: Gyurcsik, B.; Department of Inorganic and Analytical Chemistry, Dóm tér 7, Hungary; email: gyurcsik@chem.u-szeged.hu                         Chemicals/CAS: beta lactamase, 9073-60-3; histidine, 645-35-2, 7006-35-1, 71-00-1; isopropyl thiogalactoside, 26112-89-0; penicillinase, 9001-74-5; serine, 56-45-1, 6898-95-9; Anti-Bacterial Agents; beta-Lactamases; beta-Lactams; Histidine; Imidazoles; Ions; Isopropyl Thiogalactoside; Metalloproteins; Penicillinase; Protein Sorting Signals; Serine                         Funding details: Horizon 2020, 101004806, 739593                         Funding details: Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, NKFIH, 2019-2.1111-TÉT-2019-00089, GINOP-2.3.2-15-2016-00001, GINOP-2.3.2-15-2016-00020, GINOP-2.3.2-15-2016-00038                         Funding text 1: This research was supported by the Hungarian National Research, Development and Innovation Office ( GINOP-2.3.2-15-2016-00038 , GINOP-2.3.2-15-2016-00001 , and GINOP-2.3.2-15-2016-00020 , as well as 2019-2.1111-TÉT-2019-00089 ) and the EU Horizon 2020 grant No. 739593 . The authors thank Peter Baker, for the development, maintenance; the ELKH Cloud ( https://science-cloud.hu/ ) for hosting the ProteinProspector search engine; and Professor Kyosuke Nagata for providing the nuclear factor I gene. Z.H.N. thanks the support from the European Union's Horizon 2020 research and innovation program under grant agreement No 101004806 (MOSBRI–Molecular Scale Biophysics Research Infrastructure) allowing to participate at the ESC1: Circular Dichroism: best practice and data analysis course.