Temoneira-1 β-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography
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...
Elmentve itt :
| Szerzők: | |
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| Dokumentumtípus: | Cikk |
| Megjelent: |
2023
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| Sorozat: | PROTEIN EXPRESSION AND PURIFICATION
201 |
| Tárgyszavak: | |
| doi: | 10.1016/j.pep.2022.106169 |
| mtmt: | 33241868 |
| Online Access: | http://publicatio.bibl.u-szeged.hu/28465 |
| Tartalmi kivonat: | 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. |
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| Terjedelem/Fizikai jellemzők: | 9 |
| ISSN: | 1046-5928 |