| Yazarlar |
Doç. Dr. Hatice Esra DURAN
Kafkas Üniversitesi, Türkiye |
Şükrü Beydemir
Anadolu Üniversitesi, Türkiye |
| Özet |
| Human carbonic anhydrase VII (hCA VII), a cytosolic enzyme, defends against oxidative stress by preventing reactive oxygen species from forming. In our study, first, hCA VII was cloned into Escherichia coli (One Shot Mach1-T1R) strain by using cDNA of the human brain and successfully expressed. The integrity of the plasmid generated by colony PCR was checked, and after, for protein expression, the plasmid was transformed into E. coli BL21 (DE-3) strain. hCA VII expression was observed after 6 h of isopropyl-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein containing hexahistidine (6xHis) was purified with 7.02 EU/mg of specific activity, had 48.07% of purification yield, and approximately 21-folds using a ProbondTM nickel chelating resin affinity column. Then, both molecular mass determination and purity control of the purified recombinant enzyme was done by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The mass of the SUMO-hCA VII fusion protein was calculated as 46.77 kDa. As a result of Western blot analysis using anti-His G-HRP antibody, the fusion protein was detected as approximately 45 kDa. Furthermore, the characterization assays and in vitro inhibition studies were done for the recombinant enzyme. K values of these agents were found between 0.29 μM and 157.6 mM. Finally, molecular docking investigations of these antibiotics were undertaken to understand further the binding interactions on the active site of this recombinant enzyme. |
| Anahtar Kelimeler |
| carbonic anhydrase,E,coli,enzyme catalysis,metalloenzymes,molecular docking,recombinant protein |
| Makale Türü | Özgün Makale |
| Makale Alt Türü | SSCI, AHCI, SCI, SCI-Exp dergilerinde yayımlanan tam makale |
| Dergi Adı | Biotechnology and Applied Biochemistry |
| Dergi ISSN | 0885-4513 |
| Dergi Tarandığı Indeksler | SCI-Expanded |
| Dergi Grubu | Q2 |
| Makale Dili | İngilizce |
| Basım Tarihi | 02-2023 |
| Cilt No | 70 |
| Sayı | 1 |
| Sayfalar | 415 / 428 |
| Doi Numarası | 10.1002/bab.2367 |
| Makale Linki | http://dx.doi.org/10.1002/bab.2367 |
| Atıf Sayıları | |
| WoS | 5 |
| Google Scholar | 4 |
