大腺桿菌PE24分子的ADP核糖基轉移酶活性在硝基苯肼（NBAG）和體外培養的癌細胞株上進行了評估。PE24的基因編碼從大腺桿菌分離出來，克隆到pET22b （+）質粒中，在IPTG誘導下在大腸桿菌BL21（DE3）中表達。通過菌落PCR、工程構造體的消化後插入物的出現以及鈉二十硫酸鹽聚丙烯酰胺凝膠電泳（SDS-PAGE）蛋白質電泳進行了基因重組的確認。在低劑量γ射線（5、10、15、24 Gy）前後使用化學化合物NBAG進行了紫外光譜、傅里葉紅外線光譜、碳十三磁共振和高效液相色譜檢測，以確認PE24提取物的ADP核糖基轉移酶活性。PE24提取物單獨及與紫杉醇和低劑量γ射線（5 Gy和一次24 Gy）聯合對黏著細胞株HEPG2、MCF-7、A375、OEC和Kasumi-1細胞懸浮液進行了細胞毒性評估。表達的PE24分子通過FTIR和NMR揭示出ADP核糖基化了NBAG，HPLC色譜圖譜的不同保留時間出現了新的峰。重組PE24分子的照射與ADP核糖基化活性的減少有關。PE24提取物在癌細胞株上的IC50值<10 μg/ml，並且在正常OEC細胞中10 μg/ml時有可接受的細胞存活率。總體而言，PE24提取物與低劑量紫杉醇聯合起到了協同作用，IC50減少，而在低劑量γ射線照射後記錄到抗拒作用和IC50值的上升。關鍵點：• 成功表達並進行生物化學分析。• 低劑量γ射線和金屬離子降低了重組PE24的細胞毒性。• 與低劑量紫杉醇聯合起協同作用。© 2023. The Author(s).

The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro cultured cancer cell lines. Gene encoding PE24 was isolated from P. aeruginosa isolates, cloned into pET22b( +) plasmid, and expressed in E. coli BL21 (DE3) under IPTG induction. Genetic recombination was confirmed by colony PCR, the appearance of insert post digestion of engineered construct, and protein electrophoresis using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The chemical compound NBAG has been used to confirm PE24 extract ADP-ribosyl transferase action through UV spectroscopy, FTIR, c13-NMR, and HPLC before and after low-dose gamma irradiation (5, 10, 15, 24 Gy). The cytotoxicity of PE24 extract alone and in combination with paclitaxel and low-dose gamma radiation (both 5 Gy and one shot 24 Gy) was assessed on adherent cell lines HEPG2, MCF-7, A375, OEC, and Kasumi-1 cell suspension. Expressed PE24 moiety ADP-ribosylated NBAG as revealed by structural changes depicted by FTIR and NMR, and the surge of new peaks at different retention times from NBAG in HPLC chromatograms. Irradiating recombinant PE24 moiety was associated with a reduction in ADP-ribosylating activity. The PE24 extract IC50 values were < 10 μg/ml with an acceptable R2 value on cancer cell lines and acceptable cell viability at 10 μg/ml on normal OEC. Overall, the synergistic effects were observed upon combining PE24 extract with low-dose paclitaxel demonstrated by the reduction in IC50 whereas antagonistic effects and a rise in IC50 values were recorded after irradiation by low-dose gamma rays. KEY POINTS: • Recombinant PE24 moiety was successfully expressed and biochemically analyzed. • Low-dose gamma radiation and metal ions decreased the recombinant PE24 cytotoxic activity. • Synergism was observed upon combining recombinant PE24 with low-dose paclitaxel.© 2023. The Author(s).