Ant plasmid was cultured at 37 C in lysogeny broth (LB) containing one hundred /ml ampicillin and 50 /ml chloromycetin for 12 h. The cultures had been diluted (1:1000) with LB broth and subjected to further incubation till the OD600 reached about 0.eight, then induced by isopropyl -D-thiogalactoside (IPTG) at a final concentration of 0.5 mM. After induction for 12 h at 28 C, the cells had been collected and broken by an ultrasonic binding/wash buffer (1 PBS with 1 Triton X-100) at four C. Right after ultrasonication, the cell debris was removed by centrifugation at 8000g for 30 min, as well as the supernatant was retained. The recombinant proteins had been purified straight in the lysate applying GST-sefinose (TM) resin. The supernatant was applied to a Poly-Prep Chromatography Column (BIO-RAD, USA) with 1 ml GST-sefinose (TM) resin, which was pre-washed having a binding/washing buffer. The purified proteins had been dialyzed in 1 PBS at four C for 24 h, together with the 1 PBS replaced every single 12 h. The protein concentrationMar. Drugs 2021, 19,11 ofwas determined utilizing the Bradford strategy, applying BSA (bovine serum albumin) as the common. The purified proteins had been mixed having a six SDS gel-loading buffer, boiled at 100 C for 10 min, and resolved with 12 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie brilliant blue R250. Finally, the purified proteins have been stored at -80 C in aliquots, unless otherwise specified. four.five. Peptide Synthesis The peptides from Al-crus three and Al-crus 7 containing the WAP domain have been made and synthesized by GenScript Biological Technology Co., LTD. Al-crusWAP-3 from Al-crus 3: SCPPRRPLCPKFHTPPQTCGNDSKCSGTDKCCLDTCLEVCK, and Al-crusWAP 7 from Al-crus 7: RCPPVRPVCPPVRSFAPPASCSNDGACGGIDKCCYDKCLEQHTCK. The purity of these peptides was more than 98 . four.six. Antibacterial Activity Assays The examined bacteria in the -80 C stock had been first inoculated on plates, and then a single colony for PF-06873600 Technical Information culture was picked up in LB broth. To avoid contamination, the tested bacteria were further sequenced and identified. Antimicrobial activities had been examined against seven Gram-positive and six Gram-negative bacteria. The MIC was determined by a liquid development inhibition assay . The purified proteins have been consecutively diluted with sterile water in five multiples; subsequent, 0.two BSA was utilized because the damaging C2 Ceramide medchemexpress control. Aliquots (ten ) from each dilution have been transferred to a 96-well polypropylene microtiter plate (Corning, Wujiang, China), and every single nicely was inoculated with one hundred of mid-log bacterial suspension (105 CFU/ml) in poor broth (1 tryptone, 0.5 NaCl (w/v), pH 7.5). The experimental assays had been grown for 12 h with shaking at 120 rpm/hr and 37 C. The OD600 values had been measured just about every 4 h working with a microplate reader (Multiscan FC, Thermo Fisher, American). All of the experiments had been performed at the least three times for the replications. For the thermal stability evaluation, the freshly purified proteins had been kept at diverse temperatures for 48 h and then processed to perform antibacterial assays, as pointed out above. For the peptide antimicrobial activity experiment, the bacteria had been exactly the same as these pointed out above. The peptides have been centrifuged just before dissolution with ddH2 O to 550 and kept at -80 C in aliquots. Finally, the MIC50 was determined. 4.7. SEM Imaging The M. luteus, S. aureus, and imipenem-resistant Acinetobacter baumannii had been treated with Al-crus 3 and Al-crus 7 having a MIC50 concentration, respectively.