General chemical reagents were purchased from Sigma-Aldrich, Sangon Biotech Co., Ltd. (Shanghai, China). High-performance liquid chromatography (HPLC) was performed on an Agilent 1260 Infinity apparatus with a diode array detector (DAD). HPLC-MS experiments were performed on Agilent 1290 HPLC system coupled with a Thermo Electron LTQ-Orbitrap XL mass spectrometer. All chemicals and solvents were of analytical or chromatographic grade. The resuspended cells were lysed by an ultrasonic processors VCX750 (Sonics and Materials Inc, PA, USA). Common biochemicals and chemicals were purchased from standard sources for laboratory use.
All strains and plasmids used in this study are listed in Supplemental Table S2. Escherichia coli DH5α was used for general cloning. Escherichia coli Rossetta (DE3) was used as a host for protein expression. E. coli strains were routinely cultured in Luria–Bertani (LB) liquid medium at 37 °C, 220 rpm, or LB agar plate at 37 °C. When appropriate, kanamycin (Kan; 100 μg mL-1 for E. coli) and chloramphenicol(Chl; 25 μg mL-1 for E. coli) were added to the medium.
DNA manipulations were performed using standard procedures or the manufacturer’s protocols for E. coli. Plasmid extraction, DNA purification and gel extraction were carried out using commercial kits (Omega Biotech Co., Ltd., Beijing, China). Oligonucleotide synthesis and DNA sequencing were performed at Tsingke Biotech Co., Ltd. (Beijing, China). PCR reactions were carried out using Pfu DNA polymerase (TIANGEN Biotech Co., Ltd., Beijing, China). Western blot was carried out according to the protocols provided by the Shanghai Universal Biotech Co.,Ltd. (Shanghai China). His-Tag (27E8) Mouse mAb (HRP Conjugate) and Anti-mouse IgG, HRP-linked Antibody were purchased from Shanghai Universal Biotech Co.,Ltd. (Shanghai China).
Molecular docking was carried out in order to predict the binding potentiality between the mutant proteins and the molecule. The complex crystal structure of BmmI with GPA was used as the template for the docking-based screening. The protein structure was prepared using the Structure Preparation function in MOE. Typical manual processing included the geometry and electron density checks, addition of hydrogen atoms and optimization of their position and energy minimization. GPA was then docked to the active pocket of BmmI by using the Dock module of MOE. By comparing the affinity and stability of different interactions between BmmI variants and GPA, appropriate variants were chosen for the site-directed mutagenesis of BmmI.
The reverse complementary primers with mutation sites were designed and listed in Table S2. The pET28a plasmids carrying bmmI gene with mutation sites was linearized by reverse PCR using 2 × Phanta® Flash Master Mix DNA polymerase. The linear plasmids were ligated to circular molecules by seamless cloning using ClonExpress® Ultra One Step Cloning Kit (Nanjing, China). The introduced mutations were verified by DNA sequencing.
Single colonies of E. coil Rossetta (DE3) strain containing protein expression vectors were inoculated were inoculated in 100 μL of LB supplemented with 100 μg/ml kanamycin and 25 μg/ml chloramphenicol in 96-well plates and grown at 37 °C for 12 h. Then, 2% of the E. coli culture volume was transferred to 500 μL of LB supplemented with 100 μg/ml kanamycin and 25 μg/ml chloramphenicol in a 1.5 mL microtube and grown to an optical density at OD600 of 0.5–0.6 at 37 °C and 220 rpm. Next, protein expression was induced overnight by the addition of 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and culturing at 16 °C and 220 rpm. After centrifugation of the cultures at 13,000 rpm and 4 °C for 5 min, the resulting cell pellets were resuspended in 100 μL of bacterial protein extraction reagent (Cell Biolabs Inc., San Diego, CA, USA), incubated for 15 min at room temperature, and centrifuged at 12,000×g for 10 min. The resulting supernatants were used as the crude enzyme.
For the acylation reactions of GPA, a typical reaction (30 μL) consists of GPA (100 μM), acyl donors (2 mM), BmmI, and MgCl2 (10 mM) in Tris-HCl buffer (50 mM, pH 8.5). The reaction mixtures were incubated at 30 °C for 2 h, and were quenched by the addition of ACN (30 μL), and the denatured protein was removed by centrifugation. The assays were monitored by HPLC analysis, using a C18, YMC pack ODS-AQ column (5 μm, 150 × 4.6 mm) with UV detection at 260 nm with a gradient program (0-5 min, 40% B; 5-25 min, 40 % to 80 % B; 25 - 26 min, 80 % to 100 % B; 26 - 31 min, 100% B; 31 - 32 min, 100 % to 40 % B; 32 - 37 min, 40% B;1 mL min-1).
The cytotoxicities were evaluated against human fibrosarcoma cells HT1080 which could express the enhanced green fluorescent protein (EGFP). HT1080-EGFP cells were seeded in 96-well plates at 8,000 cells/well. After 12 h, the reaction mixtures of negative control (without GPA), GPA without BmmI, and GPA with 20 μM BmmI (final concentration of the compounds were 0.39 μ M, 0.78 μ M, 1.56 μ M, 3.12 μ M, 6.25 μ M, 12.5 μ M and 25 μ M) were added to the cells and treated for 24h. After incubation for 24 h, the density and morphology of fluorescent cells were observed using fluorescence microscope.
Safety is one of the most noteworthy issues in experiments. Having a good experimental environment is a prerequisite for making the ideal experimental results. Therefore, our team, OUC-Marine Drugs, implements several laboratory criteria to ensure the experimental safety and has always been practicing.
All of our experiments are performed in BSL1 laboratory. Our laboratory is equipped with emergency medicine kits, emergency showers, eye baths and other emergency measures. We have daily safety checks and carry out regular comprehensive inspections. For waste treatment, we have a complete process. The waste will be disposed of or recycled according to corresponding regulations. For recyclables, we perform high temperature steam sterilization. Our lab has only a small stockpile of contaminants. Since we are exposed to dangerous chemicals, such as staining the gel with Gold View and protein electrophoresis with SDS and other substances, there are separate contaminated areas in the laboratory for contamination experiments.
We use E. coli DH5alpha and E. coli Rossetta (DE3) which are on the RRform white list.
Before entering the laboratory, all members learned and mastered the microbiological experiment operation methods and precautions with the help of the laboratory seniors, and successfully passed the laboratory safety test. During the experiment, all members are required to wear goggles, gloves and lab coats to operate. During the epidemic prevention and control period, we ensure that masks are worn throughout the test period.
