Construction and transformation of MET-expressed cassettes

MET and precursor ProMET genes from Apis mellifera were codon-optimized toward K. marxianus and synthesized (Twist Bioscience, USA), which sequences are shown in Fig. S1. Sequence design and optimization for the two constructions (pKLAC2-a-his-MET and pKLAC2-a-his-ProMET) were inserted in a plasmid (pKLAC2-a-) between NdeI and NotI restriction enzyme sites. K. marxianus BCRC 22057 was transformed with SacII-linearized plasmids and integrated into the lac4 promoter (Fig. 1A), following a previously reported protocol (Lin et al. 2017). The peptide expression of melittin in this study was named MET and promelittin was ProMET.

Fig. 1

MET and ProMET construction and Western blotting assays in a yeast culture supernatant. A Schematic representation of K. marxianuse expressing MET and ProMET; B Western blotting analysis of MET; C Western blotting analysis of ProMET. Lane 1, day 1; lane 2, day 2; lane 3, day 3; lane 4, day 4

Transformants were screened in a YPG medium supplemental with G418 (200 μg/mL, Sigma, USA), and the wild type was used as a control. Successfully engineered colonies were validated through PCR amplification of the integrated genes, using yeast colony DNA as a template with the universal S1274F, S1276R, and gene-specific primer pairs. Yeast colony DNA was obtained through treatment with the QuickExtract DNA Extraction Solution (Lucigen, USA). Subsequently, gene stability was confirmed after three generations to obtain the stable strain.

Western blotting

Western blotting analysis was performed following the previous study with modifications to evaluate MET and ProMET production (Lee et al. 2020). Briefly, 10 mL of the yeast supernatant was harvested after culturing in YPG for 1 to 4 days, and total proteins were precipitated by adding an equal volume of 20% trichloroacetic acid (Acros Organics, Belgium) on ice for 30 min. After centrifugation at 13,200 rpm for 15 min at 4 °C, 300 μL cold acetone (Macron Fine Chemicals, USA) was added, and this step was repeated thrice. The protein sample was dried and dissolved in 50 µL LDS sample buffer (M00676-250, GenScript, USA), which was loaded onto Tris–glycine SDS–polyacrylamide gel electrophoresis (SDS-PAGE, SurePAGE™ 4–12% BT, GenScript, USA) and run through electrophoresis with the Tris-MOPS-SDS running buffer (GenScript, USA). Following electrophoresis, the SDS-PAGE was transferred to the 100% methanol-activated FluoroTrans® W3.3 PVDF transfer membrane (Pall Corporation, USA) by the Western blot transfer system (GenScript/eBlot L1, USA). The PVDF membrane was blocked in 5% skim milk (Sigma, USA) at RT for 2 h and incubated with HRP-conjugated 6 × His antibody (Mouse McAb, ProteinTech, USA) at RT for 1 h to detect MET or incubated with GAPDH antibody (Rabbit Ab, GeneTex, USA) as an internal control. Lastly, the membrane was shaken and washed with phosphate-buffered saline with Tween 20 (PBST) at RT and imaged for detecting protein on an ImageQuant LAS 4000 system (General Electric, USA) with ECL™ Western blotting detection reagents (Amersham, UK).

Histidine affinity chromatography

The successful yeast transformant was cultured in 5 mL YPG medium supplemental with G418 (200 μg/mL) and sub-cultured to 100 mL YPG. After culturing for 2 days at 30 °C, the yeast supernatant was harvested and passed through gravity-flow columns (Bio-Rad, USA) packed by 600 µL nickel (Ni) beads (Thermo Scientific, USA) and reached equilibrium with 6 mL buffer A (50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 8.0). The sample was washed with 240 mL buffer A and eluted with 2.4 mL buffer B (50 mM Tris, 150 mM NaCl, 250 mM imidazole, pH 8.0). The purified sample was evaluated by SDS-PAGE and Western blotting.

Dialysis

First, 5 mL of purified sample was loaded onto Float-A-Lyzer G2 (Spectrum Laboratories Inc., USA) equipment, the floated dialysate of 1X PBS buffer was stirred at 125 rpm, and the dialysate was replaced after 2–4, 6–8, and 10–14 h. The dialyzed sample was collected after dialysis.

TEV protease treatment and desalting

The 5 mL dialyzed sample was treated with five units of TEV protease (T4455, Sigma-Aldrich, USA) and shaken at 70 rpm for 1 h at 30 °C to cleave the N-terminal histidine tag. After TEV protease treatment, the sample was purified by nickel (Ni) beads, as previously described, to remove TEV protease and collect the flow-through sample. The sample was desalted through a Sephadex G-25 Coarse PD-10 Desalting column (Cytiva, USA) and eluted by ddH2O to obtain purified MET and ProMET. The protein concentration was quantified with the Pierce™ Detergent Compatible Bradford Assay Kit (Bio-Rad, USA).

HPLC

The following samples were prepared and qualified through HPLC: (1) ddH2O, the control; (2) BV-A4-1, natural crude BV extract (kindly provided by Dr. Ming-Cheng Wu, Chung Hsing University, Department of Entomology); (3) BV-CITEQ (19C10, Citeq Biologics, Netherlands), commercial crude BV extract; (4) MET, purified MET from this study; (5) ProMET, purified ProMET from this study; and (6) MET-no TEV, TEV protease untreated MET. Samples were centrifuged for 5 min at 10,000 rpm to remove particles. Then, the supernatant was separated by a TSKgel G2000SWXL column (Tosoh Bioscience GmbH, Germany), and the SAPPHIRE 800 UV–VIS Variable Wavelength DETECTOR (Czechia) detected absorbance under 215 nm. HPLC was operated at 25 °C and a 0.5 mL/min flow rate with a 0.1 M potassium phosphate and 1 M NaCl (pH 6.5) mobile phase for 50 min. Data was analyzed by PEAK-ABC software.

Mass spectrum

The purified samples of MET and ProMET were first desalted using C18 ZipTip and resuspended in 0.1% formic acid (FA). They were then analyzed by ultra-performance liquid chromatography (UPLC, Dionex Ultimate 3000, Thermo Fisher Scientific) coupled with a quadrupole-time-of-flight mass spectrometer (Q-TOF MS, triple 6600, SCIEX). A C18 column (bioZen 2.6 µm Peptide XB-C18, Nano Column, 250 × 0.075 mm, Phenomenex) was used to separate the peptides, with 0.1% FA in DIH2O and 0.1% FA in acetonitrile selected as mobile phases A and B, respectively. The gradient was set as follows: 0–4.5 min, 5% B; 4.5–31 min, 5–35% B; 31–32 min, 35–90% B; 32–52 min, 90% B, 52–53 min, 90–5% B; and 53–70 min, 5% B. The scan mode was configured for data-dependent acquisition (DDA) with a scan range of 350–2000 m/z and 50–1900 m/z for MS1 and MS2, respectively. The MS data were analyzed by Mascot software with the database of MET and ProMET sequences.

Establishment of NSC-34 cell standard curve

The NSC-34 cell was produced by fusing neuroblastoma and motor neurons (kindly provided by Dr. Pei-Chien Tsai, National Chung Hsing University, Department of Life Sciences). Cells were harvested after 3 days of activation and resuspended in DMEM medium (Dulbecco’s Modified Eagle Medium, Thermo, USA) containing 10% FBS (Fetal Bovine Serum, Thermo, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. After counting the cells, 100 μL of 6.25 × 102, 1.25 × 103, 2.5 × 103, 2 × 104, and 4 × 104 cells were inoculated into a 96-well cell culture dish (Nunc™ MicroWell™ 96-Well, Thermo, USA), and 10 μL Cell Counting Kit-8 (CCK-8, TargetMol, USA) was added to measure cell viability. The plates were cultured at 37 °C with 5% CO2 for 1–4 h until the color turned orange. The PARADIGMTM Selection Platform system (Beckman Coulter, USA) detected each plate’s value at a 450 nm absorbance value (Bio-Rad, Hercules, CA, USA). Cell numbers and absorbance values were taken to establish a standard calibration line and calculate the actual cell number in cell growth rate and regeneration experiments.

Evaluation of the NSC-34 growth rate

This experiment was modified based on that of Jung et al. (Jung et al. 2015). Cells were harvested after 3 days of activation and resuspened in DMEM (Dulbecco’s Modified Eagle Medium, Thermo, USA) containing 10% FBS (Fetal Bovine Serum, Thermo, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. After counting the cells, 100 μL of 2 × 104 NSC-34 cells was inoculated into a 96-well cell culture dish (Nunc™ MicroWell™ 96-Well, Thermo, USA) and cultured at 37 °C with 5% CO2 for 1 day. Fifty microliters of test samples was treated on cells with triplicate, including the following: (1) ddH2O, the control; (2) BV-A4-1, natural crude BV extract (kindly provided by Dr. Ming-Cheng Wu, Chung Hsing University, Department of Entomology); (3) BV-CITEQ (19C10, Citeq Biologics, Netherlands), commercial crude BV extract; (4) M, purified MET from this study; and (5) P, purified ProMET from this study. After treatment, cells remained in culture at 37 °C with 5% CO2 for 0, 6, 12, and 24 h. Next, 15 μL of Cell Counting Kit-8 (CCK-8, TargetMol, USA) was added into each group at each time point to measure cell viability and incubated at 37 °C with 5% CO2 for 1–4 h. When the color turned orange, each plate was shaken for 1 min, and the PARADIGMTM Selection Platform system (Beckman Coulter, USA) was used to detect values at 450 nm absorbance (Bio-Rad, Hercules, CA, USA). The cell growth rate was calculated using the formula: 100% + ((time point − 0 h baseline)/0 h baseline) × 100%.

NSC-34 regeneration after H2O2 damage

This experiment was modified based on studies by Jung et al. (Jung et al. 2015) and Maier et al. (Maier et al. 2013). One hundred microliters of activated 2 × 104 NSC-34 cells were inoculated into a 96-well cell culture dish (Nunc™ MicroWell™ 96-Well, Thermo, USA) and cultured at 37 °C with 5% CO2 for 1 day. H2O2 was added to a final 50 μM concentration, where ddH2O was added as the non-H2O2-damaged control group (control) and incubated at 37 °C with 5% CO2 for 1 day. Fifty microliters of test samples was treated in triplicate, including the following treatment groups: (1) BV-A4-1, natural crude BV extract (kindly provided from Dr. Ming-Cheng Wu, Chung Hsing University, Department of Entomology); (2) BV-CITEQ (19C10, Citeq Biologics, Netherlands), commercial crude BV extract; (3) M, purified MET from this study; and (4) P, purified ProMET from this study. The cell viability was measured by Cell Counting Kit-8 (CCK-8, TargetMol, USA) as previously described. Cell regeneration fold change was calculated with the formula: treatment (time point − 0 h baseline)/control (time point − 0 h baseline).

Gene expression

RNA was extracted with the NucleoSpin RNA kit (Macheret-Nagel, Germany). Briefly, 1 mL of 2 × 105 NSC-34 was inoculated into a 3.5-mm culture dish (Thermo, USA), cultured at 37 °C with 5% CO2 for 1 day, and then treated with 500 μL of test samples for 24 h as previously described. After pouring off the medium, 350 μL buffer RA1 and 3.5 μL β-mercaptoethanol were added and scraped off the attached cells. The total cell solution was added to the NucleoSpin® Filter and purified with the NucleoSpin® RNA kit manual. The extracted RNA’s concentration and quality were measured by a Nano-100 full-wavelength micro-spectrophotometer (CLUBIO, Taiwan) and stored at − 80 °C.

cDNA synthesis was prepared with the SuperScriptTM IV First-Strand Synthesis System Kit (Thermo, USA). One microliter of 50 μM Oligo d(T)20 primer, 1 μL 10 mM dNTP mix, 4 μL 1 μg Template RNA, and 7 μL DEPC-treated water (a total volume of 13 μL) were mixed and maintained at 65 °C for 15 min in the PCR machine (SensoQuest, model: Labcycler Gradient, Germany) for first-strand cDNA synthesis. The RT reaction mixture, including 4 μL 5X SSIV Buffer, 1 μL 100 mM DTT, 1 μL ribonuclease inhibitor, 1 μL SuperScript™ IV reverse transcriptase (200 U/μL), and first-strand cDNA, was added (a total volume of 20 μL) and reacted at 55 °C for 30 min and at 80 °C for 10 min. Then, 1 μL of E. coli RNase H (2 U/μL) was added to react at 37 °C for 20 min.

The significantly expressed genes from a different treatment were assessed through qPCR. Briefly, the qPCR reaction solution was mixed as follows: 10 μL 2X iQ™ SYBR® Green Supermix (Bio-Rad, USA), 1 μL 10 μM cell growth gene marker primer pairs (Maier et al. 2013) (Table 1), 4 μL sterile water, and 4 μL of the previously prepared 50 ng/μL cDNA template. qPCR was performed by the qPCR Biometra/846–070-000 (Germany) program at 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min as one cycle. The real-time quantitative PCR reaction was conducted for 45 cycles. The expression level was calculated with the formula: 2−(ΔΔCt). ΔΔCt = (ΔCt treated) − (ΔCt untreated); ΔCt = (sample average Ct) − (β-actin Ct).

Table 1 Primers used for growth-related gene expressionsData analysis

The experimental and control groups differed significantly regarding cell viability and gene expression results. The groups were compared using a one-way analysis of variance (ANOVA) and the least-significant difference test in SPSS version 25.0 (IBM, Armonk NY, USA). p values of < 0.05 were considered significant.