Serratia marcescens strain was purchased from MTCC, Chandigarh with the strain number MTCC 8708. Taq DNA polymerase master mix RED was purchased from Ampliqon, Phusion High-Fidelity DNA Polymerase mix, Restriction enzymes, T4 DNA ligase was purchased from New England Biolabs (NEB). Luria- Bertani (LB) media, LB broth, Ampicillin, Kanamycin, Streptomycin Ni-NTA resin with the column was purchased from Hi-Media. IPTG was purchased from SRL chemicals. Primers are ordered from Shrimpex Biotech service Pvt. Ltd. PCR clean up kit were purchased from Smart prime ltd. Huminsulin 30/70 40 IU/mL cartridge was purchased from Eli Lilly Pvt Ltd.
S. marcescens obtained from MTCC was cultured on nutrient agar and incubated at 37 °C for 24 h. A single colony from the plate was inoculated into the sterile nutrient broth and incubated at 37°C for 24 h. coliTOP10 was inoculated in LB broth containing Streptomycin (50 μg/mL) from the glycerol stock culture. E.coliBL21 was plated on LB agar and incubated at 37 °C for 24 h. A single colony from the plate was inoculated into the sterile nutrient broth and incubated at 37 °C for 24 h. All the strains were maintained in LB broth for further research work.
Isolation of plasmid from E.coli using alkaline lysis method
pET-28 a (+) vector was isolated by inoculating the single colony in 100 mL of LB broth with kanamycin antibiotic and incubated at 37°C for 16h. Further, the cells were pelleted down by centrifuging at 12,000 rpm, 4°C, for 5mins. The supernatant were discarded and the pellets dissolved in 10mL of alkaline lysis buffer I containing 50mM Glucose, 25 mM Tris HCl, 10mM EDTA, pH 8.0 and vortexed vigorously. Then 8 mL of Alkaline lysis buffer II (0.2 NaOH, 1 % SDS) was added and tubes were inverted several times with incubation in ice for 3 min to mixed down the solution. Finally, 6mL Alkaline lysis buffer III was added to the solution and the tubes were inverted several times to mix it properly. Centrifuge at maximum speed for 20 min at 4°C, residues discarded. The supernatant were transferred to a fresh micro-centrifuge tube. Then an equal volume of ice-cold isopropanol was added and incubated at 4 °C for 30 min and centrifuged at maximum speed for 15 min. Then pellet was washed using 70 % ethanol. Then the supernatant was discarded, air-dried until the ethanol smells go off. Pellet dissolved in TE buffer pH8.0 stored at -20 °C. Then the isolated plasmid visualized in 0.8 % agarose gel. RNase A treatment given for 3h and isopropanol precipitation steps were repeated, visualized in 0.8% agarose gel .
Cloning of SP gene in pET- 28 a (+)
The SP gene was amplified from isolated genomic DNA using the forward and reverse primer (Table 1) with Nde I and Hind III restriction site respectively. The primers are designed by Primer 3 software using the SP gene sequence . These enzymes determined using the NEB Cutter online tool to identify which restriction sites were not present in the desired sequence. The restriction enzymes were high fidelity in nature. It has 100 % compatibility in cutsmart buffer so double digestion could be easily performed instead of halting the reaction after one digest and continuing the next set of digestion after the first set of reactions has completed. 1μg of purified insert and vector are used for setting up a restriction digestion reaction. Single digestion with each enzyme was also set for the vector to check whether the two restriction enzymes were working. The digested vector and insert are gel eluted to obtain gene without the restriction components to prevent further acts of restriction enzymes on the DNA samples. Then the purified vector and insert are ligated using the T4 DNA ligase enzyme. The restriction and ligation reaction is performed according to the NEB Cloning protocol. The cloned gene was transformed to E.coli TOP10 competent cells and then colony PCR was performed to confirm the recombinant vector transformation. Then plasmids from the positive colonies are isolated and then transformed to the E.coli BL 21 for expression of SP gene. Cloning was confirmed by colony PCR, and restriction digestion was performed to check the release of insert form vector.
Expression of SP enzyme
The positive colony with the desired gene of interest confirmed by colony PCR and re-digestion was inoculated in LB broth with kanamycin (50 μg/mL) incubated in shaking incubator for overnight at 37 °C. From the overnight grown culture, 1 % was transformed to new LB broth of 10mL and incubated at 37°C till it reaches the OD at 600 nm in the range of 0.4-0.6. Then IPTG (HiMedia) of 1mM final concentration was added to the culture and incubate at 37 °C for 4 h and 16 h. GFP protein expression was used as a positive control.
The positive colony with the desired gene of interest was inoculated in LB broth incubated in shaking incubator for overnight at 37 °C. From the overnight grown culture, 1 % was transformed to new LB broth of 10 mL and incubated at 37 °C till it reaches the 0.4-0.6 OD value at 600nm. Then IPTG of 1 mM final concentration was added to the culture and incubate at 37 °C for 4h . GFP protein expression was used as a positive control.
Ni Affinity Chromatography
After the sonication process, the crude extract of the recombinant protein was purified using Ni Affinity chromatography method. Therefore, only an N-terminal 6XHis-tag is on the recombinant protein which will bind to the Ni in the Ni affinity resin column. The Ni Affinity resin was passed through the column and allowed the resin to settle down in the column. Then crude extract was passed through the column and flow through was collected. Wash buffer with 20mM Imidazole was used to remove the loosely bounded protein. Finally, Elution buffer with 300mM of Imidazole was used to elute out the purified recombinant protein. Again the wash buffer was used to clean the column to remove thoroughly the residual proteins from the column. Then purified enzyme was subjected to different assays to check the activity .
The crude extract, flow-through, purified enzyme, induced cell extract; wild type strain cell extract was loaded in the SDS PAGE to check the molecular weight of the protein and to show the difference in protein expression and concentration . The separating gel was 12% and the stacking gel was 5 % was used to perform the SDS PAGE. The SDS gel was strained for 4h in a staining solution containing a pinch of Coomassie brilliant blue R -250, 10 % glacial acetic acid and 50 % Leishman stain, 40 % distilled water. SDS gel was destained for overnight on gel rocker in 60 % Leishman stain 30 % glacial acetic acid and 10 % water.
The protease activity of SP was performed using casein as a substrate as mentioned in Sigma’s Non-specific protease activity assay. Briefly, 5mL of casein solution was incubated at 37 ºC for 5 min and then 1mL of the enzyme to be tested is added to it and incubated at 37 ºC for 10 min. to stop the reaction, 5 mL of TCA is added and insoluble particles are filtered using syringe filter. Then 5 mL of sodium carbonate is added and 1mL of Folin’s reagent was added to the solution immediately and OD value was measured at 660 nm. The protein content of the enzyme was determined by the Bradford method .
In vitro insulin amyloid formation and degradation
Insulin amyloids were formed by incubating Huminsulin 30/70 40 IU/mL at 65 ºC for various time intervals from 2 h to 24 h. At each interval, SP was added to the insulin amyloid formed and incubated the mixture at 37 ºC for 1h. The formation of insulin amyloid and degradation of insulin amyloid by SP was confirmed by turbidity assay and FTIR analysis .
Insoluble protein aggregates can be estimated at 600nm. Hence the insoluble insulin amyloid fibrils can be detected at 600 nm using UV VIS spectrophotometer. 10μl of formed insulin amyloids at each time interval and the SP treated samples at each time interval were diluted with 1mL of PBS (10mM, pH 7.4) and the OD was measured at 600nm using a UV spectrophotometer.
The transformed colonies of E.coli BL21 with the recombinant plasmid was expressed using the 1 mM IPTG and then sonication was to isolate intracellular expressed enzyme and the recombinant enzyme was purified by Ni-NTA column. The purified sonicated sample and supernatant was loaded in the casein plate. The plate was incubated at 37°C for 16 hours and checked for the zone clearance at patched sites .
In vitro cytotoxicity
An in vitro cytotoxicity test method was performed for the given test sample as per ISO 10993:5. The culture medium from the PC-12 cells was replaced with fresh medium. Test sample in triplicates was added on the cells. After incubation at 37±1°C for 18h, MTT (mg/mL) was added in all the wells and incubated for 4 h. After incubation, DMSO was added in the wells and read at 570 nm using a photometer. Cytotoxicity and cell viability were calculated by the below formula. (Table 2)
Cytotoxicity = [(Control – Treated)/ Control] * 100
Cell viability= (Treated / Control) * 100