Putting enzymes on pause
Such effects could render fungal cells non-viable, and if the resistance-conferring mutations are widespread in the clinical study population, they should slow the development of resistance. Such drugs might also exhibit a longer duration of clinical efficacy. It is for these reasons that my research team has concluded that Cdc14 is the best target protein to inhibit. Developing targeted antifungal treatments such as Cdc14 inhibitors could transform agricultural resilience, especially for staple crops in regions facing food insecurity. By reducing fungal infections, this approach may alleviate food shortages, increase crop yields, and help meet the nutritional needs of a growing global population.
METHODOLOGY
Recombinant E. Coli production. A plasmid E. coli BL21 containing a histidine tag, a gene for ampicillin resistance, and the gene coding for the Cdc14 protein in A. alternata was mixed into 50μL of Escherichia coli. Next, it was incubated on ice for 30 minutes before being placed in a 42 °C water bath for 1 minute, which led to the transformation . The solution was put on ice for 2 minutes and then incubated for an additional 30 minutes in a 37 °C water bath. Afterwards, the incubated E. Coli colonies were centrifuged to create a pellet. An amount of 400 μL of the supernatant was removed, and then the pellet was resuspended in 2× YT. The recombinant E. coli colony was spread out on an agar plate containing 2xYT medium and incubated overnight at 37 °C . Large-scale recombinant protein expression. 5 mL of E. coli culture was added to 2xYT medium with 100 μg/mL ampicillin. To measure the optical density at 600 nm (OD600), 1 ml of culture was taken. The solution was then placed in shaking incubators at 37°C with rotation at 220 rpm. OD600 was periodically measured until absorbance values of 0.7-1.0 AU were reached, after approximately three hours, to ensure that the E. coli cells were in the mid-log phase of growth. Finally, to induce Cdc14 expression, L-arabinose was added to a final concentration of 1 mM, and the culture was incubated overnight at 37 °C in the incubator. The solution, which contained 2xYT medium, E. coli, and L-arabinose, was centrifuged at 5,000 rpm for 30 minutes. During the first round of centrifuging, the absorbance of the medium at 600 nm was recorded. Then, the cells were spun down in a microcentrifuge for 2 minutes at 6,000 rpm. The supernatant was discarded, and the cell pellet was resuspended in 4xSDS sample dye. Heat shock was administered by heating the sample at 95°C for 5 minutes in a heat block. After the final centrifugation, the supernatant was discarded, and the pellet was frozen. Lysis of E. Coli pellet. The thawed E. Coli pellet was then resuspended in a solution of 30 mL lysis buffer, lysozyme to 1 mg/ml, leupeptin to 10 µM, 4 μL of universal nuclease, and lepstatin to 1 µM. This solution was then left on ice for 30 minutes before phenylmethylsulfonyl fluoride (PMSF) was added to a concentration of 0.5 mM. Next, the mixture was sonicated for 5 minutes on ice using a probe sonicator with a flow rate of 5 mL/min to degrade the cell membrane further, and then centrifuged at 35,000g and 4 °C for 30 minutes. The supernatant (containing Cdc14) was then removed, and the pellet was discarded. Preparation of solutions for enzyme purification. Lysis buffer contained 25 mM HEPES, 500 mM NaCl, 0.1% (v/v) Triton X-100, 10 mM imidazole, and 10% (v/v) glycerol. Nickel buffer A contained 25 mM HEPES, 500 mM NaCl, and 10% (v/v) glycerol. Nickel buffer B contained 25 mM HEPES, 500 mM NaCl, 250 mM imidazole, and 10% (v/v) glycerol.
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