Recombinant Production And Purification of Hydrophobin SC16 From Escherichia coli And Monitoring of Its Self-Assembly Using Fluorescence Assays

Our research is focused on optimizing the recombinant production of hydrophobins and using them for applications such as biocompatible films and coatings. This protocol supports hydrophobin research for industrial coatings, emulsifiers, and environmental applications, including microplastic capture and remediation systems. To begin, transform SHuffle T7 Express Escherichia coli cells by heat shock at 42 degrees Celsius for 45 seconds using a pET-21 expression plasmid encoding H-GB1-SC16.

Inoculate two milliliters of lisogeny broth containing 100 micrograms per milliliter of ampicillin with a single colony and incubate overnight at 37 degrees Celsius with shaking. Add one milliliter of the overnight culture to one liter of lisogeny broth containing 100 microgram per milliliter ampicillin in a 2.8-liter baffled round bottom flask. Grow the culture at 37 degrees Celsius until the optical density at 600 nanometers reaches approximately 0.6.

Add isopropyl beta-D-1 thiogalactopyranoside to a final concentration of 0.5 millimolar. Incubate overnight at 20 degrees Celsius while shaking. The next day, harvest the cells by centrifuging at 4, 000 g for 20 minutes and pour off the supernatant.

Re-suspend the cell pellet in 30 milliliters of lysis buffer containing 20 millimolar tris(hydroxymethyl)aminomethane at pH 8 and 250 millimolar sodium chloride. Incubate the suspension in an 80 degrees Celsius water bath for 15 minutes and mix gently every five minutes during incubation. Centrifuge the cell lysate at 25, 000 g for 20 minutes at 4 degrees Celsius.

Load the supernatant onto a gravity column containing two to three milliliters of nickel ion-charged, immobilized metal affinity chromatography resin. Wash the column with lysis buffer containing 30 millimolar imidazole. Then elute protein using 10 milliliters of lysis buffer containing 300 millimolar imidazole.

Dialyze the eluded fraction using 3, 500 kilodalton molecular weight cutoff dialysis tubing against one liter of buffer containing tris and sodium chloride for three hours while stirring at 100 RPM. Add thrombin protease at 20 units per milligram fusion protein directly to the dialysis bag. After replacing the buffer, continue dialysis overnight at 4 degrees Celsius.

Reload the dialyzed sample onto the nickel ion-charged, immobilized metal affinity chromatography column. Wash with buffer containing 30 millimolar imidazole and elute with buffer containing 300 millimolar imidazole. Analyze collected fractions by SDS page with beta mercaptoethanol in the loading buffer.

Check for a band at approximately 10 kilodaltons to identify SC16. Repeat dialysis of the wash fraction against one liter of tris buffer for three hours. Measure the absorbance at 280 nanometers to quantify SC16.

Prepare triplicate reaction mixtures containing the following components. Prepare the stock buffer ahead of time and add the protein and sodium chloride immediately before the assay. Transfer the mixtures into two to five milliliter tubes.

Rotate the tubes end over end at 20 revolutions per minute for two hours, ensuring proper mixing upon inversion. Then transfer 100 microliters of each sample into an opaque bottomed black 96-well plate. Add filtered thioflavin T to a final concentration of 20 micromolar to all samples.

Set the microplate reader to the appropriate excitation and emission wavelengths for thioflavin T fluorescence with a gain of 55 and an integration time of 20 microseconds. Measure and record the fluorescence intensity for each sample. For kinetic analysis, adjust the pH of the stock buffer to 5.5, 6.5, 7.5, or 8.5.

Then prepare 100 microliter reaction mixtures in triplicate in a black 96-well plate by adding SC16 and thioflavin T to the stock buffer as described previously. Place the plate in the microplate reader. Set excitation and emission wavelengths to 438 and 494 nanometers respectively.

Program cyclic measurements every 70 seconds with 60 seconds orbital shaking at 432 revolutions per minute followed by 10 seconds rest. Then set integration time to 20 microseconds and gain to 55. Run for 120 cycles.

In the first immobilized metal affinity chromatography a band at approximately 18 kilodaltons corresponding to H-GB1-SC16 was observed before thrombin cleavage. After thrombin cleavage, no protein was observed in the second immobilized metal afinity chromatography flow through fraction. After thrombin cleavage, a band at approximately 10 kilodaltons corresponding to SC16 was observed in the wash fraction.

In the second immobilized metal afinity chromatography residual SC16 at approximately 10 kilodaltons and a smaller band at approximately eight kilodaltons corresponding to H-GB1 were observed. Successful purification of SC16 was confirmed by the presence of the expected molecular weight band and separation from the H-GB1 fusion partner following thrombin cleavage. SC16 samples showed increasing fluorescence intensity over time under agitation across all tested pH conditions.

The kinetic self-assembly assay produced suboptimal results when linear shaking was used instead of orbital shaking. The thioflavin T fluorescence intensity increased 11-fold after two hours of end-over-end rotation in SC16 samples compared to non-rotated protein-containing controls. This protocol allow researcher to learn how to purify hydrophobin in high yield and do functional assays to make sure it's self-assembled.

Following this protocol, contact-angle assays can be used to monitor surface hydrophobicity after hydrophobin self-assembly on various materials. Future studies will engineer specific hydrophobins with tailored properties for industrial applications.