This method of solubilizing recombinant spider silks produces material forms that are not possible using traditional harsh organic solvents. Further, the process is grain, resulting in a simple protein solution in water. By utilizing this method, the materials formed or produced maintain the desirable characteristics of spider silk proteins.
This solubilization technique for recombinant spider silks allows for the solubilization of recombinant spider silks in a one step process. This is desirable because these notoriously difficult to produce proteins aren't lost through extensive processing. Further, since the solutions are just protein in water, other biologically active compounds can be added if that's desirable for the application.
Particularly, the protein structure function relationship, this system is memetic to how a spider makes a fiber in that it utilizes water and proteins at a high concentration. By using this solubilization method and a subsequent material formation, it should be possible to gain understanding into the materials that are formed, and the structures that are responsible for them. It is thought that it can be, we've used this technique for solubilizing synthetic peptides that otherwise would require an alkaline or acidic environment to solvate.
That can be advantageous in downstream applications for those peptides. Furthermore, a number of known proteins when expressed synthetically tend to end up in the insoluble fraction. Resolubilizing, and then refolding these proteins is often a laborious process that results in large losses.
By utilizing this technique, it might be possible to increase the efficiency of recovery processes for insoluble proteins. Placing a sealed vial in a microwave is a bit daunting giving the risk of overheating and thus over pressurizing the vial. Understanding the staged approach to applying heat and pressure is critical.
Second, for the recombinant spider silk in particular, having salt present will not allow the technique to work or work as effectively depending on how much salt is present. Salt removal is key. Be patient and careful, closely monitor your microwave times and temperatures inside the vial.
It is critical because there are a number of moving parts to operation that are often difficult to convey through traditional written materials and method sections. Given that there are many proteins that have solubility problems which prevent adequate study, this method has potential to be applied across a variety of protein spaces to improve, or even allow analytical techniques and characterization. To begin, select a clean and new eight milliliter autoclaveable borosilicate glass culture vial with a rubber lined screw cap, and place the empty vial on an analytical balance.
Tear the mass of the empty vial so that the balance reads zero mass. Add the desired lyophilized recombinant spider silk protein powder to the empty vial for the specific material. Then, add the desired amount of ultra pure water, at least two milliliters, to the vial.
Seal the vial cap, and briskly vortex the contents to create a dispersed and homogenous recombinant spider silk protein mixture. Perform a final check of the vial cap to ensure that it has been firmly and securely tightened. Then, transfer the suspended recombinant spider silk protein mixture to a conventional microwave oven with the power range of 700 to 1500 watts.
Begin operation of the microwave with five second bursts set full power by manually switching on and off. After each burst, briefly open the door, and carefully mix the vial to prevent settling, and maintain the suspended mixture. Occasionally allow the vial and solution to cool, and prevent the superheated solution from touching the seal.
Use an infrared thermometer to measure the temperature of the solution containing portion of the vial. Repeat the microwave process until the temperature reaches at least 130 degrees Celsius, and all of the solid particulates have been completely dissolved. Then, allow the temperature of the solution and the vial cap to cool below 100 degrees Celsius.
Prior to this solution being fully cooled, cast it from the vial into specific geometries to form a hydrogel. After the hydrogel is formed, place it in a water bath, and transfer it to the freezer at minus 20 degrees Celsius. Wait until the bath is frozen completely.
Complete the sponge formation process by removing the frozen hydrogel and water bath from the freezer, and thawing at 25 degrees Celsius. Then, remove the resulting sponge from the thawed water. To prepare a lyogel, transfer a frozen hydrogel sample to a lyophilizer.
After 24 hours, remove the final lyophilized gel material from the vessel. To produce films of recombinant spider silk protein, cast 200 microliters of the hot solubilized recombinant spider silk protein from the vial onto a PDMS form of the desired shape. After it is dried, peel it off the PDMS substrate for testing or treatment.
To prepare a coating that cannot be removed from the substrate, use an airbrush sprayer to apply the solubilized recombinant spider silk protein to perform an initial spray coating on the substrate of choice. After it is dried, submerge the coated substrate into the solubilized recombinant spider silk protein to form a dip coating. Repeat the dip coating to achieve the desired thickness.
To form adhesives, use a pipette to add the solubilized recombinant spider silk protein onto a substrate, and then apply a second substrate over the top of the solution. Firmly clamp the pieces together, and then dry the samples in an oven with a minimal temperature of 25 degrees Celsius for at least 16 hours. To generate wet spun fibers, use an 19 gauge glide needle, and load the solubilized dope solution into a concentric syringe with a Luer lock tip.
Eject air bubbles, and let the dope sit at the Luer lock end of the syringe. Insert at least 25 millimeters of PEEK tubing into the PEEK tubing's one piece finger tight fittings for one over 16 inch outer diameter, and 10 over 32 comb. Replace the 19 gauge needle with this setup on the Luer lock female adapter of the syringe.
Then, place nitrile gloves on the outside of the intermediate godets, to keep the fiber to be generated from slipping, and to avoid damaging the motors. Get a tall clear glass bath with 99%pure isopropanol to use as a coagulation bath. Fill the first stretch bath with an 80 to 20 ratio of isopropanol and distilled water.
In the second stretch bath, fill a 20 to 80 ratio of isopropanol and distilled water. Set up the godet stretch system on the computer. To adjust the main speed of the fiber removal, adjust the sliding bar for godet triplet A speed to a value between 10 to 14 millimeters per second, depending on how well and quickly the fiber is forming.
Initiate a first stretch by moving the sliding bar of the godet triple B stretch ratio to two For the final godet in stretch bath one, The middle upper godet, and the first godet in stretch bath two. Initiate the second stretch by moving the sliding bar of the godet triple C stretch ratio to two for the final godet in stretch bath two, the last upper godet, and the winder. This setup ensures that the first godet after the coagulation bath, and the first godet in the first stretch bath are rotating at the same speed.
Next, load the silk solution in the syringe of a custom spin line instrument. In the automated system, set the extrusion rate to 10 millimeters per second, in order to extrude the silk solution into the glass coagulation bath filled with isopropanol. Allow the fiber extrusion to become uniform, before pulling the fibers out of the bath with a thin metal hook.
Verify that removing the fiber from the bath creates a loop between the PEEK tubing tip and the path of the fiber leaving the bath. Guide the retrieved fiber through the series of godets, such that the fiber is submerged in the stretch baths, but drying in the air between the stretch baths, and before going onto a spool. In this protocol through a solubilization of recombinant spider silk protein, a variety of material forms can be achieved.
Seven material forms are presented here. Hydrogels, lyogels, sponge, adhesives, coatings, films, and fibers. Fibers require the most extensive processing by extruding into a coagulation bath, and then serially stretching the raw fiber in post bin stretch baths.
Given that the protein and water solutions are heated to a relatively high temperature and pressure, our experience has been is that the solutions are sterile at the point the protein is solvated. This allows any of the material forms presented here to be taken to cell culture, as long as they're handled appropriately, to study the cellular response to the materials. Certainly this technique has led to the discovery of new material forms, including adhesives and sponge material.
Material forms that are not necessarily orientated towards fiber formation, although fiber formation is one of the areas that has also seen improvement through the development of this technique. Generating heat and pressure inside of a sealed vial has inherent danger. Always wear personal protective equipment when performing these procedures.
