Name: material formation of recombinant spider silks through aqueous solvation using heat and pressure
Uploaded: 2019-05-06T12:30:00
Description: Watch this Scientific Journal Video about Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure at JoVE.com

The authors would like to gratefully acknowledge funding from the Utah Science and Technology Research (USTAR) initiative.

1. Recombinant spider silk mixture preparation from lyophilized protein stocks
<ol>
	<li>Determine the necessary formulation and volume required for the intended material formations. Typical formulations range from 3% (w/v) up to 15% (w/v). Using this selection, calculate the appropriate rSSp, concentrations, and ratios.
	<ol>
		<li>Use the following respective formulations to prepare each material described in this protocol: hydrogels/sponges/lyogels, 6% (w/v) 50:50 MaSp1:MaSp2; films/coatings, 5% (w/v) 80:20 MaSp1:Ma...

<ol>
	<li>Huemmerich, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Structure+Elements+of+Spider+Dragline+Silks+and+Their+Contribution+to+Protein+Solubility.">Primary Structure Elements of Spider Dragline Silks and Their Contribution to Protein Solubility.</a> Biochemistry. 43 (42), 13604-13612 (2004).</li><li>Schacht, K., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Hydrogel+Formation+of+a+Recombinant+Spider+Silk+Protein.">Controlled Hydrogel Formation of a Recombinant Spider Silk Protein.</a> Biomacromolecules. 12 (7), 2488-2495 (2011).</li><li>Jones, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+Than+Just+Fibers:+An+Aqueous+Method+for+the+Production+of+Innovative+Recombinant+Spider+Silk+Protein+Materials.">More Than Just Fibers: An Aqueous Method for the Production of Innovative Recombinant Spider Silk Protein Materials.</a> Biomacromolecules. 16 (4), 1418-1425 (2015).</li><li>Tucker, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+Physical+Properties+of+Recombinant+Spider+Silk+Films+Using+Organic+and+Aqueous+Solvents.">Mechanical and Physical Properties of Recombinant Spider Silk Films Using Organic and Aqueous Solvents.</a> Biomacromolecules. 15 (8), 3158-3170 (2014).</li><li>Harris, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sticky+Situation:+An+Investigation+of+Robust+Aqueous-Based+Recombinant+Spider+Silk+Protein+Coatings+and+Adhesives.">A Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives.</a> Biomacromolecules. 17 (11), 3761-3772 (2016).</li><li>Jones, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+Heat+and+Pressure+for+Solubilization+of+Recombinant+Spider+Silk+Proteins+in+Aqueous+Solution.">Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.</a> International Journal of Molecular Sciences. 17 (11), 1955 (2016).</li><li>Copeland, C. G., Bell, B. E., Christensen, C. D., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Process+for+the+Spinning+of+Synthetic+Spider+Silk.">Development of a Process for the Spinning of Synthetic Spider Silk.</a> ACS Biomaterials Science and Engineering. 1 (7), 557-584 (2015).</li><li>Arcidiacono, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+Processing+and+Fiber+Spinning+of+Recombinant+Spider+Silks.">Aqueous Processing and Fiber Spinning of Recombinant Spider Silks.</a> Macromolecules. 35 (4), 1262-1266 (2002).</li><li>Work, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Major+Ampullate+Silk+Fiber+Formation+by+Orb-Web-Spinning+Spiders.">Mechanisms of Major Ampullate Silk Fiber Formation by Orb-Web-Spinning Spiders.</a> Transactions of the American Microscopical Society. 96 (2), 170-189 (1977).</li><li>Decker, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Destruction+of+Endotoxin+in+Synthetic+Spider+Silk+Proteins.">Method for the Destruction of Endotoxin in Synthetic Spider Silk Proteins.</a> Scientific Reports. 8 (12166), 1-6 (2018).</li></ol>

After recombinant spider silk proteins are purified they have to then be prepared in a solution that can be used for material formation. By mixing lyophilized spider silk protein with water and exposing this mixture to microwave irradiation, to generate heat and pressure, it possible to prepare a rSSp solution. A wide variety of material forms can be produced from this simple and efficient method of rSSp solubilization. Each material has to be uniquely prepared and processed to achieve the desired outcome and properties....

Spider silks have garnered the interest of material scientists for their impressive combination of strength, elasticity, and biocompatibility. Recreating fibers has traditionally been the thrust of the research. This effort was hampered by the recombinant spider silk protein (rSSp) insolubility in water as well as the inability of traditional solvation techniques (chaotropic agents and detergents) to achieve aqueous solvation. Further, techniques that have been developed for solvating versions of rSSp do not work on all rSSp variants and also require substantial manipulation and time that often results in protein loss1,2. This has largely resulted in the field utilizing 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as a solvent from which to form fibers, and other limited material forms. The advantage being that all known rSSp are soluble in HFIP, providing data uniformity between each research group. The disadvantage is that HFIP is a toxic solvent that is expensive and impractical to scale due to health concerns and environmental considerations.
A novel approach to rSSp solvation was developed that bridged the technological gap between the harsh organic solvent HFIP and other techniques that selectively worked for rSSp solvation. The combination of specific heats and pressures was applied to suspensions of rSSp and water. The results were near 100% solvation and recovery of the rSSp, as well as high protein concentrations; a variety of materials forms were determined to be possible from these formulations that were not all achievable using HFIP or other organic solvents3,4,5,6. The objective of this approach is to efficiently and easily solubilize purified and dried recombinant spider proteins in an aqueous solution that can then be utilized for the production of a variety of material forms.
Fibers, films, coatings, adhesives, hydrogels, lyogels, microspheres, and sponge materials are all readily accomplishable from the same aqueous rSSp solution using this method. The continued evolution of this method, not only with additional rSSp but with other proteins, could lead to new material forms and alternative protein purification and solubilization avenues.

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			<td height="63" style="height:63px;width:216px;">3 mL Syringe with Luer-Lok Tip</td>
			<td style="width:148px;">BD</td>
			<td style="width:119px;">309657</td>
			<td style="width:215px;">Other size syringes can be used but to keep the tips on, it is advised to use luer-lok tips</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4 mL culture vial, clear with rubber lined cap</td>
			<td style="width:148px;">Wheaton</td>
			<td style="width:119px;">225142</td>
			<td style="width:215px;">Minimum dope volume is 1mL, max is 2mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">8 mL culture vial, clear with rubber lined cap</td>
			<td style="width:148px;">Wheaton</td>
			<td style="width:119px;">225144</td>
			<td style="width:215px;">Minimum dope volume is 2mL, max is 4mL</td>
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			<td height="42" style="height:42px;width:216px;">99% Isopropyl Alcohol, Reagent ACS/USP Grade</td>
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			<td height="21" style="height:21px;width:216px;">Freezone 4.5 Plus</td>
			<td style="width:148px;">Labconco</td>
			<td style="width:119px;">7386030</td>
			<td>Freeze Dryer</td>
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			<td height="42" style="height:42px;width:216px;">Luer Adapter Female Luer x 10-32 Female, Tefzel (ETFE)</td>
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			<td style="width:119px;">P-629</td>
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			<td height="60" style="height:60px;width:216px;">Microwave</td>
			<td style="width:148px;">Magic Chef</td>
			<td style="width:119px;">HMD1110B</td>
			<td style="width:215px;">120V, 60Hz AC; 1000 watts; 1.1 cu. ft. capacity; with glass turn table</td>
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			<td height="42" style="height:42px;width:216px;">One-Piece Fingertight 10-32 Coned, for 1/16&#34; OD</td>
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			<td style="width:119px;">F-120X</td>
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			<td height="42" style="height:42px;width:216px;">PEEK Tubing 1/16&#34; OD x 0.010&#34; ID</td>
			<td style="width:148px;">IDEX</td>
			<td style="width:119px;">1531B</td>
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			<td height="40" style="height:40px;width:216px;">Sprayer: Master Airbrush</td>
			<td style="width:148px;">Master Airbrush</td>
			<td style="width:119px;">TC-60</td>
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</table>

material formation of recombinant spider silks through aqueous solvation using heat and pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

From the described method of solubilization of rSSp, a variety of material forms can be achieved as seen in Figure 1. The method of solubilization is to apply heat and pressure, generated by a conventional microwave, to a suspension of rSSp and water. When critical temperatures and pressures are achieved, the protein will solubilize. From this solubilized rSSp solution, the required conditions are presented for seven material forms: hydrogels, lyogels, sponge...

Watch this Scientific Journal Video about Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure at JoVE.com

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

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