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

Summary

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.

Abstract

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’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.

Introduction

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 loss^1,

Protocol

1. Recombinant spider silk mixture preparation from lyophilized protein stocks

1. 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.
   1. 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.......

Discussion

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........

Materials

Name	Company	Catalog Number	Comments
3 mL Syringe with Luer-Lok Tip	BD	309657	Other size syringes can be used but to keep the tips on, it is advised to use luer-lok tips
4 mL culture vial, clear with rubber lined cap	Wheaton	225142	Minimum dope volume is 1mL, max is 2mL
8 mL culture vial, clear with rubber lined cap	Wheaton	225144	Minimum dope volume is 2mL, max is 4mL
99% Isopropyl Alcohol, Reagent ACS/USP Grade	Pharmco-Aaper	231000099
Freezone 4.5 Plus	Labconco	7386030	Freeze Dryer
Luer Adapter Female Luer x 10-32 Female, Tefzel (ETFE)	IDEX	P-629
Microwave	Magic Chef	HMD1110B	120V, 60Hz AC; 1000 watts; 1.1 cu. ft. capacity; with glass turn table
One-Piece Fingertight 10-32 Coned, for 1/16" OD	IDEX	F-120X
PEEK Tubing 1/16" OD x 0.010" ID	IDEX	1531B
Sprayer: Master Airbrush	Master Airbrush	TC-60