Preparation of purified silk fibroin protein solution from Bombyx mori silk cocoons
A Collaboration with Eszter Ozsvald
This project found inspiration from previous research on silk protein by Fiorenzo Omenetto and a paper; Regenerated Silk Fibroin from B. mori SilkCocoon for Tissue Engineering Applications, by M. K. Sah and K. Pramani which we based our protein preparation on.
The foundation of our project is rooted in the possibility of inserting fibroin-producing genes from B. mori into maize (corn) as a means of producing larger quantities of the protein for commercial applications. The previously mentioned research has focused primarily on the application of silk fibroin films as a bio-compatible substrate for tissue engineering particularly for epicortical electrode arrays where the substrate could be bioresorbable, —broken down by the body leaving only the electrodes in place.
Due the constraints of our facilities this portion of our project will have to remain a theoretical proposal and will be outlined in detail in Part I.
Preparation of silk fibroin solution:
(Process adapted from M.K. Sah and Pramanik, 2010)
- Bombyx mori silk cocoons from Mulberry Farms
- Lithium Bromide – ReagentPlus ( ≥ 99% ) from Sigma-Aldrich
- Sodium Carbonate – ReagentPlus ( ≥ 99% ) from Sigma-Aldrich
- Non-living pupa were extracted from the silk cocoons providing 10g of unprocessed silk cocoon material.
- Cocoons were cut into smaller pieces (approx. 1 x 1cm)
- Cocoon pieces were “de-gummed” by washing them in a 0.02M solution of sodium carbonate at 96°C and stirring continuously for 20 minutes. During this process the cocoon fibers became reconstituted into a larger mass of fiber.
- The mass of fiber was then washed with distilled water repeatedly to remove the sticky sericin protein surrounding the structural fibroin protein.
- The mass of fiber was then dried in an oven for 30 minutes at 60°C and then dried completely with continuous air flow at 65°C. At this stage the dry mass of fiber was weighed at 6g indicating a total loss of 4g or 40% from the degumming / washing stage.
- A silk fibroin solution was prepared by dissolving the de-gummed silk fiber in a highly concentrated 9.5M solution of lithium bromide at 72°C for 3 hours. The process of joint solubilization of proteins and polymers in LiBr solutions is fully described in this paper by Lev Bromberg.
- To remove the lithium bromide the fibroin solution was dialyzed with 1 inch dialysis tubing (molecular weight cutoff of 12,000 to 14,000 MW) against deionized water for 3 days, changing water every 8 hours.
- Finally the solution was centrifuged at 9000rpm for 20 minutes*
- The supernatant was decanted and the solution stored at 3°C for later use.
*With the generous help of Michael Long and his lab at NYU’s Smilow Neuroscience Program, we were able to borrow time on his centrifuge.
Stay Tuned for Part III – (Experimental Applications of Silk Fibroin Protein)