Biologic Biomaterials: Silk - Biomaterials - page 159

prepare chimaeric/hybrid silk-like proteins incorporating silkworm silk-like or spider silk-like sequences
to enhance protein solubility, improve biomineralization/cell adhesion, and related needs [24].
7.2.1 Silkworm Silk ( B. mori )
B. mori silkworm farming and use in textiles and in broader studies has facilitated an understanding
of the composite protein structure and more recently the broader potential for biomedical applications.
Structurally, B. mori silk fibroin fibers consist of two proteins: a light chain (~26 kDa) and a heavy chain
(~390 kDa) which are present in a 1:1 stoichiometric ratio and linked by a single disulfide bond [25].
These proteins are coated with the family of hydrophilic proteins called sericins (20-310 kDa) [2,25,26].
The disulfide linkage between the Cys-c20 (20th residue from the carboxyl terminus) of the heavy chain
and the Cys-172 of the light chain holds the fibroin together and a 25 kDa glycoprotein, named P25, has
been reported to be noncovalently linked to these proteins [27]. Silk fibroin can be highly purified from
sericins by boiling the silk cocoons in an alkaline solution, sometimes with a surfactant to improve the
purification process. Twenty to thirty percent of the silk cocoon mass is sericin, which is removed dur-
ing this alkali degumming (sericin removal) process.
7.2.2 B. mori Silk Fibroin Structure
Silk fibroin from B. mori consists primarily of glycine (Gly) (43%), alanine (Ala) (30%), and serine (Ser)
(12%) [2]. The heavy chain of the protein consists of 12 domains that form the crystalline (β-sheet) regions
in the silk fibers, which are interspersed with nonrepetitive and less-organized domains (noncrystalline)
in the proteins. The crystalline domains in the fibers consist of Gly-X repeats, with X being most often
Ala, followed by Ser, threonine (Thr), and valine (Val) [28]. The crystalline-forming silk domains have an
average of 381 residues (596 in size in the seventh domain to 36 in the 12th domain). Each domain con-
sists of hexapeptide subdomains: GAGAGS, GAGAGY, GAGAGA, or GAGYGA, where G is glycine, A is
alanine, S is serine, and Y is tyrosine. These subdomains end with tetrapeptides such as GAAS or GAGS
[25,28,29]. The less crystalline regions of the fibroin heavy chain are known as linkers or spacers and are
reported to have an identical 25 nonrepetitive amino acid residue sequence, not found in the crystalline
regions [28]. The primary sequence for the fibroin results in a hydrophobic protein with a natural coblock
polymer design, a design feature found in all silkworm and spider silks [9,12]. A number of silk poly-
morphs have been reported, including the glandular state prior to crystallization (silk I), the spun silk
state which consists of the β-sheet secondary structure (silk II), and an air/water-assembled interfacial
silk (silk III, with a helical structure) [2,9,30]. The silk I structure is known to be water-soluble and upon
exposure to heat or physical spinning, easily converts into a silk II structure. The silk I structure is also
observed in vitro in aqueous conditions and converts into a β-sheet structure when exposed to methanol
or certain salts [31]. The β-sheet structures are asymmetric with one side occupied with hydrogen side
chains from glycine and the other occupied with the methyl side chains from the alanines that populate
the hydrophobic domains. The β-sheets are arranged so that the methyl groups and hydrogen groups of
opposing sheets interact to form the intersheet stacking in the crystals. Strong hydrogen bonds and van
der Waals forces generate a structure that is thermodynamically stable [2], to the point that high pressure
and temperature during autoclaving do not significantly impact the structure. The inter- and intra-chain
hydrogen bonds form between-amino acids perpendicular to the axis of the chains and the fiber [2]. The
silk II structure excludes water and therefore is slow to degrade via hydrolysis or proteolytic activity.
7.3 Processing Silk Proteins
Natural silkworm silk fibers require little processing for use as textile materials (e.g., dyeing [32-34],
chemical modification to render them water proof prior [35,36]). However, in order to prepare alter-
native material morphologies with silk (e.g., films, foams, hydrogels, fibers, spheres) or composite