SDS-PAGE for Silk Fibroin Protein

Background

SDS-PAGE for protein or polypeptide is one of the most classic, basic and commonly used experimental methods for analyzing the molecular masses of protein subunits (Laemmli, 1970). Therefore, it is generally not difficult to obtain a good SDS-PAGE electrophoretic profile with clear bands for most proteins.

However, for researchers involved in electrophoresis experiments with silk protein, it seems that it is not easy to obtain a good SDS-PAGE profile with clear band of light chain, a subunit of silk fibroin. This is especially true for beginners or students who do not have a long experience with electrophoresis. Where is the problem? The main difficulty is not related to the SDS-PAGE technique itself but is related to the preparation of liquid silk fibroin from silk fiber, as well as the unique properties of the silk fibroin itself.

Silk protein is a general term for silk fibroin and sericin. Two parallel monofilaments spun by matured larvae of silkworm Bombyx mori are composed of 65%-75% fibroin, 20%-30% sericin, and 5% wax, pigments, sugars, and other impurities. Silk fibroin is a crystalline polymer-based fiber surrounded by several layers of the gum sericin protein. The outermost layer of sericin is easily solubilized in hot or boiling water. The innermost layer of sericin closest to the fibroin fiber is hardly solubilized in boiling water (Wang and Zhang, 2011). All layered sericins are easily solubilized in alkaline hot or boiling water. Therefore, boiling and degumming (removing sericin) in a 0.1%-0.5% Na₂CO₃ solution have been used most frequently in the laboratory. However, it not only causes a large amount of sericin degradation and hydrolysis but also leads to a decrease in its mechanical properties (Wang and Zhang, 2013). These layered sericins can also be solubilized in 8 M urea buffer at 80 °C (Yamada et al., 2001), aqueous neutral soap (Yuksek et al., 2012) and surfactant solution (Wang and Zhang, 2017) under repeated treatments. In general, the process of removing sericin from the surface of silk fibers is called as degumming. The use of these solvents for degumming treatments hardly results in a decrease of mechanical properties. But the overall degumming efficiency is very low and the repetition of treatments for more than 3 times barely remove all the sericin.

The degummed fibroin fibers are about 10-25 μm in diameter and consist mainly of a 391-kDa heavy chain (H) (Zhou et al., 2000) and a 26-kDa light chain (Yamaguchi et al., 1989), which are present in a 1:1 ratio and linked by a single disulfide bond (Tanaka et al., 1999a). In addition, a 25 kDa glycoprotein, named P₂₅, is non-covalently linked to these proteins (Tanaka et al., 1999b). The structure of fibroin is primarily attributed to its composition of only 3 amino acids organized in a repeating 6-residue sequence of (Gly-Ala-Gly-Ala-Gly-Ser)n. The fibroin in natural silk fiber is a semi-crystalline macromolecule in which the polypeptide chains are strongly held together by hydrogen bonds in an anti-parallel arrangement to form β-sheets which result in crystalline regions (Silk II), while the random coils and α-helix chains form the amorphous regions (Silk I).

Silk fibroin fiber is processed into an aqueous silk fibroin solution via a series of processing, degumming, dissolution, purification and concentration steps that are often referred to as silk regeneration. The resulting liquid silk fibroin is often referred to as regenerated liquid silk. The regenerated liquid silk is very unstable in aqueous solution and its molecular structure changes easily from Silk I into Silk II form due to environmental factors including physics and chemistry, such as temperature, pH, UV radiation, organic solvents, ion strength, stress and ultrasonic treatment. Due to the structural transition of silk protein, the regenerated liquid silk can be easily made into various forms of silk biomaterials, such as micro- or nano-particles (Zhang et al., 2007), regenerated fibers (Matsumoto and Uejima,1996), artificial skin(Jin et al., 2005), porous matrix or 3D scaffolds (Mandal and Kundu, 2009), biomimetic nanofibrous scaffolds (Park et al., 2006), and a platform for transistors (Capelli et al., 2011) and various classes of photonic devices (Kim et al., 2013), due to its biocompatibility.

The structure and properties of the final forms of silk biomaterials depend evidently on the molecular size of the regenerated liquid silk which is affected by a series of processing, degumming, and dissolution steps. The purification, concentration, and storage conditions easily induce regenerated liquid silk gelling or coagulating and denaturing. Therefore, determination of the molecular mass of the protein by SDS-PAGE is a necessary step before processing silk biomaterials.