Chang Seok Ki

Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications.

Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme

Silk fibroin (SF) nanofibers were prepared by electrospinning and their application as an enzyme immobilization support was attempted. By varying the concentration of SF dope solution the diameter of SF nanofiber was controlled. The SF nanofiber web had high capacity of enzyme loading, which reached to 5.6 wt%. The activity of immobilizedα-chymotrypsin (CT) on SF nanofiber was 8 times higher than that on silk fiber and it increased as the fiber diameter decreased. Sample SF8 (ca. 205 nm fiber diameter) has excellent stability at 25°C by retaining more than 90 % of initial activity after 24 hours, while sample SF11 (ca. 320 nm fiber diameter) shows higher stability in ethanol, retaining more than 45% of initial activity. The formation of multipoint attachment between enzyme and support might increase the stability of enzyme. From these results, it is expected that the electrospun SF nanofibers can be used as an excellent support for enzyme immobilization.

Molecular weight distribution and solution properties of silk fibroins with different dissolution conditions

Four regenerated silk fibroin (SF) samples were prepared under different dissolution conditions and their molecular weight (MW) distributions and solution properties in water and formic acid were examined. SFL, produced by dissolving in LiBr aqueous solution for 6h, showed the highest MW level. In the three SFC samples, produced by dissolving SF in CaCl(2)/H(2)O/EtOH solution for dissolution times ranging from 3 to 180 min, the MW of the SFs decreased with increasing dissolution time and a new band appeared at low MW. Interestingly, SFL presented as a relatively transparent aqueous solution with 10-30 nm particle size, whereas the three SFC samples exhibited a turbid solution with 100-300 nm particle size. SF formic acid solutions showed a higher viscosity than SF aqueous solutions and exhibited almost Newtonian fluid behavior, whereas SF aqueous solutions displayed abrupt shear thinning in the low shear rate region (0.1-3 s(-1)).

Multi-biofunction of antimicrobial peptide-immobilized silk fibroin nanofiber membrane: Implications for wound healing

UNLABELLED An antimicrobial peptide motif (Cys-KR12) originating from human cathelicidin peptide (LL37) was immobilized onto electrospun SF nanofiber membranes using EDC/NHS and thiol-maleimide click chemistry to confer the various bioactivities of LL37 onto the membrane for wound care purposes. Surface characterizations revealed that the immobilization density of Cys-KR12 on SF nanofibers could be precisely controlled with a high reaction yield. The Cys-KR12-immobilized SF nanofiber membrane exhibited antimicrobial activity against four pathogenic bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa) without biofilm formation on the membrane surface. It also facilitated the proliferation of keratinocytes and fibroblasts and promoted the differentiation of keratinocytes with enhanced cell-cell attachment. In addition, immobilized Cys-KR12 significantly suppressed the LPS-induced TNF-α expression of monocytes (Raw264.7) cultured on the membrane. These results suggest that a Cys-KR12-immobilized SF nanofiber membrane, which has multiple biological activities, would be a promising candidate as a wound dressing material. STATEMENT OF SIGNIFICANCE This research article reports various bioactivities of an antimicrobial peptide on electrospun silk fibroin nanofiber membrane. Recently, human cathelicidin peptide LL37 has been extensively explored as an alternative antibiotic material. It has not only a great antimicrobial activity but also a wide variety of bioactivities which can facilitate wound healing process. Especially, many studies on immobilization of LL37 or its analogues have shown the immobilization technique could improve performance of wound dressing materials or tissue culture matrices. Nevertheless, so far studies have only focused on the bactericidal effect of immobilized peptide on material surface. On the other hand, we tried to evaluate multi-biofunction of immobilized antimicrobial peptide Cys-KR12, which is the shortest peptide motif as an analogue of LL37. We fabricated silk fibroin nanofiber membrane as a model wound dressing by electrospinning and immobilized the antimicrobial peptide. As a result, we confirmed that the immobilized peptide can play multi-role in wound healing process, such as antimicrobial activity, facilitation of cell proliferation and keratinocyte differentiation, and inhibition of inflammatory cytokine expression. These findings have not been reported and can give an inspiration in wound-care application.

Effect of degumming condition on the solution properties and electrospinnablity of regenerated silk solution.

The application of silk on tissue engineering scaffolds has been studied intensively because silk has an electrospinning technique using a good blood compatibility, excellent cytocompatibility and biodegradability. Silk consists of two polymers, fibroin and sericin. In spite of importance of sericin, most studies were focused on the fibroin only and the effect of residual sericin on the electrospinning performance of silk has not been considered. In this study, regenerated silk with different residual sericin contents was prepared by controlling the degumming conditions. The effects of the degumming conditions on the solution properties and electrospinning performance of silk were examined. The fast protein liquid chromatography (FPLC) measurements confirmed that the molecular weight of the regenerated silk decreased slightly with increasing residual sericin content. More molecular aggregation of silk occurred with increasing sericin content, resulting in an increase in the solution turbidity of formic acid. All silk formic acid solutions exhibited almost Newtonian fluid behavior and the viscosity increased with increasing sericin content. Interestingly, the dope solution viscosity of silk increased remarkably at sericin contents <1% (or degumming ratio >25%) leading to significant improvements in electrospinnability and an increase in the fiber diameter of the silk web.

Silk Protein as a Fascinating Biomedical Polymer: Structural Fundamentals and Applications

Silk is a textile material, as well as one of the oldest biomaterials. However, the recent progress of biomedical science and technology has led to the replacement of silk by various biomaterials based on synthetic polymers. Despite the wide variety of biomaterials available, these materials suffer certain limitations that prevent them from meeting the various demands of the medical field. Therefore, silk continues to attract considerable interest as a promising biomaterial. This paper explains the fundamentals of silk protein, and reviews the many applications of silk biomedical polymers.

Crosslinking reaction of phenolic side chains in silk fibroin by tyrosinase

Tyrosinase oxidizes the tyrosyl residues in silk fibroin (SF) with oxygen, resulting in the production ofo-quinone residues. Subsequently, the inter- or intramolecular crosslinks are formed by reaction with amino groups in through nonenzymatic process. The measurement of oxygen consumption proved that the tyrosyl residues in SF were mostly oxidized to quinone residues by tyrosinase. The reaction mechanisms were proposed in this study and the crosslinking reaction ofo-quinone residues and the enzymatic oxidation of tyrosyl residues could be confirmed by the measurements of UV,1H-NMR and GFC.

Preparation of Silk Sericin Beads Using LiCl/DMSO Solvent and Their Potential as a Drug Carrier for Oral Administration

Silk sericin (SS) was fabricated into beads using LiCl/DMSO solution as a solvent. Up to 30 % (w/v) of SS could be dissolved within 3 hours, and the shape of solidified SS depends on the concentration of SS. Ethanol was the best coagulant among alcohols, making beads with suitable mechanical strength for further application. SS beads swell more at a pH above the isoelectric point (pl) than below the pl. The pH and the presence of an enzyme greatly affect the dissolution rate of SS beads. Whereas only 10 % of SS beads were dissolved at pH 2.2 in the presence of pepsin, more than 45 % of SS beads were dissolved at pH 7.4 in the presence of trypsin. The release of drug was suppressed in a stomach-like environment while promoted in an intestine-like environment.

Metal ion adsorbability of electrospun wool keratose/silk fibroin blend nanofiber mats

In this study, electrospun wool keratose (WK)/silk fibroin (SF) blend nanofiber was prepared and evaluated as a heavy metal ion adsorbent which can be used in water purification field. The WK, which was a soluble fraction of oxidized wool keratin fiber, was blended with SF in formic acid. The electrospinnability was greatly improved with an increase of SF content. The structure and properties of WK/SF blend nanofibers were investigated by SEM, FTIR, DMTA and tensile test. Among various WK/SF blend ratios, 50/50 blend nanofiber showed an excellent mechanical property. It might be due to some physical interaction between SF and WK molecules although FTIR result did not show any evidence of molecular miscibility. As a result of metal ion adsorption test, WK/SF blend nanofiber mats exhibited high Cu2+ adsorption capacity compared with ordinary wool sliver at pH 8.5. It might be due to large specific surface area of nanofiber mat as well as numerous functional groups of WK. Consequently, the WK/SF blend nanofiber mats can be a promising candidate as metal ion adsorption filter.

Silk Fibroin/Chitosan Conjugate Crosslinked by Tyrosinase

Two biopolymers, silk fibroin (SF) and chitosan, were conjugated by tyrosinase (EC 1.14.18.1), a polyphenolic oxidase, to improve their physicochemical properties, such as their thermal properties and morphological stabilities in organic solvents. The crosslinking between SF and chitosan took place mainly through Michael addition reactions. A main reaction between the amino groups in chitosan ando-quinone, the oxidation product of the tyrosyl residue in SF, was confirmed by UV spectroscopy. Measurements of viscosity and light scattering indicated that the crosslinked SF/chitosan conjugate was compact: it had a smaller particle size because of tight bonding forces between the SF and chitosan molecular chains. Thermal decomposition of SF/chitosan conjugates crosslinked by tyrosinase occurred at higher temperatures. The adhesiveness of the SF/chitosan conjugates decreased steadily as the crosslinking reaction progressed. We propose that this new crosslinking method be used for the preparation of silk fibroin/chitosan conjugates using tyrosinase. We expect that SF/chitosan conjugates crosslinked by tyrosinase can be used preferentially in biomedical applications because of its unique properties and non-toxicity.