Silk protein solubility

Spider silk proteins from plants remain soluble at high temperatures, allowing them to be enriched by boiling [26]. In order to enrich the spider silk-ELP fusion protein, we therefore exposed tobacco leaf extracts to heat treatment at 95°C for 60 min and then cleared the supernatant by centrifugation. In further steps, the reversible precipitation behavior of ELP fusion proteins was exploited to develop a suitable purification strategy. For the selective precipitation of SOl-lOOxELP, NaCl was added at a final concentration of 2 M and the temperature was increased to 60 °C. In this man-... 

B. Silk Proteins Stability and Solubility in Solution Sol—Gel Transition. .. 25... 

Silk proteins (spidroins in spiders and fibroins in Lepidoptera insects) are assembled into well-defined nanofibrillar architectures (Craig and Riekel, 2002 Eby et al., 1999 Inoue et al., 2000b, 2001 Li et al., 1994 Putthanarat et al, 2000 Vollrath et al., 1996). Spidroins and fibroins are largely constructed from two chemically distinct repetitive motifs or blocks (Table I), an insoluble crystalline block and a soluble less-crystalline block (Craig, 2003 Fedic et al., 2002 Hayashi and Lewis, 2000 Hayashi et al., 1999). The crystalline blocks are composed of short side-chained amino acids in highly repetitive sequences that give rise to /1-sheet structures. 

Nevertheless, despite our rapidly increasing knowledge about silk protein sequences, very little is known about the part played by gene design (Tatham and Shewry, 2000) in the stability and solubility of the prespun silk and in the final property of silk fibers across a wider variety of spiders and insects. 

Fig. 3. Solubility of silk proteins in solution as a function of time. Low solubility corresponds to protein aggregation. The fast and slow aggregations are observed in vitro (Dicko et al., 2004a), whereas the stable helical conformation (storage structure) is observed in vivo (Dicko et al., 2004b,d). This illustrates the inherent instability of silk protein in solution and shows the /(-sheet polymorph structure as the most stable form. In other words, the spiders actively control and modulate the unavoidable silk protein aggregation prior to fiber formation.

Protein materials such as hair and silk are soluble in the cold oxychloride, but cellulose is not affected. 

Whether we discuss silk, proteins embedded in membranes, or soluble complexes of cytosolic proteins, we must ask questions about interactions. A first step is to identify interactions720-730 among proteins either in vitro or in living cells.731 Proteomic methods, which include the yeast two-hybrid method (Box 29-F), are widely used for this purpose. It is possible to identify large sets of interacting proteins, to identify disease states, to observe effects of drugs, and to compare metabolism among species. 

Yeast and bacterial systems often give low levels of expression of silks, and this has led to the development of production systems in tobacco and potato. Scheller et al. (2001) have shown that spider silk proteins can be produced in transgenic plants. They inserted synthetic spider silk protein (spidroin) genes into transgenic plants under the control of the CaMV35S promoter. Using this system they were able to demonstrate the accumulation of recombinant silk proteins to a level of at least 2% of total soluble protein in the endoplasmic reticulum of tobacco leaves, and potato tubers. 

Huemmerich, D., Helsen, C.W., Quedzuweit, S., Oschmann, J., Rudolph, R., and Scheibel, T. "Primary structure elements of spider dragline silks and their contribution to protein solubility". Biochemistry 43(42), 13604—13612 (2004). 

Nexia is already working with the US army to develop bulletproof vests and surgical suture material from spider silk. The team produced soluble recombinant spider silk proteins with molecular masses of 60-140 kDa (Lazaris ef al., 2002). They were able to wet spin the silk monofilaments derived from a concentrated aqueous solution of soluble recombinant spider silk protein under conditions of low shear and coagulation. The spun fibers were water-insoluble with diameters ranging from 10 to 40 pm and exhibited toughness and modulus values comparable to those of native dragline silks but had lower tenacity. They anticipate that the manufacturing processes for these products would be more environmental friendly than the production of conventional plastics. 

Synthetic spider silk has also been produced in transgenic tobacco and potato expressing the endogenous silk protein genes of the spider Nephila davipes [147]. Proteins of up to 100 kDa in size, and with 90% identity to silk protein, were produced in tobacco leaves, potato leaves and potato tubers at up to 2% of the total soluble protein accumulation level [151]. 

D. Coleman, F.O. Howitt, Studies on silk proteins. I. The properties and constitution of fibroin. The conversion of fibroin into a water-soluble form and its bearing on the phenomenon of denaturation, Proc. R. Soc. Med. 134 (1947) 544. 

Our research group has reported in a series of papers that production of enzymes from PLLA degrading microorganisms was induced when some proteins such as silk fibroin, elastin, gelatin, peptides, and some amino acids were present in the cultivation medium [27, 44—46, 48]. For instance, a significant increase in the enzyme activity of Amycolatopsis strain HT-41 was detected when silk fibroin was added into the culture medium [44]. Moreover, various amino acids, peptides, and poly(L-amino acids) were used as enzyme inducers for the production of enzymes from T. albtm, L. waywandensis, and A. orientalis. Silk fibroin was found to be the best inducer for A. orientalis. On the other hand, the enzyme activities of L. waywandensis and T. album were stimulated by elastin but not by silk fibroin. Soluble culture substrates such as peptides and amino acids had lower induction activities in comparison to protein substrates (silk fibroin, elastin, collagen, and keratin) [48]. 

Liebmann, B., Hummerich, D., Scheibel, T., and Fehr, M. 2008. Formulation of poorly water soluble substances using self-assembling spider silk protein. Colloids Surf. A Physicochem. Eng. Aspects, 331, 126-132. 

Secondary Structure. The silkworm cocoon and spider dragline silks are characterized as an antiparaHel P-pleated sheet wherein the polymer chain axis is parallel to the fiber axis. Other silks are known to form a-hehcal (bees, wasps, ants) or cross- P-sheet (many insects) stmctures. The cross-P-sheets are characterized by a polymer chain axis perpendicular to the fiber axis and a higher serine content. Most silks assume a range of different secondary stmctures during processing from soluble protein in the glands to insoluble spun fibers. 

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