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CELL CULTURE'S SPIDER SILK ROAD
 
Jeffrey Perkel
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Even after locating the webs, Rising ran into trouble. “We struggled to catch them because they are so fast. They kept running down into this burrow, and then it was impossible to get hold of them.”

Eventually, the team found their solution: When the spider was in the web, a team member would shove a knife into the burrow, blocking the spider's retreat, at which point “you could walk up to it and capture it in a jar. So, that was really easy, once you knew how to do it.”

In the end, Rising collected about 100 specimens, from which she and her team isolated the major ampullate gland that produces dragline silk. From there, they produced cDNA libraries and cloned parts of the spider's major ampullate spindroin 1 gene, MaSp1.

Today, Rising has no spiders in her lab, save those that wander in from the outside. She sacrificed her specimens shortly after collecting them. E. australis, she says, are “very beautiful,” with “striped legs and a yellow abdomen and white head. I think they have eight eyes.” But, are they poisonous? “I didn't know then and I still don't know. I was never bitten, and nobody else [was], either.”

Not all silk is equal

Every spider silk protein is a little different, but most contain N- and C-terminal domains flanking as many as 100 alanine- and glycine-rich repeats. E. australis MaSp1 is about 10 kb long, but coding sequences can extend out to 15 kb or longer. The size and repetitive nature of the structure makes it difficult to manipulate and express intact silk proteins in transgenic organisms—bacteria will excise parts of the gene, for instance, and the proteins can aggregate—so researchers typically develop smaller custom protein designs.





Lewis says his lab has designed some 36 distinct silk protein variants ranging in size from 3 to 8 kb, each with unique material properties. Most of those are expressed in (and codon-optimized for) bacteria, but two are made in goat's milk, from which he can collect 15–20 g per week, and some are expressed in alfalfa (hay), which could yield nearly 220 kg per acre. Lewis also has expressed spider silk proteins in transgenic silkworms. Recombinant silk containing just 5% spider protein, he says, is already 50% to 70% stronger, and 40% more elastic, than silkworm silk alone.

Scheibel's lab has made more than 40 silk variants, all of which they produce in bacteria. His “workhorse,” called eADF4(C16), is a 50 kD protein comprising 16 repeats (but neither terminal domain) of the Araneus diadematus ADF4 protein, an MaSp1 homolog.

Rising's variant, called 4RepCT, was developed under the guidance of principal investigator Jan Johansson, and comprises just the four C-terminal repeats of MaSp1 plus the C-terminal domain. “What the team did was to try to find the shor test protein that we could produce in E. coli and that, after purification, formed macroscopic fibers,” Rising explains.

But being macroscopic does not make the fibers silk, exactly. 4RepCT, for instance, has only about 10% of the tensile strength and one-sixth the elasticity of natural silk.

Of course, biochemists also have different needs than spiders, so in the end it may not matter. Spiders need a fiber strong enough and elastic enough to catch an insect in flight. They need the material to be stable enough to be stored for extended periods as a liquid in their glands, and yet primed to form fibers quickly enough to catch them as they fall. And they have to be able to do all that at room temperature and pressure, more or less. Researchers only need something approximating the properties of the biological material they are studying, they can afford to wait hours for polymerization to occur, and they can prepare their materials under a variety of conditions.

Unlike spiders, researchers can also produce silk proteins in any form they want. Lewis spent $100,000 on a custom-built apparatus that extrudes spider silk through a 5 micron needle— the resulting shear force converts the material from liquid to fiber—and then stretches and spins the material into something that looks like kitchen twine, yet is pure white and surprisingly soft. But he and others can also create foams, like the milk in a latte, transparent films, hydrogels, and other formats. “We have seen [that] pancreatic cell islets maintain their specific islet architecture and stay viable for longer times when cultured on a soft and porous material compared to a flat and rigid surface,” says My Hedhammar, Associate Professor at the Swedish University of Agricultural Sciences and R&D Director of Spiber Technologies, a company she cofounded with Rising and others to commercialize spider silks.

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