When it comes to producing revolutionary new polymers, forget about vast
factories belching out fumes and toxic waste. The latest developments in
materials manufacturing involve vats of genetically engineered bacteria.
Chemists have learnt over the past few years how to equip these organisms with
genes that have been specially tailored to turn out designer proteins. The
materials have a huge range of potential uses. Chemists have already produced
a polymer that can serve as an adhesive for living tissue, and made 鈥渟mart鈥�
plastics that respond to changes in their environment. The future might even
hold a nonstick fried egg.
The key to this infant industry is the ability of bacteria to turn out
complex proteins with absolute precision. Proteins are long-chain molecules
made up of amino acids joined to each other by links known as peptide bonds.
Some of these natural proteins have properties that are a match for any
synthetic polymer. Spider silk is a protein that forms fibres stronger than
those made from the high-tech material Kevlar. Keratin, the main fibrous
component of hair, skin and claw, is a protein, too. And so is elastin, the
tough, elastic component of ligaments, arteries and lungs. The molecules of
these structural proteins are composed of quite short sequences of amino acids
that repeat again and again along the polymer chains. These repeat sequences 鈥�
involving up to 20 different types of amino-acid unit 鈥� determine way the
chains fold up, and this in turn is responsible for the bulk properties of the
substance. For instance, keratin is so strong because it contains helical
protein chains that twist around each other in a hierarchical way: strands
twist into superstrands, and superstrands into clusters and bundles to form
exceptionally strong, rope-like fibres.
The sequence of amino acid units in a natural protein is defined by the DNA
molecules in the cells that produce it 鈥� specifically by the sequence of
chemical groups called nucleotides strung along the DNA chain. In effect the
DNA supplies a template on which the protein is put together, one amino acid
at a time. 快猫短视频s now have ways of tapping into, and altering, this
template by snipping open the double-helical strands of a DNA molecule, and
replacing one nucleotide coding unit with another or splicing prefabricated
sequences 鈥� built from scratch using chemical methods 鈥� into the chain. They
know, too, how to multiply the products of genetic manipulation to obtain
plentiful supplies of any stretch of DNA.
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Armed with this knowledge researchers have been exploring ways of inserting
the blueprints for artificial structural proteins into bacterial DNA. It
doesn鈥檛 matter much to a bacterium whether an artificial gene in its DNA codes
for a protein like one that the bacterium produces naturally or one that is
entirely different: if its in the genome, the bacterium will make it. In the
late 1980s, Joseph Cappello and his coworkers at Protein Polymer Technologies
(PPTI) in San Diego, California, pioneered the techniques that are now
generally used for making artificial proteins. The first step is to make an
artificial DNA sequence that encodes one repeat unit of the protein polymer
the researchers are aiming for. These stretches of synthetic DNA are generally
made by attaching nucleotides one by one to build up a chain anchored to a
solid support. Once these DNA sequences have been synthesised they can be
replicated in large numbers using the polymerase chain reaction (PCR). They
are then joined end to end to produce the synthetic 鈥済ene鈥� that will encode
the polymer. The linking process is helped along by the enzymes, called
ligation enzymes, that do this job in nature.
The next step is to insert the artificial gene into bacterial DNA. Whereas
the DNA of multicellular organisms is packaged into chromosomes and sits in a
cellular nucleus, a bacterium鈥檚 DNA takes the form of several double-stranded
rings that float about freely in the cell. As well as a main ring, bacteria
have smaller rings of DNA called plasmids. It is these that are used as the
vehicles for the artificial genes. Using enzymes it is possible to snip open a
plasmid and stitch in the artificial gene. When the plasmids are put back
inside the bacteria, the cell鈥檚 molecular machinery then goes about its
business of translating the DNA blueprint embodied in the plasmid into
individual protein molecules 鈥� only now within the blueprint is a plan for a
designer protein specified by the altered DNA. It is hard to imagine a
cleaner, greener way of making materials. The bacteria are cultured in water
at around body temperature and fed on a diet of amino acids. No potentially
toxic organic solvents are needed, and the very nature of the process requires
conditions in the fermentation vessels that support life.
Sticking point
In 1990 the PPTI team used this technique to persuade the common gut
bacterium Escherichia coli to make an artificial silk-like protein. Cappello
and his colleagues then aimed for a more ambitious target: a designer protein
that would combine the strength of their artificial silk with the useful
properties of a biological protein called fibronectin, which helps cells to
stick together in the body. The researchers hoped to come up with a way to
make cells grown in tissue culture stick to normal human cells. Artificially
grown tissue is already proving valuable in promoting wound healing, and it
might eventually be possible to grow entire organs this way to replace damaged
or malfunctioning ones.
Existing methods for making human cells stick to synthetic substrates such
as cultured tissue cells use either natural cell-adhesion proteins extracted
from blood or from animals, or purely synthetic peptides which contain certain
key amino acid sequences. But there are difficulties with both approaches:
natural products are much stickier than the artificial ones, but they are also
much less stable. The PPTI team hoped to make a protein that combined the
stability of artificial agents with the stickiness of natural ones. To do
this, they devised an artificial protein in which silk-like structural blocks
of six amino acids appeared repeatedly along the chain, interrupted every
ninth block by a single fibronectin-like block of 16 amino acids. This entire
sequence was itself repeated 13 times to form the complete protein molecule
containing more than 900 amino acid units. To polymer chemists this is a
large, complicated structure, and it would be extraordinarily difficult to
reproduce it exactly by standard chemical synthesis. But compared with the
complex proteins that bacteria make routinely, the structure is pretty
simple.
Perhaps too simple. For reasons not yet understand, cells do not take
kindly to repetitive DNA sequences in their genomes. The cell鈥檚 molecular
machinery has a tendency to rearrange such sequences by cutting and pasting
the DNA, thereby scrambling the repetitive structure. So Cappello鈥檚 team took
advantage of the fact that the genetic code has a degree of redundancy:
because there are only 20 types of amino acid in natural proteins but 64
possible three-nucleotide combinations of the four nucleotides that encode
them, each amino acid can be represented by more than one triplet. So by
varying the triplet combination that represents a given amino acid, it is
possible to generate repetitive amino acid sequences from a DNA sequence that
is not itself quite as repetitive.
Home and dry
After mastering tricks like this, and by providing the bacteria with
various 鈥減romoter鈥� molecules to enhance the efficiency with which they convert
the DNA to proteins, Cappello and his colleagues were able to obtain their
artificial protein as a dry powder that was about 85 per cent pure. The PPTI
researchers found that a coating of their synthetic protein would indeed
enable cells to become attached to a variety of substrates, sometimes giving
better results than fibronectin itself. The structural blocks, meanwhile,
ensured that the protein stayed stable in temperatures at which the natural
proteins would lose their activity. The protein is now marketed commercially
by PPTI as an adhesive for attaching mammalian cells to tissue cultures, under
the name ProNectin F.
Other research groups have since used this approach to make a variety of
synthetic proteins with structures similar to those of natural fibrous
proteins. Last year, David Kaplan at the US Army鈥檚 Natick Research,
Development and Engineering Center in Massachusetts used bacteria to express
artificial genes based on the ones that the orb-weaving spider uses to make
its silk. From this it might be possible to make silk-like materials much more
plentiful, as they could be grown in fermentation vats rather than being
harvested from the spiders themselves. Spider silk is extremely strong, and
the possible applications for artificial materials with silk-like properties
range from aircraft engineering to reinforcement for bullet-proof vests.
Dan Urry at the University of Alabama, meanwhile, has used the bacterial
method to make synthetic proteins similar to elastin. The molecules of the
natural protein have a coiled structure, and their spring-like behaviour makes
the material elastic. But this structure changes with temperature: as the
protein is warmed, water is stripped from around the chains and they collapse
into a dense, sticky mass. Urry has made artificial polypeptides built up from
the five-unit aminoacid sequence valine-proline-glycine-valine-glycine 鈥� the
pattern that is characteristic of elastin. He found that, like elastin, these
artificial peptides change their structure at certain temperatures. When
heated, the loosely coiled chains switch to a more ordered, more tightly
coiled state, in which much of the water surrounding the chain is squeezed
out; this causes sheets of the material to contract. As the peptide chains
coil up they exert a substantial force 鈥� some of Urry鈥檚 proteins can lift more
than 1000 times their dry weight as they contract. The temperature at which
the conformation of the chains switches depends on their precise amino acid
composition, so by tinkering with the genes that make the protein Urry can
vary the synthetic polymer鈥檚 properties. He believes that his synthetic
elastin-like polymers might prove useful for wound repair. More prosaically,
there is the possibility of producing a super-absorbent material for
disposable nappies.
One apparent limitation of the bacterial method is that it is restricted to
just the 20 amino acid building blocks found in nature. Or is it? David
Tirrell of the University of Massachusetts at Amherst is interested in using
unnatural fluorinated amino acids to form polypeptide chains that might have
properties similar to polytetrafluoroethylene (PTFE), the basis of the
nonstick coating Teflon. Chemical groups containing fluorine are usually keen
to avoid water. So Tirrell reasoned that if a polypeptide could be made with a
backbone of water-soluble units interspersed with fluorinated amino acids, it
might fold up into a shape in which the fluorinated groups sat on the outside,
yielding a kind of nonstick protein.
To put this plan into practice, however, requires a touch of bacterial
husbandry as well as genetic engineering, because most bacteria will turn up
their noses at fluorine-containing compounds. Tirrell鈥檚 answer has been to
breed a new strain of bacteria that could use the fluorinated amino acids for
protein synthesis and that would not synthesise their own unfluorinated amino
acids instead. The carrier the researchers chose for the fluorine atoms was
phenylalanine, which has a benzene ring sidegroup to which fluorine atoms can
be attached. They starved the bacterial culture of phenylalanine and fed it
the fluorinated variant instead. Sure enough, the resulting polymer turned out
to be insoluble in water, suggesting that the fluorinated groups did indeed
migrate to the surface of the folded polymer.
While he was pondering the possible uses for such a material, Tirrell
discovered on his desk one day a mischievous suggestion from his graduate
students. The note showed a picture of a fried breakfast in the making: an egg
frying in a nonstick pan. Next to it was a picture of a breakfast of the
future 鈥� which was being made in an old iron frying pan, because the egg
itself was nonstick. Its protein component, the albumin that makes up most of
egg white, had been fluorinated.
Although Tirrel doesn鈥檛 expect to be making non-stick fried eggs in the
near future, he sees huge opportunities in this new-found ability to redirect
the protein-synthesising abilities of bacteria. He imagines tagging natural
proteins with a synthetic polypeptide chain, by inserting the DNA sequence for
an artificial polypeptide right next to the sequence for the natural protein
in the bacterial plasmid. For example, a protein might be given an artificial
side chain that will fold up into a crystalline film (Tirrell鈥檚 group has
already built synthetic proteins that will do this). Then such hybrid proteins
might spontaneously form thin films on a substrate, which would immobilise
them for use in devices such as bioreactors and biosensors. Or a protein might
be equipped with an optically or electrically active substituent, made up of
suitably modified, non-natural amino acids, that could make it possible to
control the protein鈥檚 function with light or electricity.
Tirrell鈥檚 researchers have already made a synthetic protein containing 3-
thienylalanine in place of the natural amino acid alanine. The sulphur-
containing thienyl group was used because it is the building block of
polythiophene, a polymer that conducts electricity. Tirrell aims to link up
the thienyl groups protruding from a film of this synthetic protein to make a
biocompatible conducting material for possible use in biomedical devices.
Ultimately, the researchers hope that the worlds of synthetic and natural
materials will start to merge, as they find ways to extract the best from both
of them.
Plastics with a life of their own
Bacteria have already chalked up a success as a source of polymers on a
commercial scale. The British biotechnology company Zeneca, a subsidiary of
ICI, manufacturers and markets a biodegradable polymer called Biopol, which is
synthesised in fermentation vats by a bacterium Alcaligenes eutrophus. Biopol
is used in moulded plastic items such as bottles and for the controlled
release of drugs.
The bacteria are fed a diet of propionic acid and glucose, which they turn
into a polyester. This acts as an energy reserve for the bacteria and is
stored as granules inside the cell, just as our own cells store fat. When
extracted from the cells and collected, it provides a flexible material
similar to polypropylene 鈥� except that it is fully biodegradable into harmless
compounds. ICI is now using A. eutrophus and other bacteria to develop a range
of different biodegradable plastics.
While researchers can exercise some control over the nature of these
plastics by altering the chemicals in the feedstock, the synthetic procedure
within the cells is dictated by the bacterial enzymes and cannot easily be
changed. Furthermore, the products will always be something of a mxied bag,
with slight variations in the detailed molecular structure, since the bacteria
are concerned only with producing an energy store, and don鈥檛 need to be
perfectionist about the precise structure of the polymer chain.