Scientists Create 22-Amino Acid Bacterium
By Jason Socrates
A team of investigators at The Scripps Research Institute
and its Skaggs Institute for Chemical Biology in La Jolla,
California has modified a form of the bacterium Escherichia
coli to use a 22-amino acid genetic code.
"We have demonstrated the simultaneous incorporation of
two unnatural amino acids into the same polypeptide," says
Professor Peter G. Schultz, who holds the Scripps Family Chair
in Chemistry at Scripps Research. "Now that we know the genetic
code is amenable to expansion to 22 amino acids, the next
question is, 'how far can we take it?'"
In an upcoming issue of the journal Proceedings of the
National Academy of Sciences, the team describes how they
engineered this modified form of E. coli to make myoglobin
proteins with 22 amino acidsincorporating the unnatural
amino acids O-methyl-L-tyrosine and L-homoglutamine
in addition to the naturally occurring 20.
Scientists have for years created proteins with such unnatural
amino acids in the laboratory, but until Schultz and his colleagues
began their work in this field several years ago, nobody had
ever found a way to get organisms to add unnatural amino acids
into their genetic code. Earlier studies by Schultz's group
described the incorporation of a number of single unnatural
amino acids with a variety of uses in chemistry and biology
into E. coli and into the yeast Saccharomyces cerevisiae.
This latest result is a boon because it demonstrates that
multiple unnatural amino acids can be added to the genetic
code of a single modified organism. This proof-of-principle
opens the door for making proteins within the context of living
cells with three, four, or more additional amino acids at
Why Expand the Genetic Code?
Life as we know it is composed, at the molecular level,
of the same basic building blocks. For instance, all life
forms on earth use the same four nucleotides to make DNA.
And with few exceptions, all known forms of life use the same
common 20 amino acidsand only those 20to make
The question is why did life stop with 20 and why these
While the answer to that question may be elusive, the 20-amino
acid barrier is far from absolute. In some rare instances,
in fact, certain organisms have evolved the ability to use
the unusual amino acids selenocysteine and pyrrolysineslightly
modified versions of the amino acids cysteine and lysine.
These rare exceptions aside, scientists have often looked
for ways to incorporate unnatural amino acids into proteins
in the test tube and in the context of living cells because
such novel proteins are of great utility for basic biomedical
research. They provide a powerful tool for studying and controlling
the biological processes that form the basis for some of the
most intriguing problems in modern biophysics and cell biology,
like signal transduction, protein trafficking in the cell,
protein folding, and proteinprotein interactions.
For example, there are novel amino acids that contain fluorescent
groups that can be used to site-specifically label proteins
with small fluorescent tags and observe them in vivo.
This is particularly useful now that the human genome has
been solved and scientists are now turning their attention
to what these genes are doing inside cells.
Other unnatural amino acids contain photoaffinity labels
and other "crosslinkers" that could be used for trapping proteinprotein
interactions by forcing interacting proteins to be covalently
attached to one another. Purifying these linked proteins would
allow scientists to see what proteins interact with in living
cellseven those with weak interactions that are difficult
to detect by current methods.
Unnatural amino acids are also important in medicine, and
many proteins used therapeutically need to be modified with
chemical groups such as polymers, crosslinking agents, and
cytotoxic molecules. Last year, Schultz and his Scripps Research
colleagues also showed that glycosylated amino acids could
be incorporated site-specifically to make glycosylated proteinsan
important step in the preparation of some medicines.
Novel hydrophobic amino acids, heavy metal-binding amino
acids, and amino acids that contain spin labels could be useful
for probing the structures of proteins into which they are
inserted. And unusual amino acids that contain chemical moieties
like "keto" groups, which are like LEGO blocks, could be used
to attach other chemicals such as sugar molecules, which would
be relevant to the production of therapeutic proteins.
Combining Amber Suppression with Frame Shift Suppression
Schultz and his colleagues succeeded in making the 22-amino
acid E. coli by exploiting the redundancy of the genetic
When a protein is expressed, an enzyme reads the DNA bases
of a gene (A, G, C, and T), and transcribes them into RNA
(A, G, C, and U). This so-called "messenger RNA" is then translated
by another protein-RNA complex, called the ribosome, into
a protein. The ribosome requires the help of transfer RNA
molecules (tRNA) that have been "loaded" with an amino acid,
and that requires the help of a "loading" enzyme.
Each tRNA recognizes one specific three-base combination,
or "codon," on the mRNA and gets loaded with only the one
amino acid that is specific for that codon.
During protein synthesis, the tRNA specific for the next
codon on the mRNA comes in loaded with the right amino acid,
and the ribosome grabs the amino acid and attaches it to the
growing protein chain.
The redundancy of the genetic code comes from the fact that
there are more codons than there are amino acids used. In
fact, there are 4x4x4 = 64 different possible ways to make
a codonor any three-digit combination of four letters
in the mRNA (UAG, ACG, UCC, etc.). With only 20 amino acids
used by the organisms, not all of the codons are theoretically
But nature uses them anyway. Several of the 64 codons are
redundant, coding for the same amino acid, and three of them
are nonsense codonsthey don't code for any amino acid
These nonsense codons are useful because normally when a
ribosome that is synthesizing a protein reaches a nonsense
codon, the ribosome dissociates from the mRNA and synthesis
stops. Hence, nonsense codons are also referred to as "stop"
codons. One of these, the amber stop codon UAG, played an
important role in Schultz's research.
Schultz and his colleagues knew that if they could provide
their cells with a tRNA molecule that recognizes UAG and also
provide them with a synthetase "loading" enzyme that loaded
the tRNA with an unnatural amino acid, the scientists would
have a way to site-specifically insert the unusual amino acid
into any protein they wanted.
They needed to find a functionally "orthogonal" paira
tRNA/synthetase pair that react with each other but not with
endogenous E. coli pairs. So they devised a methodology
to evolve the specificity of the orthogonal synthetase to
selectively accept unnatural amino acids.
Starting with a tRNA/synthetase pair from the organism Methanococcus
jannaschii, they created a library of E. coli cells,
each encoding a mutant M. jannaschi synthetase, and they changed
its specificity so that it could be use to recognize the unnatural
amino acid O-methyl-L-tyrosine .
To do this, they devised a positive selection whereby only
the cells that load the orthogonal tRNA with any amino acid
would survive. Then they designed a negative selection whereby
any cell that recognizes UAG using a tRNA loaded with anything
other than O-methyl-L-tyrosine dies.
In so doing, they found their orthogonal synthetase mutants
that load the orthogonal tRNA with only the desired unnatural
amino acid. When a ribosome reading an mRNA within the E.
coli cells encounters UAG, it inserts the unnatural amino
acid O-methyl-L-tyrosine .
Furthermore, any codon in an mRNA that is switched to UAG
will encode for the new amino acid in that place, giving Schultz
and his colleagues a way to site-specifically incorporate
novel amino acids into proteins expressed by the E. coli.
Similarly, Schultz and his colleagues made an engineered
tRNA/synthetase orthogonal pair from the polar archean organism
Pyrococcus horikoshii that recognizes the four-base
The tRNA has a four-base anticodon loop, and when a ribosome
reading an mRNA within the E. coli cells encounter
AGGA, it inserts the unnatural amino acid L-homoglutamine
at that site.
By placing both of these systems within the same E. coli
cell, Schultz and his colleagues have demonstrated, as a proof
of principle, that it is technically possible to have mutually
orthogonal systems operating at once in the same cell. This
opens up the possibility of doing multiple site substitution
The article, "A twenty-two amino acid bacterium with a functional
quadruplet codon" is authored by J. Christopher Anderson,
Ning Wu, Stephen W. Santoro, Vishva Lakshman, David S. King,
and Peter G. Schultz and will be posted online during the
week of May 10-16, 2004 by the journal Proceedings of the
National Academy of Sciences. See: http://www.pnas.org/cgi/content/abstract/0401517101v1.
The article will appear in print later this year.
This work was supported by the Department of Energy and
the Skaggs Institute for Research. Individual scientists involved
in this study were sponsored through a National Science Foundation
Predoctoral Fellowship, a Canadian Institutes of Health Research
fellowship, and a Career Award in the Biomedical Sciences
from the Burroughs Wellcome Fund.
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