Doing Nature One Better: Expanding The Genetic Code In Living Mammalian Cells

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Doing Nature One Better: Expanding The Genetic Code In Living

Mammalian Cells

http://www.sciencedaily.com/releases/2007/07/070702084251.htm

Researchers at the Salk Institute for Biological Studies have

developed a novel strategy to expand the natural repertoire of 20

amino acids in mammalian cells, including neurons, and successfully

inserted tailor-made amino acids into proteins in these cells. In a

powerful demonstration of the method's versatility, they then used

unnatural amino acids to determine the operating mechanism of

the " molecular gates " that regulate the movement of potassium ions

in and out of nerve cells.

" In the past, this type of engineering has been mainly restricted to

bacteria or in yeast, and it was very challenging to efficiently

incorporate unnatural amino acids in mammalian cells. But most

biomedical questions have to be studied in the cells of higher

organisms and animal models to arrive at meaningful answers, "

explains Lei Wang, Ph.D., an assistant professor in the Chemical

Biology and Proteomics Laboratory, who led the current study

published in the July issue of Nature Neuroscience.

The genetic code, which is shared by plants, animals and bacteria,

includes 64 codons encoding 20 different amino acids and three stop

signals. Being able to expand the code and insert non-natural amino

not only greatly enhances researchers' ability and precision, but

also provides novel tools for addressing challenging questions

insurmountable with conventional means.

" We had tried using conventional mutagenesis to introduce mutations

into the potassium channel but it didn't give us any answers, " says

A. Slesinger, Ph.D., an associate professor in the Peptide

Biology Laboratory, who collaborated with Wang on the current

study. " Being able to incorporate bulky unnatural amino acids into

living mammalian cells really made all the difference, " he adds.

During his graduate studies, Wang pioneered a method to accommodate

additional amino acids in bacteria. His approach mimicked the

strategy every cell relies on to incorporate conventional amino

acids into proteins: During protein synthesis, amino acids are

brought out one by one by molecules known as transfer RNAs (tRNA)

and added to the growing protein chain according to the instructions

spelled out in the genetic code till a stop codon -- for which no

corresponding tRNA/amino acid pair exists -- lets everybody know

that this particular job is done.

From a large pool of mutated aminoacyl-tRNA synthetases -- the

enzyme that loads tRNAs with their corresponding amino acids -- Wang

selected the one that would attach a desired artificial amino acid

to a tRNA that recognizes one of the stop codons. Every time the

stop codon appeared in the genetic code, the new tRNA would insert

the artificial amino acid.

But doing the same trick in mammalian cells becomes way more

complicated. Simply transferring the bacterial genes into mammalian

cells doesn't work since they flat out refuse to produce bacterial

tRNAs. While it is easy to screen large numbers of mutated aminoacyl-

tRNA synthetases in bacteria and yeast, it can't be done in

mammalian cells in the same way. But Wang and his team got around

both obstacles.

" We found that we could coerce mammalian cells to express bacterial

tRNAs by using the H1 promoter, " says first author Wenyuan Wang,

Ph.D., a postdoctoral researcher in Wang's laboratory. Relying on

yeast to do the dirty job of finding a synthetase that recognizes

tRNA and attaches the right unnatural amino acid helped them to

overcome the second challenge. " Using yeast for the selection

process and then transferring the enzyme for use in mammalian cells

may sound like a naïve idea, but members from the same kingdom

behave very similarly in terms of tRNA synthetases and it worked, "

he adds.

After a green fluorescence protein-based functional assay in various

mammalian cells and neurons literally gave them the green light,

Wang teamed up with Slesinger, who studies ion channels in the

brain, to illustrate that this technology can solve otherwise

intractable biological questions.

When a signal travels along a nerve cell, the potassium channel

Kv1.4, which belongs to a class of so-called fast-inactivating ion

channels, opens briefly and then quickly shuts down. Structural

studies had suggested that in a process similar to threading a

needle the channel's flexible head feeds through a small portal and

blocks the central pore of the channel. Wang and Slesinger used the

new unnatural technology as a molecular ruler to answer the question

whether increasing the size of the thread had an effect on the speed

of inactivation "

" We introduced mutations into the thread, so it would be too big to

fit through the hole, " says Wang, " but we couldn't see a difference

with natural amino acids. " Adding even bulkier, artificial amino

acids provided the answer. " Now the process of inactivation was

really slow, supporting the hypothesis that the diameter of the

flexible head plays a crucial role in the fast inactivation of this

channel, " adds Slesinger.

Researchers who contributed to the study include K.

Takimoto, a graduate student in Wang's laboratory, staff scientist

Gordon V. Louie, Ph.D., staff chemist J. Baiga and

Medical Investigator ph P. Noel, all in Jack H. Skirball

Center for Chemical Biology and Proteomics, and Kuo-Fen Lee, Ph.D.,

professor in the Peptide Biology Laboratory.