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| An overview of the new protein production process. Protein is
produced in a tube via a cell-free transcription and translation
reaction (left). This reaction is performed in the presence of
lipids and detergent, which protect hydrophobic protein segments
and help initial folding along (middle). When detergent is removed
by dialysis, the properly folded proteins end up embedded within
liposomes, replicating their normal integration into cell membranes
(right). enlarge image |
| Copyright : (2009) RIKEN |
Membrane-embedded proteins execute many of the most important
cellular functions, detecting external environmental cues,
mediating communication and signaling, and facilitating molecular
import and export. Accordingly, they also represent important
targets for drug development, and an estimated 60% of currently
available drugs are believed to target membrane proteins.
On the other hand, experimental investigation of the structure and
function of individual membrane proteins is routinely thwarted by
the general difficulty of preparing large quantities of properly
folded protein. This is largely due to the chemical composition of
these proteins, which have large hydrophobic surfaces that are
chemically averse to being exposed to water; these protein segments
are stable when embedded within the equally hydrophobic lipid-based
cell membrane, but tend to assemble into irregular, improperly
folded clumps when prepared in solution.
Most water-soluble proteins can readily be produced either within
modified cell lines or through the use of cellular extracts that
contain the full complement of machinery required for protein
synthesis. However, the yield of membrane proteins obtained from
cultured cells is generally inadequate, and scientists have had to
tinker extensively with extract-based production methods to obtain
usable quantities of functional protein.
Now, a research team led by Shigeyuki Yokoyama at the RIKEN Systems
and Structural Biology Center in Yokoyama has developed an
efficient preparation method that produces high yields of
functional membrane proteins1. “Up until now, aggregation and
misfolding of membrane proteins during cell-free protein synthesis
have been avoided by adding either lipid-based liposomes or
detergents,” explains Yokoyama. However, he adds that such
workarounds were not a complete solution, and challenges remained
in selecting the appropriate detergent for a given protein, or
ensuring that newly synthesized proteins were properly folded and
inserted into the membranes of liposomes.
Best of both worlds
Yokoyama and colleagues solved this problem by developing a hybrid
technique that incorporates elements of both preparation methods,
synthesizing their proteins in a bacteria-derived extract
containing both detergent and lipid molecules. During production,
hydrophobic stretches of the newly formed protein are protected by
this lipid-detergent mixture; once synthesis is complete, the
detergent is removed from the sample via dialysis and the remaining
lipids subsequently assemble into bubble-shaped liposomes with the
mature proteins securely embedded within these artificial
membranes.
The researchers then demonstrated the efficacy of their technique
with bacteriorhodopsin (BR), an archaea-derived photosynthetic
pigment protein. The light-responsive properties of BR made it a
particularly useful test subject for this method—improperly
folded or aggregated BR appears yellow, while preparations of
mature BR appear purple. In initial experiments, they were able to
demonstrate successful production of properly folded BR with a
variety of detergents, and although the overall folding efficiency
was somewhat lower than previously described preparations, the
overall quantity of protein produced was significantly
greater—as much as 80-fold greater, depending on the
detergent. Subsequent analysis confirmed that much of this properly
folded BR was successfully integrated into the membranes of
liposomes following detergent removal.
Yokoyama and his colleagues' protein preparations also retained
proper functional characteristics. Naturally occurring BR undergoes
a series of chemical transitions known as a photocycle, in which
excitation by light at particular wavelengths induces a series of
subtle structural changes; these shifts can in turn be quantified
by changes in the light-absorption properties of the protein. They
found that their synthetic BR mirrored the photocyclic
characteristics of native protein, and even displayed the capacity
to act as a light-activated proton pump—a key component of
its photosynthetic activity.
Making a big production of it
Whether researchers are looking to derive protein in crystalline
form for structural analysis or in solution for functional
characterization, the ability to obtain high-purity preparations is
essential. In this regard, the Yokoyama team’s technique
passed with flying colors, with a 75-fold greater yield of
functional BR protein relative to a previously described high-yield
liposome-based method.
Yokoyama indicates that since the initial publication of this work,
his group has had the opportunity to test their method on a variety
of membrane-bound receptors and channels, and they are currently in
the process of characterizing the efficiency of these preparations.
Fortunately, the system is sufficiently flexible that a number of
adaptations can be introduced to optimize production for any given
protein. “We will test other lipids or steroid detergents for
the functional overproduction of other types of membrane
proteins,” he says. “Moreover, by controlling lipid
bilayer formation and protein synthesis speed through changes in
reaction conditions such as temperature, we can improve this system
to be suitable for many types of membrane proteins.”
Even as Yokoyama and his colleagues work out the details of
refining their overexpression system, they have already begun to
contemplate future targets for which this method might prove
valuable—and with so many poorly characterized membrane
proteins, the field is wide open. “We hope to apply this
system to biochemical, biophysical and structural studies of human
G-protein coupled receptors, which are important targets for drug
design, and proteins that transport small molecules such as
nutrients, across the membrane,” he says, “as well as
complexes incorporating membrane proteins, because the cell-free
system makes it easy to co-express more than one
protein.”
Shigeyuki Yokoyama
Shigeyuki Yokoyama was born in Tokyo, Japan, in 1953. He received
his BS and PhD degrees from the University of Tokyo in 1975 and
1981, and following completion of five years of postdoctoral work,
became an associate professor in 1986 and a professor in 1991 in
the Department of Biophysics and Biochemistry, University of Tokyo.
In 1993, he was appointed chief scientist of the RIKEN Cellular
Signaling Laboratory, and later project director of the Protein
Research Group in the Genomic Sciences Center. He played a pivotal
role as science director of the RIKEN Structural Genomics /
Proteomics Initiative (RSGI) and director of the Highthroughput
Factory supporting the Protein 3000 project. Since 2008, he has
acted as director of the Systems and Structural Biology Center
(SSBC) at the RIKEN Yokohama Institute. |