|
Naohiro
Kameta, Organic Nanotube Material Team, the Nanotube Research Center of
the National Institute of Advanced Industrial Science and Technology
(AIST), has developed organic nanotube gels that restore the activity of
denatured proteins by folding them into the native three-dimensional
structure (refolding). The organic nanotube gels can protect proteins
from heat and chemicals.
This
technology has been realized by forming organic nanotube gels in which
the inner and outer surface structures and diameter of the nanochannel
are precisely controlled for the protein of interest. The denatured
proteins are encapsulated in the process of the organic nanotube gel
formation and are then recovered from the gel by using a change in pH.
In this way, only proteins with restored activity can be obtained at
high purity. Use of the organic nanotubes as the gel media has the
benefit of allowing easy removal of the denaturant by water washing and
easy separation and recovery of the protein of interest; these can be
achieved without filtration or centrifugal separation. In addition, when
proteins are encapsulated in the nanochannel of the organic nanotube,
the activity of the proteins is not inactivated by heating or by adding a
high concentration of denaturant. Expected applications of this
technology include efficient preparation of high-purity proteins and use
in nanoreactors and enzyme sensors by combining an enzyme with the
organic nanotube.
Details
of the results have been published online in ACS Nano, a scientific
journal of the American Chemical Society, on May 23, 2012.
Social background of research
From
a green innovation perspective, enzymes that promote chemical reactions
in the body in a specific and highly selective manner with a high yield
are attracting substantial attention as energy-efficient,
low-environmental-impact catalysts in chemical industrial processes. An
enzyme, which is a protein, is folded into a specific three-dimensional
structure by the interaction of amino acids (protein components) and
exhibits catalytic activity that is based on the structure.
Recombinant
technology using Escherichia coli is commonly used in the industrial
production of proteins. However, an enzyme considerably changes its
structure from the native three-dimensional one in the expression of a
recombinant protein and aggregates of denatured proteins with no
catalytic activity are formed. The restoring efficiency of proteins with
a native three-dimensional structure and original catalytic activity
from the aggregate is very low. Consequently, various additives to help
suppress protein aggregation or to encourage protein refolding into the
native three-dimensional structure have been developed. However, the
additives have low yield and versatility.
In
the body, there is a protein group called molecular chaperones that
have nanospaces to encapsulate denatured proteins for isolation and to
help with protein refolding. In recent years, porous inorganic materials
and polymer nanoparticles that can mimic molecular chaperones have
attracted attention. However, an additive is required for desorption of
proteins from the porous inorganic materials or polymer nanoparticles
and both the additive and incompletely refolded proteins contaminate the
aimed protein. Therefore, a complex separation process is required and
such a process may cause redenaturation of the protein.
History of research
Over
more than 10 years, AIST has been working to develop fibrous organic
nanomaterials and tubular ones (i.e. organic nanotubes) formed by
self-organization in solvents of amphiphilic molecules synthesized from
renewable, natural products, such as sugars, amino acids, nucleic acids,
and fatty acids. In recent years, AIST has established a process for
mass production of organic nanotubes and has been developing various
applications of these organic nanotubes.
The
outer surface of conventional bilayer-membrane organic nanotubes has
the same structure as the inner surface on the nanochannel side.
Recently, by using molecular design and molecular arrangement control,
AIST has created monolayer-membrane organic nanotubes with an
inner-surface structure that differs from that of the outer surface.
Providing the nanochannel-side surface with a structure that allows
interaction with drugs, protein, DNA, and nanoparticles (called
“guests”), the nanotube can encapsulate guests efficiently and
selectively. Storage and release of the guests can be regulated by
externally controlling the interaction.
The
researcher formed organic nanotube gels in which the surface structure
and the diameter of the nanochannel were precisely controlled to enable
denatured protein molecules to be encapsulated as guests. He also aimed
to develop new functions, such as the promotion of protein refolding and
the protection of protein activity.
Details of research
Denatured
proteins in which hydrophobic regions are exposed to the surface are
prone to aggregation in aqueous solution. A hydrophobic structure was
introduced to the nanochannel-side surface to efficiently encapsulate
denatured proteins in organic nanotubes and suppress protein
aggregation. An amphiphile 1 (Fig. 2a) and its derivative with a
hydrophobic benzyl group (shown in green in Fig. 2a) at the end were
designed and synthesized, and an organic nanotube gel (L-10-NTG) of the
amphiphile 1 and the derivative (molar ratio of 9:1) was formed by
self-organization of these two compounds. The gel was formed by
controlling pH of the solution with sodium hydroxide at room temperature
(Fig. 2b). The organic nanotube consists of a monolayer membrane with
molecules of the amphiphile 1 and the derivative arranged in parallel
and has an inner diameter of 10 nm (Fig. 2b, c, and d). For comparison
an organic nanotube gel H-10-NTG was prepared by self-organization of
only amphiphile 1, and an organic nanotube gel H-20-NTG (inner diameter:
20 nm) was prepared by self-organization of only amphiphile 2.
Chemical formulae of the amphiphiles and the derivative
Green
fluorescent protein (GFP, molecular weight about 30,000) as a model
protein was chemically denatured with guanidinium chloride (GdmCl,
concentration 6 M). Denatured GFP was added to the solution during the
self-organization of L-10-NTG and H-10-NTG and were encapsulated in the
nanochannels when the organic nanotube gels were formed. The amount of
denatured GFP encapsulated in 5 mg of L-10-NTG was 38 µg—about three
times more than that encapsulated in H-10-NTG (13 µg). This was probably
due to the hydrophobic interaction between the benzyl groups in
L-10-NTG and the hydrophobic regions of the denatured GFP.
When
the organic nanotube gel encapsulating the denatured GFP was washed
with water to dilute the denaturant GdmCl to less than 1 mM, some of the
encapsulated GFP restored fluorescence activity, and protein refolding
was promoted in the nanochannels.
In
addition, when a recovery solution (a buffer solution with a pH of 7.8)
was added, the glycine amino groups of the amphiphile 1 on the
nanochannel surface were uncharged and the electrostatic interaction
with the GFP vanished. Consequently, GFP refolded into a native
structure in the nanochannels, as well as GFP incompletely refolded in
the nanochannels, was recovered in the refolded state from the organic
nanotube gel. The total refolding ratio of the encapsulated GFP was 49%
for H-10-NTG and 85% for L-10-NTG. Introduction of the hydrophobic
structure (benzyl groups) into the nanochannels strongly induced
refolding, particularly in the release and recovery process. The
denatured GFP molecules that were not refolded (51% for H-10-NTG and 15%
for L-10-NTG) remained encapsulated in the organic nanotube gel; only
the refolded GFP was selectively recovered into the recovery solution
with adjusted pH without the use of a special additive. The refolding
ratio of GFP obtained by the conventional dilution method was 14%,
showing that protein refolding was promoted by the organic nanotube gel.
H-10-NTG
promoted refolding of carbonic anhydrase (CAB, with a molecular weight
of about 30,000–comparable to that of GFP), but it did not efficiently
induce refolding of citrate synthase (CS, with a molecular weight of
about 100,000–higher than that of GFP). H-20-NTG with an organic
nanotube diameter of 20 nm exhibited a high ability to promote refolding
of CS, but it did not induce refolding of GFP and CAB at all. Instead,
it promoted aggregation of these proteins in the nanochannels. This
shows that an organic nanotube gel with an inner diameter appropriate
for each protein has a high level of ability to promote protein
refolding.
The
organic nanotube gels have the ability to promote protein refolding, as
well as a protective ability that strongly suppresses the thermal and
chemical denaturations of proteins. Whereas free CAB molecules in water
lost almost all of their enzyme activity owing to thermal or chemical
denaturation by heat or a high concentration of urea (denaturant), the
CAB encapsulated in the nanochannels of L-10-NTG were protected from
heat or the denaturant and retained 90% of their enzyme activity under
the same conditions.
Future plans
The
researcher intends to create organic nanotube gels in which the surface
structure and nanochannel diameter are controlled appropriately for a
variety of proteins with different properties. He aims to develop an
artificial molecular chaperone system through collaborative research,
including provision of samples. He will also apply enzyme-conjugated
organic nanotubes to nanoreactors and enzyme sensors.
Source: AIST