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Precision Polymers Through
Protein Templated Synthesis
A research proposal for the
Radboud Honours Academy
2014-2015
Kayleigh van Esterik
Joëlle Klop Lennart van Melis Jonas Würzinger
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“I was born not knowing and have had only
a little time to change that here and there.”
-Richard P. Feynman
We would like to thank everyone
who supported us for their help,
especially Dr. Oren Sherman and Dr. Amid Sachdeva
and of course our supervisor Dr. Dennis Löwik.
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1a. Details of proposal
Title: Precisely Defined Polymers through Protein Templated Synthesis.
Area: ● (bio)Chemistry Ο Physics and Mathematics Ο Health Ο Other (please, specify)
1c. Details of the applicants
Name: Kayleigh van Esterik
Gender: Ο Male ● Female
Name: Joëlle Klop
Gender: Ο Male ● Female
Name: Lennart van Melis
Gender: ● Male Ο Female
Name: Jonas Würzinger
Gender: ● Male Ο Female
1d. Supervisor
Name: Dennis Löwik
Tel: +31 (0)24 36 52382
Email: [email protected]
Institute and Department: Radboud University Nijmegen; Department of Bio-Organic Chemistry
3a. Scientific summary
Nature is capable of producing precisely defined polymers in length and design, however this is not yet easily
possible with chemical synthesis. With nature as inspiration, we would like to produce precisely defined polymers
with the use of an anti-parallel β-sheet protein as a template. Unnatural amino acids (uaa's) with polymerizable
side chains will be incorporated at each upper turn of this protein, in E. coli using amber suppression and a
PylRS/tRNACUA tRNA synthetase pair. This will be done in the C321ΔPRfa strain of E. coli in which release factor 1
(RF1) has been deleted and all naturally occurring amber codons have been replaced for high efficiency.
Two systems are proposed with uaa's containing side chains that are able to cover the distance (4.7 Å)
between two β-sheet strands and contain ester or peptide bonds to enable separation of the polymer from the
protein template. In the styrene-maleic anhydride system, only one uaa (a styrene/lysine derivative) will be
incorporated to create monodisperse copolymers. The second system needs the incorporation of an initiator for
ATRP, which will be done using quadruplets and an altered ribosome (Ribo-Q1). Different forms of poly-
pentadienoic acid will be produced.
In our opinion the rapid developments in the field of incorporating uaa's will make it possible to
incorporate multiple distinct uaa's with high efficiency. This proof of principle study could eventually lead to more
freedom in the design of the desired polymer. The ability to produce precisely defined polymers creates endless
possibilities for new polymers and materials.
3b. Summary for the broad scientific committee
Polymers play a big role in everyday life. Not only do we use synthetic polymers for packing products, our cells
use polymers as well. A human cell contains several kinds of polymers: the best known are DNA and proteins.
The DNA strands are used for storage of information for the production of proteins.
The production of a polymer with a precisely defined length and sequence is not yet easily possible via
chemical routes. The ability to do so would create endless opportunities and possibilities for the production of
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new polymers with new properties. In this research proposal nature is used as an inspiration for the production
of precisely defined polymers. Different techniques that manipulate the process of translation exist and are
constantly being improved. In our opinion, the latest developments in this field, especial considering the
incorporation of unnatural amino acids (the building blocks of proteins) will enable us to create these precisely
defined polymers.
By incorporating unnatural amino acids in a protein with a specific fixed structure in a way that they can
polymerize after the production of the protein, the precision of nature’s polymerization techniques is used to our
advantage. Two systems are proposed, the first is focused on obtaining monodisperse copolymers, with one type
of sequence. The second system is appropriate for a complex sequence and if the progression in the
incorporation of unnatural amino acids continues as it does, the production of every imaginable polymer will be
in reach.
3c. Summary for the general public
Synthese van precisiepolymeren door eitwitsjablonen
Eiwitten bestaan uit aminozuren en kunnen door cellen exact volgens een vooraf bepaalde sequentie worden
opgebouwd. Synthetisch is het echter nog lastig om polymeren (zoals plastics) van exact één lengte en met een
ingewikkelde sequentie te produceren. Door onnatuurlijke aminozuren in een eiwit in te bouwen die achteraf
polymerizeerbaar zijn, kan de precisie van de natuur gebruikt worden om wel polymeren te maken met een
ingewikkelder sequentie en een exacte lengte. Polymeren komen overal voor in onze samenleving, dit onderzoek
zou ervoor kunnen zorgen dat er nieuwe (biologische) materialen gemaakt kunnen worden met compleet nieuwe
eigenschappen, die precies afstelbaar zijn.
4. Description of the proposed research
See page 5.
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1 Introduction Polymers play a big role in everyday life. Not only do we use synthetic polymers for packing products, our cells
use polymers as well. A human cell contains several kinds of polymers: the best known are DNA and proteins.
The DNA strands are used for storage of information for the production of proteins. Every kind of protein has its
own sequence and length, which are defined in the corresponding gene in a DNA strand. Through transcription,
the part of the DNA strand that contains a certain gene is transcribed into an mRNA strand. Next, a protein is
produced, based on the codons in the mRNA strand. An enzyme, the ribosome, translates every codon to the
corresponding amino acid, in a process called translation.
The production of a polymer with a precisely defined length and sequence is not easily possible via
chemical routes. Normally a certain sequence is repeated, for example ABC. Synthesis of a polymer with a more
complex sequence such as ABCAABCCABABC can't be controlled, and therefore these more complex polymers
cannot be routinely produced. Currently, when polymers are produced, a certain dispersity in length is obtained.
This dispersity is however not a large problem for the application of most polymers, since the properties will not
vary that much.
When it would be possible to easily produce precisely defined polymers, new materials with new
properties could be obtained. Also, with
precisely defined polymers, the properties of
these materials would be adjustable by
altering the sequence of monomers. This
would create endless possibilities for the
synthesis of macromolecules and materials.
In proteins, only twenty amino acids
account for proteins with numerous
functions and properties. So even a limited
number of available monomers could result
in many different polymers.
In this research proposal, nature is used as an inspiration to achieve the production of precisely defined
polymers. Nature’s system of transcription and translation has evolved over thousands of years and will be used
to our benefit. Different techniques that manipulate the process of translation exist and are constantly being
improved, with the ultimate goal of using the natural protein-producing system for the production of unnatural
polymers (Figure 1).3 Since complex sequences can be stored in DNA and mRNA, it is possible to transmit this
information to a complex sequence of monomers by the incorporation of polymerizable unnatural amino acids in
a protein. In this way polymers with an exact length and sequence can be synthesized.
We will first introduce a system in which maleic anhydride and styrene will be polymerized for the production of a
monodisperse copolymer by using protein templated synthesis. This will be achieved by incorporating lysine
based unnatural amino acids with a styrene sidechain at the upper turns of an anti-parallel β-sheet protein
through amber suppression in E.coli. After purification, the copolymer will be synthesized and obtained by
separating protein and polymer (Figure 2). This system will be further discussed in section 2.
To work towards controlling the sequence of the polymer, a second system is introduced in which a poly-
pentadienoic acid will be formed through an initiator based radical polymerization. The monomer will be
incorporated in the same manner as proposed in the first system. The initiator is incorporated at the first position
to gain more control over the polymerization. This will be accomplished by using a quadruplet technique in which
an altered ribosome is used to efficiently incorporate both unnatural amino acids. After successful incorporation
of both unnatural amino acids and purification, the polymer is obtained by atom transfer radical polymerization
(ATRP) and the consecutive separation of protein and polymer. This system will be further discussed in section 3.
Figure 1: It is possible to alter the central paradigm for the production of proteins based on natural amino acids (coloured circles) into a process that uses unnatural amino acids (coloured stars) for the production of unnatural proteins and ultimately unnatural polymers using nature’s systems for transcription and translation. Adapted from Chin et al.
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The field of genetic reprogramming is developing extremely fast. To use these new techniques to the fullest this
proposal is presented. With the use of these techniques cell machinery normally used for protein expression
could be used for the synthesis of many desired polymers. In this way nature’s techniques are used to their full
potential. Recombinant DNA techniques are also often used for the expression of proteins, so this is a good way
to further explore the possibilities of these techniques.
Native chemical ligation is a technique commonly used for the production of synthetic proteins.5,6.
Although this technique is also suitable for the execution of the proposal, the maximum length of the amino acid
chain is limited.5 Genetic code expansion techniques are used in this proposal to find more applications for this
rapidly developing field. Also, it might be possible to form longer proteins and therefore, also longer polymers.
Native chemical ligation could be used however to form smaller synthetic proteins, to test certain properties and
polymerization conditions. The synthesis of longer and more complex polymers with native chemical ligation is a
laborious process and differs a lot from genetic code expansion techniques. Using both techniques is therefore
not desired. However, if the formation of longer proteins in E. coli would not be successful, expressed protein
ligation could be used to connect smaller expressed proteins.6
A precise length becomes more important when synthesizing precisely defined polymers, since every
part of the polymer can uniquely contribute to certain properties of the material. Although our proposed systems
will not produce complex polymers, they include import steps necessary for the production of precisely defined
polymers through the concept of protein templated synthesis and monodisperse polymers will be produced. The
consecutive systems have increased complexity to work towards the synthesis of precisely defined polymers.
In the second system ATRP will be used as a polymerization technique, since the Radboud University
Nijmegen has a lot of expertise regarding this technique. This system will also be easily adjustable for the
production of more complex monomers, when the efficiency of the incorporation of unnatural amino acids is high
enough. Since the field of genetic reprogramming and the incorporation of unnatural amino acids is developing
very fast, in our opinion the production of precisely defined polymers of exact length and sequence soon will be
in reach.
Figure 2: Precisely defined polymers through protein templated synthesis. After the incorporation of polymerizable unnatural amino acids (blue) in an anti-parallel β-sheet protein (red) polymerization takes place and the protein and polymer will be separated. Altered from Jordano S.
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2 Copoly-Maleic anhydride-Styrene
Maleic anhydride and styrene form quite an extraordinary
polymerization pair. Since the monomers of maleic anhydride do not
polymerize with themselves, the copolymer will be formed (Figure 3).
This radical copolymerisation has been studied extensively and has
proven itself to be very efficient. It is therefore a good starting point for
this research.7,8
To synthesize monodisperse copolymers, a lysine based
unnatural amino acid with a styrene group (Sty-Lys) will be incorporated
in a protein through amber suppression in E. coli. This will be done in a
protein with a fixed secondary structure, to space the monomers in such
a way radical polymerization can take place (Figure 5 & 6). After
growing E. coli cells, production of the unnatural protein is induced and
fully produced proteins are selected. Polymerization of these templates
is initiated by adding maleic anhydride and an initiator. The protein and
polymer will then be separated and analysed.
2.1 Incorporation of unnatural amino acids
In cells, the sequence of a protein is anchored in DNA. The DNA is transcribed into mRNA and the assembly of
the protein is based on this mRNA through translation by the ribosome. The codons in the mRNA interact with
the complementary anticodons in tRNA. These tRNA’s deliver the correct amino acid and are loaded by amino
acyl tRNA synthetases (AARS). The incorporation of unnatural amino acids uses the degeneracy of the
assignment of all 64 possible triplet codons to the twenty natural amino acids by the tRNA system.9
By introduction of an additional tRNA/AARS pair which corresponds to a natural codon, the decoding of
this codon could be altered. It is important however that the tRNA/AARS pair is orthogonal to the host's amino
acids, tRNA and AARS, so there will be no interference. This was first achieved in 1998 by Further and colleagues
using the yeast PheRS/tRNACUAPhe pair.10 By optimizing this AARS toward new unnatural amino acids, the recoding of
the genetic code came in reach. The group of Peter Schultz was the first to achieve this and by now over a
hundred unnatural amino acids have been incorporated in various organisms using this approach.9,11
Different techniques and strategies have been developed over time and especially the efficiency of
amber suppression has increased immensely lately. It is this technique that is proposed to incorporate the
unnatural amino acid with the reactive side chain that will serve as the monomer for polymerization.
2.1.1 Amber suppression
For the incorporation of unnatural amino acids the stop codons (UAG, UAA or UGA) can be used. This is
convenient, since there is no competing natural amino acid. It is also possible to use sense codons, hence the
name for this technique: sense suppression.12 Because of the competition with natural amino acids, this
technique is less efficient. Therefore the focus will lie on using stop codon suppression.
Since a natural translation system usually consists of three different stop codons halting the translation,
theoretically two codons can be used to incorporate unnatural amino acids. Using the stop codon UAG (the
‘amber codon’), which is called amber suppression, is the technique most used in E. coli to incorporate unnatural
amino acids.13 This codon is used because it is the rarest stop codon. It uses an orthogonal AARS/tRNACUA pair
that is optimized toward the recognition, binding and incorporation of the desired unnatural amino acid.
Competition of the tRNACUA with releasing factor 1 (RF1) affects the efficiency of the incorporation of unnatural
amino acids, since RF1 will bind to the ribosome to stop the translation in response to the amber stop codon.9,14
Other factors could also possibly limit the efficiency of this technique, such as the number of plasmids for the
introduction of the necessary enzymes15, the amino acetylation efficiency of the amino-acyl tRNA synthetase
Figure 3: A) Styrene, B) Maleic anhydride, C) Copolymer of styrene and maleic anhydride.
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(AARS)/tRNA system and the efficiency of the incorporation of the unnatural amino acid by the ribosome.16 Due
to all these factors, the efficiency of amber suppression can differ greatly for every unnatural amino acid.
The typical efficiency obtained when using amber suppression was about 20-30%, with efficiency
dropping about a ten-fold when inserting more than one unnatural amino acid.13 Chin and colleagues showed
that using an orthogonal ribosome (ribo-X) increased the efficiency to more than 60% for the insertion of one
unnatural amino acid, while the efficiency for the insertion of two unnatural amino acids was increased to more
than 20%.14
Orthogonal ribosomes (such as ribo-X) have been evolved to efficiently execute the amber suppression
technique. The first orthogonal ribosome, ribo-O, was developed by altering the 16S subunit of the ribosome to
recognize a changed Shine Delgarno sequence in the leader sequence of the O-mRNA.17 This sequence makes
sure the ribosome recognizes only to the mRNA that is to be translated.9 Since then, the orthogonal ribosome
has been further optimized for the reading of an amber codon as a sense codon by selecting out of a library of
ribosomes with different mutations in the 530-loop region. This enhanced orthogonal ribosome was called ribo-X
and its fidelity is comparable to natural translation.18,19
Knocking out all RF1 in the E. coli strain JX33 further enhanced the efficiency of amber suppression to
>99.8%.20 This could be done by improving the expression of releasing factor 2, which took over some of the
functions of RF1, making sure the deletion of RF1 was not lethal. Another approach that has led to the deletion of
RF1 is replacing all known UAG stop codons in E. coli strain MG1655 with UAA codons by using multiplex
automated genome engineering (MAGE). This permitted the deletion of RF1, which led to the transformation of
the UAG codon to a sense codon.21 Since all UAG stop codons were replaced with UAA stop codons, normal
proteins were still correctly produced. This is necessary for the viability of the cell, which is needed for the
production of the protein template.
The high efficiency that is achieved by knocking out RF1 and the fact that it is most used for the
incorporation of unnatural amino acids led to the choice to use amber suppression to form the protein template.
In our research we will use the C321ΔPRfa strain of E. coli, in which RF1 has been deleted and all amber codons
have been replaced to create a high efficiency. Furthermore, this strain is fit for the incorporation of unnatural
amino acids through both amber suppression and the quadruplet-technique, which will be described in section
3.1.1.22,23
2.1.2 tRNA synthetase pairs
For the incorporation of an unnatural amino acid it is
necessary to have a proper aminoacyl-tRNA synthetase that
loads the unnatural amino acid onto its cognate tRNA. This
set of tRNA and tRNA synthetase should be entirely
orthogonal, to prevent loading of the unnatural amino acid
onto natural tRNA or natural amino acids onto unnatural
tRNA (Figure 4). Both options would result in erroneous
incorporation of amino acids into the protein (Figure 4).3,16
Multiple of these orthogonal pairs have already
been discovered and used for the incorporation of unnatural
amino acids. These pairs are usually derivatives of the
tRNA/tRNA synthase pairs of other organisms, where this
organism incorporates an amino acid not incorporated by
the host.9,16
For this research, pyrrolysyl-tRNA synthetase
(PylRS) and its cognate tRNAPyl are selected for the selective loading of Sty-Lys. PylRS is derived from
Methanosarcina species and naturally incorporates pyrrolysine (Pyl) at the amber stop codon.16,22 PylRS is
favoured as tRNA synthetase, because this aminoacyl-tRNA synthetase has some quite unique properties that
make it very suitable for the incorporation of unnatural amino acids. One of these properties is the high
promiscuity for the side chains of the substrate.22 Therefore, it is very likely that enzyme evolution16 of PylRS is
Figure 4: Two orthogonal sets of tRNA's (blue and grey) and tRNA synthetases (yellow) for the ligation of two distinct amino acids (red) to the tRNA's.
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not required for the efficient incorporation of Sty-Lys. Since the pyrrolysine side chain is quite bulky, PylRS has a
large pocket and is usable for large unnatural amino acids, like Sty-Lys. PylRS has already been used to
incorporate a wide variety of unnatural amino acids derived from lysine, phenylalanine and pyrrolysine
derivatives and is therefore expected to successfully incorporate Sty-Lys.16,22
2.1.3 Protein template
The information for the production of the protein template will be introduced in E. coli via plasmids. These
plasmids will contain an antibiotics resistance gene, so the cells containing the desired plasmids can be isolated.
Prior to the gene for the production of the protein template will be an inducible promoter. In this way the cells
can grow until production of the protein is induced. The cell will be likely to die because it uses all of its energy
for the production of the protein template. At that point the proteins can be harvested from the cell.
To make sure enough loaded tRNA is present to incorporate the unnatural amino acid, sufficient time is
needed between the incorporation of each unnatural amino acid. This can be achieved by incorporating natural
amino acids between the incorporation of two unnatural ones, which will further enhance the efficiency. To make
sure all the Sty-Lys unnatural amino acids will be polymerizable, a secondary structure is needed that will set the
unnatural amino acids in a fixed position.
This fixed secondary structure is found in fibroin, a protein naturally produced by the silkworm Bombyx
mori.2,24 Large parts of this protein (about 95%) are crystalline and form a highly ordered anti-parallel β-sheet
structure.24 The simple repeated amino acid sequence
-[(AG)xEG]n- (A = alanine, G = glycine, E = glutamic acid) is inspired by this fibroin and adopts an anti-parallel
β-sheet structure.25 This protein has a known DNA sequence and has been produced by recombinant DNA
techniques for use in various studies.2,24-27 The number of AG repeats (indicated with x in the sequence) can be
varied, to find the optimal amount of natural amino acids between the incorporation of the unnatural amino
acids. We expect that a larger value for x will result in a higher yield of complete proteins, since there is more
time for the unnatural tRNA synthetases to load its cognate tRNAs.
Glutamic acid is a poor β-sheet former and induces the turn in the anti-parallel β-sheet structure (Figure
5).2 Because it sticks out of the lamellar surface, the position of this amino acid is expected to be accessible for
polymerization.2 By inserting the unnatural amino acid Sty-Lys at each upper turn of the fibroin analogue, a
polymerizable structure would be formed (Figure 6). The amino acid located at the lower turns will not be altered
and remain glutamic acid, because this amino acid also enhances the solubility and enhances the formation of
turns.2 If the desired solubility is not reached by this method, it is possible to construct a fusion protein to
enhance the solubility even further.
By altering the amount of turns in the anti-parallel β-sheet protein the length of the polymer would be
unambiguously determined. To ensure that every protein template is complete, a His6-tag will be incorporated at
the C-terminus. The complete proteins will be purified with the use of a Ni-column.20 Lysis of the cell is possible,
since we have no interest in keeping the protein producing cell alive 20. Because the His6-tag is on the protein’s
C-terminus, the final yield only consists of completely synthesised proteins, since the protein production initiates
at the N-terminus. The His6-tag could also be used for determining the yield of the protein synthesis.
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For polymerization, the distance between two turns of
the anti-parallel β-sheet structure is an important property. Stress in the protein induced by polymerized side
chains of the unnatural amino acids could disadvantage the polymerization. The distance between two β-sheet
strands is approximately 4.7 Å.25,27 The distance seems to be independent of the type of amino acid at the turns,
but is slightly affected by the amount of AG repeats. 25,27 The distance in the protein is favourable, since it almost
perfectly corresponds with multiple different possible monomers, as will be further discussed in section 2.1.4 and
3.1.3. Two unnatural amino acid will be incorporated at each turn to cover the distance. Since a -[AG]x-
sequence is a strong β-sheet former, it is expected that the increase from one amino acid at each turn to two
amino acids will not stop the formation of the anti-parallel β-sheet structure. This system is therefore proposed
to be able to polymerize different kinds of monomers and give more freedom is the design of polymers.
An alternative protein could be an alpha helix. This protein also has a fixed secondary structure, with
amino acid side chain pointing outwards. A so called 'leucine zipper' consists of two alpha helical structures which
are stabilized by hydrophobic and electrostatic interactions.4 The unnatural amino acids are suggested to be
incorporated at position seven of the left alpha helix (Figure 7), since the unnatural amino acid is a lysine
analogue and an unnatural amino acid at this position is expected to interfere the least with the electrostatic and
hydrophobic interactions. When the unnatural amino acids are polymerized, it is expected that het alpha helical
structure is further stabilized, since the turns of the alpha helix would be held together by the polymerized
unnatural amino acids. The distance between two turns in an alpha helix is 5.4 Å.28
Figure 5: Lamella formation for -[(AG)3EG]-. The AG (alanylglycine) repeats form anti-parallel β-sheet and E (glutamic acid) is positioned at the turns. The thickness d depends on the number of AG repeats and is optimizable. The distance x between two β-sheet is appoximately 4.7 Å. Adapted from Smeenk.
2
Figure 6 : Model for the incorporation of an unnatural amino acid (blue) at the upper turns in an anti-parallel β-sheet (red). A His6-tag will be used for purification (yellow). Altered from Jordano S.
1
Figure 7: Helical wheel representation of the leucine zipper. The wide arrows represent hydrophobic interactions, the thin arrows represent electrostatic interactions. Position K7 is proposed for the incorporation of the unnatural amino acid, since this amino acid is a lysince analogue. Adapted from Hodges and Litowski.
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2.1.4 Unnatural amino acid
The unnatural amino acid proposed should be a lysine analogue to be compatible with the PylRS/tRNAPyl system.
In combination with the optimized amber suppression this system will provide the best chance for the
incorporation of our unnatural amino acid. The high efficiency is needed since the unnatural amino acid has to be
incorporated numerous times. The polymerizable group should cover a distance of approximately 4.7 Å after
polymerization, to prevent strain on in the protein. The distance covered by a linked pair of maleic anhydride and
styrene covers a distance of about 4.9 Å and is therefore a good option for the protein templated polymerization.
Figure 8 shows the proposed unnatural amino acids. Styrene is incorporated, and maleic anhydride will
later be added in solution for the polymerization, since this monomer is incapable of polymerizing with itself. The
unnatural amino acids contain a peptide linkage, for the separation of protein and polymer by hydrolysis. An
ester linkage might also possible if a milder separation method is favourable.
If the peptide bond is directly connected to the styrene group (Figure 8B) the polymerizing carbon will
be more electron-deficient and is therefore less reactive. This might prevent homopolymerization of styrene
within the protein. On the other hand, it might cause a less efficient polymerization of styrene with maleic
anhydride. An extra carbon between the peptide bond and the styrene group could be added to have less of
these electronic effects. Experiments are necessary to find the monomer with the most favourable properties.
2.2 Polymerization
In this section, the process of polymerization between the incorporated Sty-Lys amino acids and the added
maleic anhydride is explained. It can be expected that styrene in the protein template will not polymerize with
itself, due to the large distance. The reaction between the incorporated Sty-Lys amino acids and the added
maleic anhydride is a free-radical polymerization.8 The mechanism involves a free radical that propagates from
one monomer to the other by attacking double bonds. The reaction conditions are expected to be similar to the
polymerization of free styrene with maleic anhydride. After the free radical polymerization is discussed we will
focus on the separation of the polymer from the protein and its purification.
Figure 8: Lysine based unnatural amino acid with a styrene based group. A) An extra carbon between the peptide bond and styrene group for a less electron deficient carbon. B) The peptide bond and styrene group directly connected, causing a more electron deficient carbon. C) Pyrrolysine.
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2.2.1 Free radical polymerization
No initiator is need to polymerize the incorporated Sty-Lys monomers with maleic anhydride, because thermal
initiation is possible within a range from 76oC to 172°C.8 This may cause a problem since the natural protein
from Bombyx Mori denaturates at 67°C,29 although the denaturation temperature is likely to depend on the
length of the β-sheet protein. Benzoyl peroxide can be added to initiate the reaction without high temperatures,
when the protein template and maleic anhydride are in a solution of methyl ethyl ketone.8 The protein adopts its
β-sheet structure in ethanol and glycerol.30 If the protein is not soluble in methyl ethyl ketone, it might be able
to enhance the solubility with fusion proteins. and is therefore expected to maintain its β-sheet structure in this
solvent. Although the polymerization is not initiated in a controlled manner, it is expected that the polymerization
of styrene and maleic anhydride will proceed towards the end of each side of the template, due to the efficient
copolymerization of maleic anhydride and styrene. To prevent different polymers from polymerizing to another,
the protein templates should be diluted. The process from protein template to polymer is shown in Figure 9.
The particular reaction is very efficient and thus will lead to a relatively high yield. It is convenient to use
a polymerization that has proven to be successful, a low yield will show that the protein template is not optimal.
Small synthetic proteins with the desired unnatural amino acids could be produced using native chemical ligation
to help optimize the reaction conditions for polymerization.5,31
A B C
Figure 9: A) Protein template with styrene-lysine analogues incorporated and added maleic anhydride. The red arrows indicate the β-sheet protein. B) The result after polymerisation. C) The copolymer of maleic anhydride and styrene after separation from the protein. Altered from Jordano S.
1
2.2.2 Separation polymer and protein, purification & analysis
After the monomers have been polymerized, the polymer has to be detached from the protein. This could be
achieved with the use of acid hydrolysis. In this process the protein template will be lost, because it degenerates
into its constituent amino acids. Since it will be very difficult to reuse the protein template, losing the template in
this stage is not regarded as a significant loss.
The product after the separation consists of the polymer, amino acids and small peptide chains. The
smaller molecules can be separated by dialysis or size exclusion chromatography.8 Another option could be to
purify the polymer with the use of solvent extraction.
When the polymer is successfully purified it has to be determined if indeed the desired product has
formed. MALDI-TOF mass spectrometry and possibly size exclusion chromatography are suggested for this
analysis. These techniques will enable us to determine the monodispersity of the polymers present in the
product. Also 1H-NMR spectroscopy can be used to confirm that the polymer consists of only the maleic
anhydride and styrene monomers and their ratio.
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2.3 Challenges and future research
The method suggested would be a very good starting point in exploring the possibilities of using a protein
template for the synthesis of precisely defined polymers. The amber suppression technique is very efficient, so a
high enough protein yield is expected. Also, the distance between the monomers (4.9 Å) and the two β-sheet
strands (4.7 Å) are very alike, this indicates that also the polymerization will go well. If this protein template
would not work as desired, it might be possible to use an alpha helix protein. The distance between two helical
turns is approximately 5.4 Å. Since less natural amino acids are incorporated between each unnatural amino
acids in the alpha helix than in the β-sheet protein, it would be easier to use methods such as native chemical
ligation to make synthetic proteins to test the different properties for the polymerization. However, it is possible
that the incorporation of unnatural amino acids is more difficult, since the unnatural amino acids are more
frequently incorporated. On the other hand, in the β-sheet protein, two consecutive unnatural amino acids will
have to be incorporated.
The main problem with this technique might be the initiation of the polymerization. If the proposed
method for initiation, using thermal activation, degrades the protein, the use of an initiator is required as an
alternative method. For this purpose benzoyl peroxide will be added in free solution and initiation will be able to
occur throughout the whole protein template. If the polymerization does not proceed through the entire template
in both directions and no other initiation methods are possible, this approach will be abandoned. The second
proposed system (section 3) will not have this problem.
Although a complex design is not possible with the maleic anhydride and styrene method, the basics of
using a protein template will be examined. The experience gained by testing this system can then be used for
the development of more complex systems. A second more complex system will be discussed in chapter 3.
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3 Poly-pentadienoic acid
The copolymer of maleic anhydride and styrene is only adjustable in length, while the goal of using a protein as
template is to be able to design the complete polymer, in length and sequence. In a more flexible system, this
goal could be reached. Since the polymerization should be controlled to prevent undesirable cross-reactions, an
initiated polymerization should be used. Atom transfer radical polymerization (ATRP) is a controlled
polymerization technique, which uses an initiator to start the polymerization and is therefore selected for this
system.32
To execute this type of polymerization, at least two different unnatural amino acids should be
incorporated in the protein: the initiator and a monomer. With this system poly-pentadienoic acid will be
produced. Although no complex sequence will be formed, this system is easily adjustable for the synthesis of
more complex polymers, since ATRP is also compatible with multi-monomer systems.32 This could be reached by
incorporating multiple different unnatural amino acids as monomers.
3.1 Incorporation of multiple different unnatural amino acids
Two unnatural amino acid will be incorporated per turn of the anti-parallel β-sheet, just as in the first proposed
system (section 2.1.3). Polymerization via atom transfer radical polymerization requires an initiator. To maintain
control over the polymerization, this initiator should also be incorporated in the protein. Because two different
unnatural amino acids have to be incorporated, two orthogonal incorporation methods are required to do so.9 For
the unnatural amino acid that will be polymerized we will use amber suppression technique as is described in
section 2.1.1. For the initiator the quadruplet system as is developed by Chin and co-workers is selected.33
Since the systems for the incorporation of two distinct unnatural amino acids should be orthogonal, the
AARS/tRNA pairs should also be orthogonal. Therefore the unnatural amino acids should be based on two distinct
amino acids and the AARS should be derived from two different species.34 An alternative for the anti-parallel β-
sheet is an alpha helical structure, as discussed in section 2.1.3.
3.1.1 Quadruplets
The quadruplet system is an elegant translation system that uses four nucleotides instead of three for a single
codon. Because of this, the quadruplet system provides 44 = 256 possible combinations for encoding, instead of
43 = 64 combinations with triplet codons. Since all these 256 combinations are ‘blank codons’, the theoretical
amount of different unnatural amino acids that could be incorporated in single translational system increases
immensely with the use of quadruplets.
Since the natural translation system does not accept quadruplet codons, an orthogonal system had to be
created. For this purpose, Ribo-Q1 was developed, an orthogonal ribosome that accepts orthogonal tRNA’s with
an extended anticodon loop. Furthermore, special orthogonal AARS/tRNA pairs were developed.33-37. Natural
ribosomes cannot be evolved, since this would enhance misreading of the proteome. While the highest efficiency
used to be 20-30% for quadruplet codons15, Jason Chin and co-workers improved the Ribo-Q1 system so it is
able to incorporate unnatural amino acids responding to a quadruplet with high fidelity and efficiency up to 80%
for amber codon derivatives.23,33,34
The Ribo-Q1 system will be used to incorporate the initiator and the polymerizing unnatural amino acid.
The polymerizing unnatural amino acid will be incorporated in response to amber codons (as described in section
2.1.1). For the initiator a tyrosine based quadruplet will be used, since quadruplets using amber codon
derivatives will interfere with the triplet based amber suppression. The Ribo-Q1 system is very well able to
respond to quadruplets as well as amber codons.34 When Ribo-Q1 is used in the C321ΔPRfa strain of E. coli the
efficiency is comparable to natural translation.23 The efficiency for quadruplet decoding is significantly enhanced
up to 30% for non-amber codon derivatives.23 Although this efficiency is not high enough to effectively
incorporate multiple unnatural amino acids through the use of quadruplets, we deem it high enough for the
incorporation of the initiator. When the incorporation of the initiator fails due to the low efficiency, it will not
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Figure 10: a) Proposed unnatural amino acid to serve as monomer in polymerization (lysine analogue). X: NH or O, R-group: H, alkyl group, cyanide, ethoxy, Cl or F. b) Proposed unnatural amino acid as initiator (tyrosine analogue). X: NH or O.
cause errors in the ultimately synthesized polymer, since the atom transfer radical polymerization will not start
without an initiator.32 In our opinion, the combination of this quadruplet system and amber suppression gives
overall the highest efficiency for the incorporation of our desired unnatural amino acids.23
3.1.2 tRNA synthetase pairs
For the incorporation of two distinct unnatural amino acids, two different, orthogonal sets of tRNA/tRNA
synthetase are needed. To incorporate the proposed monomer (section 3.1.3) the same AARS/tRNA pair
(PylRS/tRNACUA) as described in section 2.1.2 will be used.
The tRNA/tRNA synthetase used for the incorporation of the initiator would be derived from the tyrosine
tRNA synthetase from M. jannaschi (TyrRS).38 This AARS/tRNA pair is also orthogonal and has already been used
for the incorporation of various unnatural amino acids based on tyrosine.11 For the use of this pair in the
quadruplet technique a four base codon derived from a codon for tyrosine will be used.15
One favourable property of TyrRS is that is AARS is not capable of proofreading a unnatural amino acid
ligated to the synthetase, since it has no editing system.38 The pocket of TyrRS is not as large as in PylRS, and
therefore it is likely that enzyme evolution is necessary to efficiently incorporate our desired unnatural amino
acid.18,16,23
3.1.3 Unnatural amino acid
The unnatural amino acid that serves as the monomer incorporated in this system is derived from lysine, just as
the unnatural amino acid proposed in the maleic anhydride/styrene system (Figure 9a). This unnatural amino
acid has a conjugated double bond and covers the distance between two β-sheet strands of the protein (4.7 Å)
quite well, as it is able to cover a distance of 4.9 Å. A conjugated double bond is a quite regular sequence for
polymerization, and pentadienoic acid well available. It's likely that also other derivatives of the proposed
monomer are polymerizable through this system. At the start of the research, multiple monomers will be tested
for polymerization, so the effect of different monomers in this system can be discussed. Different R-groups might
have an interesting influence on the polymerization due to electronic effects. A cyanide group would create a
more electron deficient neighbouring carbon, whereas an ethoxy group would be more electron donating and
therefore cause a higher electron density on the carbon. R-groups easily available would for example be: H, alkyl
groups, cyanide, ethoxy group, chloride or fluoride.
As discussed before, the initiator will be incorporated by using a tyrosine based system. The unnatural
amino acid is therefore a tyrosine analogue and also contains a peptide linkage for hydrolysis after
polymerisation (Figure 9b). An ester linkage might also be possible for milder separation.
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A B C
Figure 11: A) Protein template with initiator and incorporated polymerizable unnatural amino acids. The red arrows indicate the β-sheet protein. B) The result after polymerization. C) The synthesized polymer after separation from the protein. Altered from Jordano S.
1
3.2 Polymerization
For this method we choose to use atom transfer radical polymerization (ATRP)This technique is widely used in
the production of all different kinds of polymers. With ATRP it is possible to produce polymers with a special
design, such as polymers with alternating monomers or a gradient.32 However more complicated design in
sequence is not yet possible. In our opinion complex sequences will be possible using protein templates. In the
following, the exact procedure of polymerization of the incorporated unnatural amino acids will be explained.
3.2.1 ATRP
In atom transfer radical polymerization (ATRP) an initiator and catalyst are necessary to start the polymerization.
A common used catalyst in ATRP is a copper(I) complex32 , e.g. [CuCl-2,2’-bipyridine].39 The optimal conditions
for the polymerization, such as concentrations, solvents and temperature, have to be determined. It is also
important to investigate which initiator, monomer and catalyst cause for the highest yield. Another significant
factor is that the protein template remains intact in the process of polymerization. The initiator has to contain a halogen that is easy removable. This halogen will associate with the catalyst and a radical is left at the initiator. This radical will then react with a double bond that is nearby to form a new single bond. A new radical will then form the next bond, etc., until a polymer has formed.
32 The initiator has to be placed at the first loop in the protein to
maintain control over the polymerization. In this way polymerization will occur in only one way through the protein template. The process from protein template to polymer is shown in A B C
Figure 11.
3.3 Challenges and future research
This proposed system creates great flexibility in the synthesis of monodisperse polymers. Since an incorporated
initiator is used, polymerisation will only start at the beginning of the protein template. A difficulty arises with the
incorporation of the initiator as a second unnatural amino acid. While this is achievable with a combination of
amber suppression and tyrosine based quadruplets the efficiency and thus the yield will decrease to about
30%.23 As the ultimate goal is to synthesize polymers with a complex sequence, multiple different unnatural
amino acids will have to be incorporated. The current efficiency will make this a challenge, however, seeing the
rapid improvement in the incorporation of unnatural amino acids, this might not be a problem in the near future.
In 2006 researchers introduced the four-base codon/anticodon pairs to mammalian cells40, in April 2014 it was
already possible to optimize orthogonal translation with the use of amber suppression and the quadruplet
technique.33 If these incorporation techniques are available, the current proposed system could directly be used
for the synthesis of precisely defined polymers with more complex sequences.
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4 Challenges & Innovations
This research proposal presents an entirely new technique for the synthesis of precisely defined polymers. With
this innovative approach some challenges will be encountered. While some of the challenges have already been
mentioned or explained in earlier sections, in this section they will be summarized and possible solutions will be
given. Furthermore, the innovative elements of this research are mentioned.
4.1 Challenges
4.1.1 Possible early release of the protein
When translation is hindered, the protein that is translated will not completely form and it will be released too
early from the ribosome. This could lead to unintended errors in length of the polymer. To ensure that these
unfinished proteins will not be part of the polymerization, a His-tag is incorporated at the C-terminal of the
protein, so selection is possible as is discussed in section 2.1.3.
4.1.2 Distance between reactive groups
The method of using a β-sheet protein as a template for polymerization solves the problem of having a too low
efficiency of the incorporation of unnatural amino acids. The protein also provides a fixed structure, more
suitable for polymerization. However, the length of one monomer after the polymerization should ideally be
exactly equal to the distance between the turns of the β-sheet. If this is not the case stress will be induced in the
protein and polymer, which will disadvantage the polymerization. This would most likely result in the
polymerization to stop or the template to be destroyed. To avoid this, the length of the polymerizable side chains
has to be examined, so that they are likely to fit, just as done in sections 2.1.4 and 3.1.3. If one wants to
incorporate other unnatural amino acids than the ones discussed in this proposal, this limitation always needs to
be considered.
4.1.3 Solubility of the template
To execute the polymerization and to purify the ultimately formed polymer, different separation methods are
proposed. However, these methods depend on the properties of the formed protein. It is possible that the
formed protein is not soluble in aqueous solution. The incorporated unnatural amino acids could alter the
properties of the formed protein. To enhance the solubility glutamic acid is incorporated at the lower turns
(section 2.1.3). To further enhance the solubility it is possible to use well soluble fusion proteins.
4.1.4 Possible reaction of the styrene monomers
As already discussed in section 2.3, it is possible that the styrene monomers polymerize with each other. The
maleic anhydride monomers, however, cannot form homopolymers.8 Despite the distance between to styrene
monomers there is still a chance that styrene homopolymers are formed. Optimizing the reaction conditions,
such as the concentration of maleic anhydride are proposed to solve the possible undesired reaction.
4.1.5 Possible reactions between polymers
Another problem we might encounter is the possible reactions between different polymer strains. To make it
unlikely for the polymers to polymerize to another strain, the proteins could be diluted. This solution could be
applied in any polymerization method.
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4.1.6 Incorporation in vitro
If it is not possible to produce the entire desired protein through expression in E. coli, it might be possible to use
expressed protein ligation to connect multiple small protein elements to gain larger protein templates.6 It is also
possible to use flexizymes to produce the protein in vitro.41 Native chemical ligation might be used to synthesize
smaller proteins, for larger proteins this might be quite laborious.31,5 This way it is still possible to produce
precisely defined polymers. The polymerization of both of the systems of unnatural amino acids is therefore
going to be tested using small templates prepared via native chemical ligation before trying to incorporate them,
in order to make sure the polymerization works.
4.2 Innovations
First of all, this research will increase the knowledge about the incorporation of unnatural amino acids, in
particular using amber suppression and the quadruplet technique. Especially the incorporation of this many
unnatural amino acids in one protein has to our knowledge not been shown before. Furthermore, the research
uses a protein as a template for polymerization in a unique and new way. It also provides new applications for
the use of an expanded genetic code. But the most important innovative element of this research is that it is an
enormous step towards synthesis of polymers exactly defined in length and design.
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5 References
1 S., J. Lecture 4: Protein Folding and Dynamics, <https://www.studyblue.com/notes/note/n/lecture-4-protein-folding-and-dynamics/deck/1667628> (2011).
2 Smeenk, J. M. Recombinant production of periodic polypeptides, Introducing structural order in polymeric materials Phd. thesis, Radboud University Nijmegen, (2006).
3 Chin, J. W. Reprogramming the genetic code. The EMBO journal 30, 2312-2324, doi:10.1038/emboj.2011.160 (2011).
4 Litowski, J. R. & Hodges, R. S. Designing heterodimeric two-stranded alpha-helical coiled-coils. Effects of hydrophobicity and alpha-helical propensity on protein folding, stability, and specificity. The Journal of biological chemistry 277, 37272-37279, doi:10.1074/jbc.M204257200 (2002).
5 Raibaut, L., Ollivier, N. & Melnyk, O. Sequential native peptide ligation strategies for total chemical protein synthesis. Chemical Society reviews 41, 7001-7015, doi:10.1039/c2cs35147a (2012).
6 Hackenberger, C. P. & Schwarzer, D. Chemoselective ligation and modification strategies for peptides and proteins. Angewandte Chemie 47, 10030-10074, doi:10.1002/anie.200801313 (2008).
7 Chen, G. Q., Wu, Z. Q., Wu, J. R., Li, Z. C. & Li, F. M. Synthesis of Alternating Copolymers of N-Substituted Maleimides with Styrene via Atom Transfer Radical Polymerization. Macromolecules 32, 232-234 (2000).
8 Moore, E. R. & Pickelman, D. M. Synthesis of Styrene/Maleimide Copolymers and Physical Properties Thereof. Industrial & Engineering Chemistry Product Research and Development 25, 603-609 (1986).
9 Neumann, H. Rewiring translation - Genetic code expansion and its applications. FEBS letters 586, 2057-2064, doi:10.1016/j.febslet.2012.02.002 (2012).
10 Furter, R. Expansion of the genetic code:. Protein Science 7, 419-426 (1998). 11 Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annual review of biochemistry
79, 413-444, doi:10.1146/annurev.biochem.052308.105824 (2010). 12 Link, A. J. & Tirrell, D. A. Reassignment of sense codons in vivo. Methods 36, 291-298,
doi:10.1016/j.ymeth.2005.04.005 (2005). 13 Krishnakumar, R. & Ling, J. Experimental challenges of sense codon reassignment: an innovative
approach to genetic code expansion. FEBS letters 588, 383-388, doi:10.1016/j.febslet.2013.11.039 (2014).
14 Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nature biotechnology 25, 770-777, doi:10.1038/nbt1314 (2007).
15 Lammers, C., Hahn, L. E. & Neumann, H. Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes. Chembiochem : a European journal of chemical biology 15, 1800-1804, doi:10.1002/cbic.201402033 (2014).
16 O'Donoghue, P., Ling, J., Wang, Y. S. & Soll, D. Upgrading protein synthesis for synthetic biology. Nature chemical biology 9, 594-598, doi:10.1038/nchembio.1339 (2013).
17 An, W. & Chin, J. W. Synthesis of orthogonal transcription-translation networks. Proceedings of the National Academy of Sciences 106, 8477-8482, doi:10.1073/pnas.0900267106 (2009).
18 Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 292, 498-500, doi:10.1126/science.1060077 (2001).
19 Chin, J. W. Reprogramming the genetic code. Science 336, 428-429, doi:10.1126/science.1221761 (2012).
20 Johnson, D. B. et al. RF1 Knockout Allows Ribosomal Incorporation of Unnatural Amino Acids at Multiple Sites. Nature chemical biology 7, 779-786, doi:10.1038/nchembio.657 (2011).
21 Lajoie, M. J. et al. Genomically Recoded Organisms Expand Biological Functions. Science 342, 357-360 (2013).
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22 Wan, W., Tharp, J. M. & Liu, W. R. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochimica et biophysica acta 1844, 1059-1070, doi:10.1016/j.bbapap.2014.03.002 (2014).
23 Sachdeva, A. (Cambridge, February 2015). 24 He, Y. X. et al. N-Terminal domain of Bombyx mori fibroin mediates the assembly of silk in response
to pH decrease. Journal of molecular biology 418, 197-207, doi:10.1016/j.jmb.2012.02.040 (2012). 25 Krejchi, M. T. et al. Chemical Sequence Control of β-Sheet Assembly in Macromolecular Crystals of
Periodic Polypeptides. Science 265, 1427-1432 (1994). 26 C., Z. et al. Silk Fibroin: Structural Implications of a Remarkable Amino Acid Sequence. PROTEINS:
Structure, Function, and Genetics 44, 119-122 (2001). 27 Cantor, E. J. et al. Effects of Amino Acid Side-Chain Volume on Chain Packing in Genetically
Engineered Periodic Polypeptides. The Journal of Biochemistry 122, 217-225 (1999). 28 Crick, F. H. C. The Packing of a-Helices: Simple Coiled-Coils. Acta Crystallographica 6, 689-697 (1953). 29 Vollrath, F., Hawkins, N., Porter, D., Holland, C. & Boulet-Audet, M. Differential Scanning Fluorimetry
provides high throughput data on silk protein transitions. Scientific Reports 4, 1-6 (2014). 30 Flanagan, K. E., Tien, L. W., Elia, R., Wu, J. & Kaplan, D. Development of a sutureless dural substitute
from Bombyx mori silk fibroin. Journal of biomedical materials research. Part B, Applied biomaterials 103, 485-494, doi:10.1002/jbm.b.33217 (2015).
31 Dawson, P., Muir, T., Clark-Lewis, I. & Kent, S. Synthesis of proteins by native chemical ligation. Science 266, 776-779, doi:10.1126/science.7973629 (1994).
32 Matyjaszewski, K. Atom Transfer Radical Polymerization (ATRP): Current Status and Future Perspectives. Macromolecules 45, 4015-4039 (2012).
33 Wang, K. et al. Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nature chemistry 6, 393-403, doi:10.1038/nchem.1919 (2014).
34 Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. & Chin, J. W. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441-444, doi:10.1038/nature08817 (2010).
35 Atkins, J. F. & Bjork, G. R. A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment. Microbiology and molecular biology reviews : MMBR 73, 178-210, doi:10.1128/MMBR.00010-08 (2009).
36 Magliery, T. J., Anderson, J. C. & Schultz, P. G. Expanding the Genetic Code: Selection of Efficient Surpressors of Four-base Codons and Indentification of "Shifty" Four-base Codons with a Library Approach in Escherichia.coli. Journal of molecular biology 307, 755-769 (2001).
37 Stahl, G., McCarty, G. P. & Farabaugh, P. J. Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding. TiBS 27, 178-183 (2002).
38 Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the Genetic Code of Escherichia coli. Science 292, 498-500 (2001).
39 Spijker, H. Nucleobase functionalized polymers prepared by ATRP. Toward DNA mimetic materials. 32-36 (2007).
40 Taki, M., Matsushita, J. & Sisido, M. Expanding the genetic code in a mammalian cell line by the introduction of four-base codon/anticodon pairs. Chembiochem : a European journal of chemical biology 7, 425-428, doi:10.1002/cbic.200500360 (2006).
41 Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chemistry & biology 14, 1315-1322, doi:10.1016/j.chembiol.2007.10.015 (2007).
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5. Timetable of the project
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6. Knowledge utilisation
The possibility to synthetize precisely defined polymers creates endless opportunities for the production of new
materials. The properties of these materials could be precisely altered and designed. Naturally produced proteins
exhibit numerous properties and functions with only a limited number of amino acids. Freedom in design thus
creates enormous possibilities for the production of new functional materials. This could, for example, be
important in the development of medicine. Polymer based nanoparticles for instance, used for drug delivery,
should be unambiguously produced for approval for human usage. The proposed method might eventually
contribute to the production of these particles.
This technique could be taken even further and be expanded to bacteria producing polymers and
plastics. This could lead to plastic producing cells, for production or self-healing materials. The genome provides
lots of control over the synthesis of any desired polymer.
When the efficiency of quadruplet coding is further enhanced it might soon be possible to incorporate a
whole series of different unnatural amino acids into a protein. With this freedom it could be possible to design
the structure of a polymer in any way desired, with great possibilities in length and sequence. Eventually, the
method of using the ribosome to directly produce the polymers might even become the most efficient technique
to make precisely defined polymers.
Furthermore, if the β-sheet method proposed in this research proposal is used, it is also possible to
produce polymers with non-peptide bonds, since the bonds are not created by the ribosome, but by the
polymerization. Eventually, it might be possible to alter the ribosome in such way that polymers could be directly
produced through the natural translation system.
8. Statements by the applicant
YES/NO I endorse and follow the Code Openness Animal Experiments (if applicable).
YES/NO I endorse and follow the Code Biosecurity (if applicable).
YES/NO By submitting this document I declare that I satisfy the nationally and internationally accepted
standards for scientific conduct as stated in the Netherlands Code of Conduct for Scientific
Practice 2012 (Association of Universities in the Netherlands (VSNU)).
YES/NO I have completed this form truthfully.
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