precision polymers through protein templated synthesis · polymers through the concept of protein...

RHA Research Programme

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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.

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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.

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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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