A genetically encoded protein polymer for uranyl binding and

Subscriber access provided by Kaohsiung Medical University

Letter

A genetically encoded protein polymer for uranyl binding and extraction based on the SpyTag-SpyCatcher chemistry Xiaoyu Yang, Jingyi Wei, Yuqing Wang, Changru Yang, Shijun Zhao, Cheng Li, Yiming Dong, Ke Bai, Yuexuan Li, Huaiyuan Teng, Dingyu Wang, Nayun Lyu, Jiamian Li, Xuyao Chang, Xin Ning, Qi Ouyang, Yihao Zhang, and Long Qian ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00223 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

A genetically encoded protein polymer for uranyl binding and extraction

2

based on the SpyTag-SpyCatcher chemistry

3 4

Xiaoyu Yang1,4, Jingyi Wei1,4, Yuqing Wang1,4, Changru Yang1, Shijun Zhao1,

5

Cheng Li1, Yiming Dong1,2, Ke Bai1, Yuexuan Li1, Huaiyuan Teng1, Dingyu

6

Wang1, Nayun Lyu1, Jiamian Li1, Xuyao Chang1, Xin Ning1, Qi Ouyang2,3,

7

Yihao Zhang1,2,*, Long Qian2,*

8 9

1

Peking University Team for the International Genetically Engineered

10

Machine Competition (iGEM), Beijing, 100871, China;

11

2

12

Sciences, Peking University, Beijing, 100871, China;

13

3

14

Physics, School of Physics, Peking University, Beijing, 100871, China.

15

4

Center for Quantitative Biology and Peking-Tsinghua Joint Center for Life

The State Key Laboratory for Artificial Microstructures and Mesoscopic

These authors contributed equally to this work.

16 17

* Correspondence should be addressed to Y.Z. ([email protected]),

18

and L.Q. ([email protected]).

19 20 21

1


Page 2 of 28

Page 3 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Abstract A defining goal of synthetic biology is to develop biomaterials with

3

superior performance and versatility. Here we introduce a purely genetically

4

encoded and self-assembling biopolymer based on the SpyTag-SpyCatcher

5

chemistry. We show the application of this polymer for highly efficient uranyl

6

binding and extraction from aqueous solutions, by embedding two functional

7

modules – the super uranyl binding protein and the monomeric streptavidin –

8

to the polymer via genetic fusion. We further provide a modeling strategy for

9

predicting the polymer’s physical properties, and experimentally demonstrate

10

the auto-secretion of component monomers from bacterial cells. The potential

11

of multi-functionalization, in conjunction with the genetic design and

12

production pipeline, underscores the advantage of the SpyTag-SpyCatcher

13

biopolymers for applications beyond trace metal enrichment and

14

environmental remediation.

15 16

Keywords

17

SpyTag-SpyCatcher, green mining, protein polymer

18 19

2



1 2 3 4 5 6 7 8 9 10 11

For Table of Contents Use Only

A genetically encoded protein polymer for uranyl binding and extraction based on the SpyTag-SpyCatcher chemistry Xiaoyu Yang1,4, Jingyi Wei1,4, Yuqing Wang1,4, Changru Yang1, Shijun Zhao1, Cheng Li1, Yiming Dong1,2, Ke Bai1, Yuexuan Li1, Huaiyuan Teng1, Dingyu Wang1, Nayun Lyu1, Jiamian Li1, Xuyao Chang1, Xin Ning1, Qi Ouyang2,3, Yihao Zhang1,2,*, Long Qian2,*

12 13 14

3


Page 4 of 28

Page 5 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

With the rapid development of bioengineering and synthetic biology, the past

2

two decades have witnessed a bloom of biomaterials fabricated from natural

3

and synthetic polymers for designs of scaffolds1, reaction membranes2 and

4

hydrogels3. The self-assembling, eco-friendly, biocompatible and inexpensive

5

natures make biomaterials a promising solution in applications such as

6

automatic bioreactor for fine chemicals4, drug delivery capsules5,6, and waste

7

filter and biosorbents7-10. However, to fully unleash their potentials, polymer

8

platforms with stable and controllable structures and accommodating multiple

9

functional modules are needed.

10

In this article we report the design of a fully genetically encoded

11

multifunctional protein polymer platform based on the SpyTag-SpyCatcher

12

chemistry. SpyTag (a 13-residue peptide) and SpyCatcher (a 116-residue

13

domain) are engineered split fragments of the second immunoglobulin-like

14

collagen adhesin domain (CnaB2) of the fibronectin-binding protein (FbaB) of

15

Streptococcus pyogenes11. The spontaneous formation of covalent isopeptide

16

bonds between Asp117 of SpyTag and Lys31 of SpyCatcher has made the

17

system a powerful “glue” for bio-conjugation12. Outstandingly, the Spy duo

18

have been used to functionalize blank-slate polymers with desired protein

19

ligands to produce biomaterials with independently tuned biophysical and

20

biochemical properties13,14. Previous studies have also demonstrated the

21

ability of SpyTag-SpyCatcher to directly cross-link peptide fibers forming

22

protein hydrogels15-17 or a multilayer polymer material18. In the present work, a

23

soluble protein polymer network was made from elastin-like peptide

24

monomers containing multiple Spy motifs.

25

We demonstrated the use of the Spy network in gathering and harvesting 4



1

uranyl ions (UO22+) from aqueous solutions, by genetically integrating two

2

functional modules – the super uranyl-binding protein, a rationally designed

3

protein with extremely high affinity and selectivity for uranyl19, and the

4

streptavidin-biotin affinity system20,21. We showed that multiple functions

5

embedded in the network through direct protein fusions to the monomers

6

were unaffected by each other, and were enhanced by the polymeric network

7

structure. Further improvement was made to the bio-production pipeline using

8

bacterial signal peptides that direct the auto-secretion of protein monomers.

9

The uranyl gather and harvest system can be used for uranyl

10

decontamination in natural waters, and for uranyl enrichment from the vast

11

oceanic reserve, which was estimated to be 1,000 times larger than the

12

uranium reserved on land22. This is, however, merely one example of the

13

many potential applications of our Spy network platform. With an ever-

14

increasing number of engineered proteins performing enhanced or novel

15

functions, our design strategy enables the rapid genetic programming of multi-

16

functional biomaterials. Moreover, our Spy network itself represents a purely

17

biological polymer that can be produced and assembled in situ, can

18

orchestrate a series of functions, and whose generation and removal can be

19

genetically controlled. One can imagine the application of such smart

20

polymers in numerous aqueous environments ranging from large water bodies

21

to the human blood.

22 23

Results

24

Design of the Spy-SUP polymer network

5


Page 6 of 28

Page 7 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

To construct a protein-based three-dimensional polymer network that

2

works in aqueous environments such as the sea water, we employed the

3

elastin-like polypeptide (ELP) based SpyTag-SpyCatcher chemistry. ELPs are

4

composed of tandem pentapeptides of the form (VPGXG)n, where X is any

5

amino acid but proline. They were chosen as the polymer backbone for the

6

high solubility, strength and resilience they bring to the material. Practically,

7

ELPs are easily produced in Escherichia coli and work under a wide range of

8

temperature and pH conditions23. Our monomers contain multiples of SpyTag

9

(therein referred to as A) or SpyCatcher (referred to as B) connected by 4x

10

and 15x of the ELP units VPGVG, respectively. The A monomer is further

11

linked to a super uranyl binding protein (SUP) at its C-terminus (Fig. 1a). The

12

highly reactive covalent bonding between SpyTag and SpyCatcher is thus

13

expected to promote extensive cross-linking between the A and B monomers,

14

forming stable polymeric networks within which the functional module, SUP, is

15

immobilized (Fig. 1b).

16

We designed three variants of the A monomer containing three, four,

17

and six SpyTag motifs separated by ELPs (nA-SUP, n = 3,4,6), respectively,

18

and mixed them with the B monomers containing three SpyCatcher motifs

19

(3B). Figure 1c shows the SDS-PAGE analysis for the end products after two-

20

hour incubation at 25°C of 1mg/mL nA-SUP with equimolar 3B. As expected,

21

high molecular weight products were observed when both A and B monomers

22

were present, indicative of the formation of cross-linked polymers, and the

23

compatibility of SUP with polymerization.

24 25

Gelation and working conditions of the Spy-SUP polymer 6



1

With more than two Spy motifs in each monomer, the polymeric network

2

adopts a three-dimensional structure, and its characteristic pore size is

3

determined by the average cross-liking degree between the SpyTag and

4

SpyCatcher motifs. An ideal pore size will properly balance between the

5

solubility and the mechanical strength of the polymer, as well as provide an

6

accommodating micro-environment for SUP stabilization while achieving a

7

maximal surface-to-volume ratio for uranyl adsorption. However, depending

8

on the number of Spy motifs in each monomer and the molar ratio between A

9

and B monomers, a critical cross-linking degree (CLD), i.e. the gel point CLD,

10

exists beyond which the polymer turns into insoluble gel. To allow for

11

optimization of pore size within a wide range, we would like to choose a

12

monomer configuration with a high gel point CLD. We built a mathematical

13

model based on the Flory-Stockmayer Theory24,25 to predict the gel point CLD

14

specifically for three-dimensional hydrogels (Supplemental Information). Our

15

analytical results indicated that gel point CLD decreases with an increasing

16

number of Spy motifs in each monomer, and has a non-monotonic

17

relationship with the molar ratio between the A and B monomers (Fig. 2a).

18

Based on this result, and the observation that 2A-SUP did not cross-link with

19

3B efficiently (data not shown), we selected the 3A-SUP SpyTag monomer for

20

further experiments. Theoretically, a maximal gel point CLD of 50% is

21

achieved at equimolar 3A-SUP and 3B monomers. In other words, as long as

22

cross-linking is kept under 50% complete (e.g. by adjusting monomer

23

concentrations), the polymeric network remains in the colloidal sol state.

24

Meanwhile, a 3A-SUP configuration maximizes the concentration of SUP

7


Page 8 of 28

Page 9 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

within, and hence the adsorption efficiency per unit mass of, the polymeric

2

network.

3

Cross-linking between monomers, especially when the reaction system

4

is far below gel point, can result in a series of polymers of different sizes, the

5

distribution of which affects the efficiency of functional modules such as SUP

6

for uranyl adsorption or mSA for polymer retrieval (monomeric streptavidin,

7

see later sections). By enumerating polymer configurations at reaction

8

equilibrium based on a probabilistic model, we further derived the molecular

9

weight distribution of sol polymers at equilibrium as a function of monomer

10

concentrations. Building on Flory’s classic work26, we introduced a correction

11

term to account for the inhibitory or recruiting effect of reacted groups to later

12

cross-linking reactions for the A and B monomers (Supplemental Information).

13

We found a general agreement of molecular weight distribution between

14

theory and quantitative SDS-PAGE analyses of reaction products (Fig. 2b and

15

Fig. S1). When fitting the correction terms to experimental results, we found

16

that the attachment of functional modules affected cross-linking in different

17

ways. Reaction systems containing SUP had a lower chance of secondary

18

cross-linking on the free A groups but a higher chance on the free B groups.

19

For mSA, the situation was reversed (Supplemental Information).

20

We next studied the temperature and pH conditions for polymerization of

21

the Spy system. Mixtures of equimolar (50µM) 3A-SUP and 3B was incubated

22

at three different temperatures or buffer pH values. Reaction progress was

23

monitored by measuring gel band intensity of free 3B monomers from buffers

24

taken at serial time points (Fig 2c&d). Polymerization was consistently

25

observed within the ranges of pH 6.3-7.3 and temperature 16°C-37°C, with a 8



1

preference for slightly acidic buffers and temperatures above 25°C.

2

Furthermore, under these conditions the reaction plateaued within 2 hours of

3

incubation, reaching >80% maximal reaction extent (data not shown). These

4

results suggested that our Spy network self-assembled promptly and robustly

5

in diverse environments. Lastly, as a test of stability, prolonged incubation of

6

reaction systems up to four days did not lead to significant decay of the

7

polymer products (Fig. S2).

8 9 10

Uranyl adsorption efficiency of the Spy-SUP network We analyzed the uranyl sequestration efficiency of the Spy-SUP network

11

assembled from 3A/4A/6A-SUP and 3B monomers, by an Arsenazo III-based

12

uranyl binding assay19 (Fig. 3a). 10 µM SUP, either in the free nA-SUP form

13

(n = 3,4,6) or the Spy network immobilized form, was incubated with

14

equimolar uranyl nitrate in TBS buffer and allowed for reaction for 1 min. After

15

centrifugation, the remaining uranyl concentration in the filtrate was measured

16

as an indicator of uranyl-sequestration efficiency. We found that depending on

17

gel point CLD, SUP function was either enhanced (3A-SUP+3B) or weakened

18

(6A-SUP+3B) in the immobilized form (Fig. 3a), supporting our hypothesis

19

that excessive polymerization jeopardizes function by obstructing uranyl

20

passage through the polymer network, reducing the chance of uranyl-SUP

21

collision, and/or posing steric hindrance to the adsorption reaction. For the

22

3A-SUP monomer we selected, uranyl sequestration increased from 87% by

23

the free form to 96% by the immobilized form. Further increasing the

24

concentration of 3A-SUP+3B network to ten-fold of uranyl did not result in a

25

significant improvement above the 96% sequestration efficiency, indicating 9


Page 10 of 28

Page 11 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

the 3A-SUP+3B network configuration was most permissive for uranyl

2

adsorption (Fig. 3b).

3

In natural waters, divalent metal ions such as Ca2+ and Mg2+, carbonate,

4

and organic molecules exist in great excess and may significantly interfere

5

with uranyl adsorption by the SUP protein27. To test the applicability of our

6

Spy-SUP network, water samples were taken from a local lake (Lake

7

Weiming, Beijing, China) or the Bohai Bay (Yellow Sea, northeast China), and

8

10 µM uranyl was added as a “pollutant”. At a SUP:uranyl molar ratio of 10:1,

9

the 3A-SUP+3B polymer could sequester 93%, 67% and 83% of total uranyl

10

in polluted TBS buffer, lake water and seawater, respectively (Fig. 3c). We

11

further tested our network’s adsorption of trace amount of uranyl. Natural sea

12

water is estimated to contain uranyl at a 13 nM concentration22. According to

13

inductively coupled plasma mass spectrometry, the 3A-SUP+3B polymer, at

14

6,000 equiv. molar concentration, sequestered 48% and 35% of the 13 nM

15

uranyl added in TBS buffer and artificial seawater (reconstituted with

16

commercial marine salt), respectively (Fig. 3d).

17 18

Retrieval of the Spy-mSA polymer network

19

The above results demonstrated that immobilization of a functional

20

module on the Spy network did not interfere with its function. We went on to

21

test another function frequently coupled to uranyl adsorption. A second

22

monomer was designed to enable retrieval of the polymeric network from

23

aqueous solutions based on the streptavidin-biotin affinity system. A

24

monomeric form of streptavidin (mSA)20 was genetically fused at the C-

25

terminus of the 3A monomer to make the harvest monomer 3A-mSA. As 10



1

reported previously, mSA was highly stable and had a Kd value less than 1nM

2

for biotin binding21. We used biotinylated magnetic beads and a magnetic

3

shelf to retrieve 3A-mSA monomers or the polymeric network formed by 3A-

4

mSA and 3B (Fig. S3). Briefly, the reaction solution was mixed with an excess

5

molar amount of biotinylated magnetic beads and shaken vigorously for an

6

hour before being run through a magnetic shelf. Bradford assay was

7

conducted to measure the concentration of residual proteins in the

8

supernatant after treatment. As shown in Fig. 4a, up to 86% of the 3A-mSA

9

monomer and >75% of the polymeric Spy-mSA network were retrieved, in

10

contrast to a 21% retrieval rate for the 3A monomer without mSA. The

11

difference in retrieval rates of monomeric mSA and polymeric mSA was not

12

significant, suggesting polymer tethering did not affect mSA-biotin

13

interactions. For the Spy-mSA networks, we further applied SDS-PAGE to

14

reaction supernatant after magnetic beads treatment, and found a universal

15

reduction in band intensity across all molecular weights (Fig. 4b), indicating

16

that the embedded mSA was indeed able to extract large polymer networks.

17 18 19

Uranyl retrieval by a bi-functional Spy-SUP-mSA network We hypothesized that multiple functional modules can be tethered to the

20

network by reactions of a combination of distinct 3A-X monomers (X =

21

functional module) with the 3B monomers, achieving multi-functionality for the

22

polymer system. However, as our model showed that different functional

23

groups slightly biased the probabilities of monomer reactions in different

24

ways, and that the functional groups may impart a preference for homotypic

25

monomers for further conjugation, we tested bi-functionality in two 11


Page 12 of 28

Page 13 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

experimental designs.

2

In the first design, monomers 3A-mSA, 3A-mRFP (monomeric red

3

fluorescent protein), and 3B were used to generate retrievable fluorescent

4

polymers. Embedding the mRFP groups alone made the Spy polymer visible

5

under UV light (Fig. S4). We replaced half of the 3A-mSA monomer with 3A-

6

mRFP in the polymer retrieval reaction system, and measured the reduction in

7

fluorescence before and after magnetic beads treatment. A 78% reduction,

8

comparable to the retrieval rate of Spy-mSA (Fig. 4c), was observed, even

9

though the effective mSA concentration in the bi-functional system was

10

halved. This result proved the free association of mRFP with mSA on the

11

polymer backbone and thus the formation of bi-functional polymers. If

12

homotypic polymerization were preferred, retrieval efficiency would have been

13

compromised by a factor representing the fraction of mRFP in single

14

functional Spy-mRFP polymers in the reaction products.

15

In the second design, we made bi-functional Spy-SUP-mSA polymers to

16

achieve uranyl adsorption and extraction. The polymer products of 3A-SUP,

17

3A-mSA and 3B at 1:1:2 molar ratio were used to extract equimolar (10 µM)

18

uranyl contained in buffer, lake water or sea water. After incubation and

19

magnetic beads treatment, 50%-60% uranyl was removed from all samples

20

(Fig. 4d). Given that results in Fig. 3c provided an upper limit for adsorption

21

efficiency of 10 µM uranyl in the respective solutions, the retrieval efficiency

22

by mSA groups in the bi-functional Spy-SUP-mSA polymers achieved at least

23

54%-89%.

24 25

Bacterial Auto-secretion of components for the Spy-SUP network 12



1

Because the procedures of cell lysis and protein purification in extracting

2

intracellular proteins are tedious and costly, and that an automated in situ

3

production pipeline is desirable for environmental applications, we sought to

4

engineer auto-secreted monomer proteins by introducing bacterial signal

5

peptides28. As the ability of signal peptides to facilitate secretion of specific

6

proteins is generally idiosyncratic, we tested four different signal peptides –

7

LTIIb, PhoA, PelB and OmpA – combinatorically with the 3A-SUP, 3A-mSA,

8

and 3B monomers (Fig. 5a). The signal peptides were linked to the N-

9

terminus of the target proteins. For the secretion assay, we employed anti-

10

histag western blot and examined protein concentration in the medium and

11

the cytoplasm (Fig. 5b). Protein bands were observed in the medium for all

12

four signal peptides, but their relative intensities varied between proteins.

13

Nevertheless, for all monomers, OmpA and LTIIb consistently promoted better

14

secretion than PhoA and PelB. For the 3A-SUP monomer, we analyzed its

15

localization at a finer resolution, and found a considerable amount of protein

16

retained in the periplasm. This suggested that increasing cell wall permeability

17

of the expression host, E. coli, e.g. by expressing bacteriocin release protein

18

genes at a subdued level29, may provide further improvements.

19 20 21

Discussion In this study, we demonstrated the formation of a multi-functional

22

biopolymer based on the SpyTag-SpyCatcher chemistry. All components of

23

our network are biomaterials produced directly from living cells and are thus

24

non-toxic and easily degradable. With the secretion strategy that we

25

proposed, autonomous production and assemblage can be achieved, and 13


Page 14 of 28

Page 15 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

requirements for chemical processing and the associated time and financial

2

costs as well as the environmental impacts may be lifted and mitigated.

3

The two basic components of our Spy network, ELPs and the Spy

4

motifs, are both adapted from naturally occurring proteins. ELPs provides high

5

solubility, strength, and resilience, with tunable working temperature by the

6

flexible X residue. SpyTag and SpyCatcher form covalent bonds rapidly within

7

at least 4-37°C, at pH 5-8, and without specific ion requirements11. Our

8

network self-assembled at pH values 6.3-8.3 and at temperatures 16°C-37°C,

9

both in buffer and natural water samples, and thereby may fit in most

10

application scenarios.

11

Our Spy-SUP network adopts a three-dimensional structure with a

12

relatively low degree of cross-linking to exist in a colloidal sol state. The

13

functional module, SUP, was terminally fused to an ELP linker. This is in

14

contrast to a previous study, where the hydrogel form was used for cell

15

encapsulation, and the functional module, the pluripotency maintenance factor

16

LIF, is linked in between ELP15. We argue that our network is more permissive

17

to the passage of reactants (here UO22+) in solution, and the functional

18

module has flexibility in finding an ideal reaction microenvironment within the

19

network, whereas in the other study, an in-between functional module is more

20

tightly coupled to the gel structure, allowing for local maintenance of cells

21

confined in the hydrogel. Indeed, the same hydrogel-based design was used

22

in a recent study to adsorb uranyl or chromate by replacing LIF with SUP or

23

ModA (a chromate binding protein) in the Spy-ELP polymer17. However, a

24

dense structure in the gel or the microbeads form led to significant non-

25

specific adsorption by the ELP backbone due to the large amount of 14



1

negatively charged Glu residuals used at ELP’s flexible position. We did not

2

observe such an effect for our soluble polymer designed to have a low cross-

3

linking degree.

4

As our quantitative models predicted, the physical state and molecular

5

weight distribution of the Spy network can be modulated through changes of

6

Spy motif numbers in each monomer, and the molar ratio between monomers.

7

Likewise, properties such as the characteristic pore size in our network awaits

8

further experimental characterization and optimization. These emphasize the

9

ease, for a genetically-encoded system such as ours, of changing/modifying

10

polymer components and configurations through standard genetic

11

manipulations to make designer polymers specialized for each individual

12

application.

13

Our Spy polymer design displayed great modularity, in that all three

14

functional modules (SUP, mSA and mRFP) embedded into the network

15

remained stable and functional under proper conditions. Moreover, for SUP,

16

immobilization to the polymer network enhanced uranyl adsorption by 10%

17

(96% vs. 87%). This may have been attributed to the electrostatic interaction

18

and hydrodynamic resistance of the polymer in solution, which, as was

19

discussed in detail by Paul J. Flory30, leads to stronger interactions between

20

uranyl ions and the polymer network, and thus a relatively high local uranyl

21

concentration. Previous work also demonstrated that immobilized enzyme

22

was more stable31 due to the buffering and shielding effect of the polymer

23

network. Compared with previous approaches using SUP embedded in

24

hydrogel microbeads17,32, our soluble polymer achieved comparable or better

25

sequestration in shorter time from buffer or seawater samples supplemented 15


Page 16 of 28

Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

with 5-10 µM uranyl. At the 13 nM uranyl concentration as in natural sea

2

water, it was shown that sequestration efficiency reached 90% by SUP

3

immobilized on amylose resin and 60% by SUP displayed on host cell

4

surface, respectively19. Our system sequestered 35% currently; further

5

improvements such as adjusting polymer linkage density and incubation time

6

are likely to increase this number to a similar range.

7

We showed examples of bi-functional Spy networks, namely, Spy-mSA-

8

mRFP and Spy-SUP-mSA. In both cases no interference between functional

9

modules was observed. In particular, the embedding of mSA allowed uranyl

10

retrieval without the need for centrifugation. It should be noted that

11

incorporation of multiple functional modules may dilute each one’s effective

12

concentration. This negative effect however may be overcome by adjusting

13

their relative ratios or by sequential induction of functional modules through

14

genetic control. Beside SUP, currently available are many engineered metal

15

binding proteins targeting e.g. lead, cadmium and mercury33-35 with high

16

affinity and selectivity. These, alone or in combination, can be linked into the

17

Spy network to deal with complex heavy metal contaminations. Furthermore,

18

the Spy network may serve as a reaction platform integrating sequestration

19

(binding modules), in situ processing (enzymatic modules), and retrieval (e.g.

20

mSA). Lastly, such genetically encoded reaction platform may be put under

21

control of other synthetic circuits in cells to achieve conditional functions. Our

22

Spy polymer design add to the synthetic biology toolbox, and we hope it

23

would inspire the development of many biological smart polymers for diverse

24

environmental and biomedical applications

25 16



1

AUTHOR INFORMATION

2

Corresponding Authors

3

*

Email: [email protected]

4

*

Email: [email protected]

5 6

Author Contributions

7

4

8

and L.Q. conceived the project. X.Y., J.W., Y.W., C.Y., S.Z., C.L., Y.D., K.B.,

9

Y.L., D.W., N.L., J.L., X.C. and X.N. designed and performed experiments.

10

C.Y., H.T. and L.Q. constructed the model. L.Q., Y.Z., X.Y. and J.W. created

11

figures and wrote the manuscript.

These authors contributed equally to this work. X.Y., J.W., C.L., Q.O., Y.Z.

12 13

NOTES

14

The authors have filed a provisional patent application related to this work.

15 16

ACKNOWLEDGEMENT

17

We thank Prof. Wenbin Zhang (Peking University), Prof. Lu Zhou (Fudan

18

University), Prof. Chunbo Lou (Chinese Academy of Sciences), Dr. Haoqian

19

Zhang, and Dr. Daqi Yu for constant advice, discussion and technical

20

supports. We are grateful to Prof. Chunli Liu (Peking University) for providing

21

necessary reagents and equipment for uranyl assays. This work was

22

supported by the National Natural Science Foundation of China (No.11434001

23

and No.11774011).

24

17


Page 18 of 28

Page 19 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Material and Methods Cloning and protein purification The genes of SpyTag and SpyCatcher with ELPs were kind gifts from Prof. Wenbin Zhang (Peking University, China), and the gene of SUP was from Prof. Lu Zhou (Fudan University, China). Phusion® High-Fidelity DNA Polymerase (NEB) was used for all PCR amplifications. The coding sequences for each recombinant protein were cloned into plasmid pET21a by Golden Gate assembly. Amino acid sequences of proteins/peptides used in this study are listed in Fig. S5. Plasmids were transformed into E. coli strain BL21 (DE3) for protein monomer expression. Cells were induced with 1 mM IPTG and grown overnight, then centrifuged at 8000 rpm for 15 min at 4°C. Cell pellets containing recombinant proteins were re-suspended in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol, pH7.4) containing SIGMAFAST™ Protease Inhibitor Cocktail Tablets (SIGMAALORICH®). The supernatant was filtered through a 0.22 µm filter and then passed through a HisTrap™ column (GE Healthcare, Inc.). The target protein monomer was eluted with a linear gradient starting with binding buffer and ending with the same buffer containing 500mM imidazole and then concentrated. SDS-PAGE was used to confirm protein purification and Braford analysis was used for quantification. Temperature and pH gradient assays For the temperature gradient assay, protein solutions were prepared and diluted to 1 mg/mL in 20 mM TBS (pH = 7.3), respectively. 3A-SUP and 3B were mixed at equimolar concentrations and incubated for 2 hours at different temperatures (16°C, 25°C, 37°C). For the pH gradient assay, protein solutions were prepared and diluted to 1 mg/mL in 20 mM TBS buffer at different pH (pH = 6.3/7.3/8.3). 3A-SUP and 3B were mixed at equimolar concentration and incubated for 2 hours at RT. 20 µL samples were taken every 30 minutes during the reaction and then boiled at 95°C for 7 minutes. After boiling, samples were placed at 25°C for 10 minutes and then centrifuged briefly. Finally, samples were applied to SDS-PAGE at 150 V, followed by Coomassie Brilliant Blue staining. After the process, SDS-PAGE gels were scanned and analyzed by software Lane 1D to find out the surplus content of monomers (compared with negative control groups) and the mass distribution of oligomers. Uranyl binding assay 3A/4A/6A-SUP and 3B were pre-incubated for 2 hours for polymerization. They were then mixed with uranyl samples and diluted to reach the final concentration of 10 µM each (pH 7.0). Solutions were incubated for 1 min with rotation before transferred into ultrafiltration tubes (MWCO: 3 kDa) and centrifuged at 5000 rpm for 15 min. A modified Arsenazo III assay19 was employed to determine the residual uranyl concentration in the filtrate. For samples containing trace amount of UO22+ (13 nM), the residual uranyl concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS)19. 18



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Biotinylated magnetic beads preparation Biotin was firstly dissolved in DMSO to a final concentration of 50 mg/mL. Incubation buffer was prepared with DMSO, biotin solution, MES buffer to a volume ratio of 0.5:0.5:9. The mixture was shaken vigorously until clear. EDC was added to the mixture to a final concentration of 1%. 1 ml aminoconjugated magnetic beads were pipetted to a microfuge tube. The supernatant was removed when the beads were precipitated using magnetic shelf. Then beads were washed in pre-cooled MES buffer for three times, then incubated in the incubation buffer and shaken for 2 hours at 0°C. Biotinylated magnetic beads were finally transferred and stored in PBST buffer containing 1 M NaCl at 4°C. mSA retrieval efficiency assay 1 mg/ml 3A-mSA and 10 mg/ml biotinylated magnetic beads were mixed at a volume ratio of 10:1 and were shaken vigorously for 1 hour at 37°C. Supernatant and biotin-coated beads were separated by a magnetic shelf. The concentration of residual mSA in supernatant was measured by Bradford assay. Uranyl retrieval efficiency by the bi-functional Spy-SUP-mSA polymer Mixture of 3A-SUP, 3A-mSA and 3B was incubated for 1 hour and then transferred into uranium-polluted water samples. The reaction samples contained 10 µM uranium, 10 µM 3A-SUP, 10 µM 3A-mSA and 20 µM 3B, and was incubated for 1 min at RT. 15-fold molar amount of magnetic beads were added into solution for retrieval. The reaction system was shaken for 1 hour. Uranyl concentration of the supernatant was measured by Arsenazo III assay. Isolation of cellular fractions of the secreted proteins Medium fraction: cells were harvested by centrifugation at 10,000 × g for 10 min at 4°C, and then a filter with 3 kDa cutoff was applied to concentrate 10 ml of medium to approximately 1 mL, yielding a concentration factor of approximately 10×. Periplasmic fraction: the cell pellet (generated from the centrifugation) was re-suspended thoroughly in 30 mL of 30 mM Tris-HCl, 20% sucrose, at pH 8. Then 60 µL 0.5 M EDTA at pH 8 was added to reach a final concentration of 1 mM. The solution was stirred slowly by a magnetic stir bar at RT for 10 min. The cells were then collected by centrifugation at 4°C for 10 min at 10,000 × g. The pellet was thoroughly re-suspended in 30 mL of icecooled 5 mM MgSO4 and the cell suspension was stirred slowly for 10 min on ice. During this step, the periplasmic proteins were released into the buffer. The solution was then centrifuged at 4°C for 10 min at 10,000 × g to pellet the shocked cells. A filter with 3 kDa cutoff was used to concentrate 10 mL of buffer (periplasmic fraction) to approximately 1 mL, yielding a concentration factor of approximately 10×. Cytoplasmic fraction: the cytoplasmic fraction was acquired using the same method as the pellet processing procedures in protein purification. All of the isolated fractions were mixed with 5× SDS-loading buffer, and immediately heated for 3 min at 85°C to denature the proteins. They were store at –20°C until SDS-PAGE analysis. 19


Page 20 of 28

Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Western Blot for Protein Secretion Assay 10 µL of different components of the cell (medium, cytoplasmic and periplasmic components) were mixed with 10 µL 2× SDS loading buffer and separated by SDS/PAGE. Proteins were transferred to the PVDF membrane (Merck®) at 100 mA for 30 min. The membrane was rinsed with 15 mL PBST containing 0.1% Tween-20 followed by blocking with 15 mL of 5 wt% non-fat milk in PBST at RT. The membrane was washed three times with PBST and incubated overnight at 4°C with 10 mL mouse anti-Histag antibody (Abcam®) at 1:10,000 dilution in PBST containing 5 wt% non-fat milk. The membrane was then washed three times with 15 mL PBST and incubated with goat antimouse IgG HRP conjugate (Abcam®) at 1:8,000 dilution at RT for 1 hour, and then washed three times with 15 mL PBST before adding the DAB substrate to the membrane for imagining. Modeling and calculation of gel point CLD and molecular weight distribution For model details see Supplemental Information. A software to calculate gel point CLD and molecular weight distribution of the polymer products is available at http://2016.igem.org/Team:Peking/Workplace

SUPPORTING INFORMATION Supplemental Figures S1-S5 Supplemental Text

20



1

References

2

(1) Zohora, F. T., and Azim, A. (2014) Biomaterials as porous scaffolds for

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

tissue engineering applications: A review. European Scientific Journal 10， 186-209. (2) Muffler, K., and Ulber, R. (2014) Productive biofilms. Adv. Biochem. Engin./Biotechnol. 146: V-VI (3) Yang, J., Yao, M.-H., Jin, R.-M., Zhao, D.-H., Zhao, Y.-D., and Liu, B. (2017) Polypeptide-engineered hydrogel coated gold nanorods for targeted drug delivery and chemo-photothermal therapy. ACS Biomater. Sci. Eng. 3, 2391-2389. (4) Gross, R., Lang, K., Bühler, K., and Schmid, A. (2010) Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnol. Bioeng. 105, 705–717. (5) Zhao, X., Kim, J., Cezar, C. A., Huebsch, N., Lee, K., Bouhadir, K., and Mooney, D. J. (2011) Active scaffolds for on-demand drug and cell delivery. Proc. Natl. Acad. Sci. U.S.A. 108, 67–72. (6) Ren, D., Kratz, F., and Wang, S.-W. (2014) Engineered drug-protein nanoparticle complexes for folate receptor targeting. Biochem. Eng. J. 89, 33– 41. (7) Courchesne, N.-M. D., Duraj-Thatte, A., Tay, P. K. R., Nguyen, P. Q., and Joshi, N. S. (2016) Scalable production of genetically engineered nanofibrous macroscopic materials via filtration. ACS Biomater. Sci. Eng. 3, 733–741. (8) Farooq, U., Kozinski, J. A., Khan, M. A., and Athar, M. (2010) Biosorption of heavy metal ions using wheat based biosorbents – A review of the recent literature. Bioresour. Technol. 101, 5043–5053. (9) Lin, S., and Juang, R. (2009) Adsorption of phenol and its derivatives from water using synthetic resins and low-cost natural adsorbents: A review. J. Environ. Manage. 90, 1336–1349. (10) Srinivasan, A., and Viraraghavan, T. (2010) Decolorization of dye wastewaters by biosorbents: a review. J. Environ. Manage. 91, 1915–1929. (11) Zakeri, B., Fierer, J. O., Celik, E., Chittock, E. C., Schwarz-Linek, U., Moy, V. T., and Howarth, M. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. U.S.A. 109, E690–7. (12) Reddington, S.C. and Howarth, M. (2015) Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr. Opin. Chem. Biol. 29, 94-99 (13) Gao, X., Lyu, S., and Li, H. (2017) Decorating a blank slate protein hydrogel: a general and robust approach for functionalizing protein hydrogels. Biomacromolecules 18, 3726-3732. (14) Liu, X., Yang, X., Yang, Z., Luo, J., Tian, X., Liu, K., Kou S., and Sun, F. (2018) Versatile engineered protein hydrogels enabling decoupled mechanical and biochemical tuning for cell adhesion and neurite growth. ACS Appl. Nano Mater. 1, 1579-1585. (15) Sun, F., Zhang, W., Mahdavi, A., Arnold, F. H., and Tirrell, D.A. (2014) Synthesis of bioactive protein hydrogels by genetically encoded SpyTagSpyCatcher chemistry. Proc. Natl. Acad. Sci. U.S.A. 111, 11269–11274. (16) Gao, X., Fang, J., Xue, B., Fu, L., and Li, H. (2016) Engineering protein 21


Page 22 of 28

Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

hydrogels using spycatcher-spytag chemistry. Biomacromolecules 17, 28122819. (17) Kou, S., Yang, Z., Luo, J., and Sun, F. (2017) Entirely recombinant protein-based hydrogels for selective heavy metal sequestration. Polym. Chem. 8, 6158–6164. (18) Zhang, X., Wang, X., Da, X., Shi, Y., Liu, C., Sun, F., Yang, S., and Zhang, W. (2018) A Versatile and Robust Approach to Stimuli-Responsive Protein Multilayers with Biologically Enabled Unique Functions. Biomacromolecules 19, 1065-1073. (19) Zhou, L., Bosscher, M., Zhang, C., Ozçubukçu, S., Zhang, L., Zhang, W., Li, C. J., Liu, J., Jensen, M. P., Lai, L., and He, C. (2014) A protein engineered to bind uranyl selectively and with femtomolar affinity. Nat. Chem. 6, 236–241. (20) Lim, K. H., Huang, H., Pralle, A., and Park, S. (2013) Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol. Bioeng. 110, 57–67. (21) Demonte, D., Drake, E. J., Lim, K. H., Gulick, A. M., and Park, S. (2013) Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins 81, 1621–1633. (22) Nozaki, Y. (1997) A fresh look at element distribution in the North Pacific Ocean. Trans., Am. Geophys. Union 78, 221–221. (23) MacEwan S.R., Chilkoti A. (2010) Elastin-like polypeptides: biomedical applications of tunable biopolymers. Biopolymers 94, 60-77. (24) Flory, P. J. (1941) Molecular Size Distribution in Three Dimensional Polymers. I. Gelation. J. Am. Chem. Soc. 63, 3083–3090. (25) Stockmayer, W. H. (1944) Theory of Molecular Size Distribution and Gel Formation in Branched Polymers II. General Cross Linking. J. Chem. Phys. 12, 125–131. (26) Flory, P. J. (1952) Molecular size distribution in three dimensional polymers. VI. Branched polymers containing A—R—Bf-1 type units. J. Am. Chem. Soc. 74, 2718-2723 (27) Leggett, C.J., Endrizzi, F., Rao, L. (2016) Scientific Basis for Efficient Extraction of Uranium from Seawater, Ii: Fundamental Thermodynamic and Structural Studies. Ind. Eng. Chem. Res. 55, 4257. (28) Choi, J. H., and Lee, S. Y. (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 64, 625–635. (29) Beshay, U., Miksch, G., Friehs, K., and Flaschel, E. (2007) Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter. Biotechnol. Lett. 29, 1893–1901. (30) Flory, P. J. (1953) Principles of polymer chemistry. Configurational and frictional properties of the polymer molecule in dilute solution, pp 595-639, Cornell Univ. Press, New York. (31) Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., and Fernandez-Lafuente, R. (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451– 1463.

22



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

(32) Kou, S., Yang, Z., and Sun, F. (2017) Protein hydrogel microbeads for selective uranium mining from seawater. ACS Appl. Mater. Interfaces 9, 20352039. (33) Chen, P., Greenberg, B., Taghavi, S., Romano, C., van der Lelie, D., and He, C. (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew. Chem. Int. Ed. Engl. 44, 2715–2719. (34) Tao, H.C., Peng, Z. W., Li, P.S., Yu, T. A., and Su, J. (2013) Optimizing cadmium and mercury specificity of CadR-based E. coli biosensors by redesign of CadR. Biotechnol. Lett. 35, 1253–1258. (35) Song, L., Caguiat, J., Li, Z., Shokes, J., Scott, R. A., Olliff, L., and Summers, A. O. (2004) Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site. J. Bacteriol. 186, 1861–1868.

23


Page 24 of 28

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Figures

2 3 4 5 6 7 8 9 10 11 12

Fig 1. A protein polymer platform based on SpyTag-SpyCatcher chemistry. (a) Protein structure for 3A-SUP and 3B. A, SpyTag; B, SpyCatcher; ELP, elastin-like protein; SUP, Super Uranyl-binding Protein. (b) A schematic of polymer formed by 3A-SUP and 3B. Spontaneous covalent bonding between SpyTag and SpyCatcher motifs on different monomers led to the formation of cross-linked polymeric networks. (c) SDS-PAGE analysis showing cross-linking between 3A/4A/6A-SUP and 3B as high molecular weight gel bands.

24



1 2 3 4 5 6 7 8 9 10 11 12

Fig 2. Polymerization properties of the Spy system. (a) Theoretical gel points for a number of monomer configurations. The x-axis represents the molar ratio of nA-SUP over the amount of all monomers, and the y-axis represents the critical linkage degree (CLD) at gel point. (b) Molecular weight distribution of polymer products of 3A-SUP and 3B at a total concentration of 1 mg/mL. Bars represent analytical prediction by the model (see Supplemental Information), and the green line represents densitometric profile obtained from SDS-PAGE of products after 2 hr incubation of the reaction system at 25˚C (inset). (c)&(d) Polymerization reaction progress at various pH and temperatures, as measured by the fraction of unreacted, free 3B monomers. Error bars represent standard deviation of triplicate experiments.

25


Page 26 of 28

Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fig 3. Spy-SUP networks adsorb uranyl with high efficiency in various aqueous solutions. (a) Uranyl adsorption capacities of various monomers or networks containing SUP. The mixture of proteins (with 10 µM SUP) and UO22+ (10 µM) was subject to 3 kDa centrifugation filters immediately after 1 min reaction. Concentration of UO22+ in filtrates was measured by Arsenazo III chromogenic reaction. (b) Uranyl adsorption capacities at different proteinuranyl ratios. An excess of 10-fold protein polymer (formed by 3A-SUP and 3B) did not show significant enhancement of adsorption. (c) Adsorption capacities in different uranyl-containing samples. Each sample solution contained 100 µM 3A-SUP, 100 µM 3B and 10 µM UO22+. (d) Adsorption capacity for trace amount of UO22+ analyzed by ICP-MS. 13 nM UO22+ was mixed with pre-incubated 3A-SUP+3B network at a molar ratio of UO22+:SUP 1:6000. In(c) and (d), unfilled bars represent control experiments without Spy monomers in the respective samples. In all figures, error bars represent standard deviation of triplicate experiments; ****p

A genetically encoded protein polymer for uranyl binding and

Recommend Documents