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Biological and Environmental Phenomena at the Interface
Saccharides create crowding environment for gene expression in cell-free systems Lihui Bai, Xiaocui Guo, Xue Zhang, Wenting Yu, and Dayong Yang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03744 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019
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Saccharides create crowding environment for gene expression in cell-free systems Lihui Bai#, Xiaocui Guo#, Xue Zhang, Wenting Yu, Dayong Yang*
School of Chemical Engineering and Technology, Key Laboratory of Systems
Bioengineering (Ministry of Education), Collaborative Innovation Center of Chemical
Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
*E-mail:
[email protected] (Dayong Yang)
Keywords: crowding effect, saccharides, cell-free system, synthetic biology
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ABSTRACT
Cellular physical microenvironment such as crowding shows great influence on
enzymatic reactions. Herein, we report a new finding that saccharides with low-
molecular-weight create effective crowding microenvironment for gene expression in
cell-free protein synthesis (CFPS) which provides valuable implication for living
systems. Four saccharides including sorbose, galactose, sucrose and cellobiose are
screened out as effective crowders. At low concentration range of saccharides, both the
mRNA and protein amounts present an upward trend with the concentration increment
of saccharides; when the concentrations exceed a critical value, mRNA and protein
amounts turn to decrease. A mechanism is proposed that at low concentrations of
saccharides, the effective concentrations of reactants increase due to the coexistence
of crowders and reactants in a finite volume; when the concentrations exceed a critical
value, the molecular diffusion of reactants is dominantly restricted due to the increased
viscosity. Our finding opens a new view that saccharides with low-molecular-weight
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could be crowders and provides new insight that substances with low-molecular-weight
in cells would produce crowding effect on biochemical reactions in living systems.
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Introduction
Cellular physical environment has significant impacts on its internal reaction
networks. From a physicochemical point of view, all cells are concentrated in small
volumes and filled with high concentration of biomolecules (proteins, nucleic acids,
ribosomes and saccharides, etc.), which makes crowding phenomenon an ubiquitous
and significant feature of cellular environment1, 2. For instance, the macromolecular content of E. coli was calculated to be 300-400 g/L, which generally occupied 20-30% cell volume3, 4. Therefore, investigation of crowding effect will provide a more
comprehensive understanding of cellular physical environment. Crowding effect on
reaction networks mainly includes two aspects. Each of the crowding agents and
reactants has a finite molecular volume and cannot occupy the same space at one
time3. The effective volume for reactants decreases when the crowding agents and
reactants coexist. As a result, the association of the enzymes and substrates is
facilitated owing to the increased effective concentration of these biomolecules5, 6. For
example, it has been demonstrated that the crowding agents could increase the
association between DNA and enzymes7, 8. Besides, crowding molecules increase
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viscosity of solutions, which slows down the rates of diffusion-controlled reactions3, 9.
For example, Klumpp and coworkers found that the slow diffusion of the tRNA
complexes in the cytoplasm generated a physical limit on the speed of translation10.
Kapral and coworkers found that small molecule diffusion and reaction rate coefficients
decreased in crowded environment11. Zhao and colleagues found that the rotational
diffusion of protein was influenced by bulk concentration and the polymer structure12.
However, it’s challenging to study the crowding effect inside cells due to the
extremely complex reaction networks and sophisticated intracellular environments.
Instead, CFPS allow activating biological protein expression without using living cells, and provide a simplified and well-controlled in vitro model for studying the physicochemical basis of cells and even constructing artificial cells13-15. Crowding effect
has been extensively studied in CFPS, in which crowders were macromolecules such
as polyethylene glycol (PEG) and ficoll. For example, Yamane and colleagues added
PEG in CFPS and protein expression was enhanced16. Huck and coworkers reported
that diffusion coefficient of RNA and protein decreased due to the macromolecular
crowding caused by ficoll 70 in cell-free system17. Xu and coworkers found that cell-free
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transcription was improved by the addition of PEG-8000, ficoll-70 or ficoll-400, but
translation was inhibited18.
Crowding molecules inside cells not only include macromolecules such as proteins
and nucleic acids, but also comprise substances with low-molecular-weight. For
example, Sharp found that the crowding effect was weakly dependent on crowder size
and medium-sized molecules had the same effect as much larger macromolecules,
such as proteins and RNA19. Li and coworkers employed sucrose and ficoll as crowders
to investigate the crowding effect on I4%
structure20. These two studies
suggested that small molecules might create crowding environment via excluded
volume effect. In this study, we report a new finding that saccharides with lowmolecular-weight create effective crowding microenvironment in E. coli extract-based
CFPS. Ten typical monosaccharides and disaccharides were tested and four
saccharides were screened out as crowders. A mechanism figure is proposed to
elucidate the crowding effect of saccharides. The experimental results are in
accordance with our mechanism diagram.
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Experimental Section
Construction of in vitro transcription and translation Transcription and translation were carried out by mixing 25 J feeding buffer, 15 J E.
coli extract, 200 ng pRset-eGFP plasmid or 200ng pRset-YFP plasmid and nucleasefree water and crowding agents to a final volume of 50 J 2 Unless stated otherwise, the
reaction volume was kept at 50 J 2 The feeding buffer contained 50 mM hepes, 90 mM
potassium glutamate, 15 mM magnesium glutamate, 1.5 mM amino acid mixture, 20
mM 3-phosphoglycerate, 5 mM cyclic adenosine monophosphate, 2.5 mM nicotinamide
adenine dinucleotide, 2.5 mM coenzyme A, 0.5 mM folinic acid, 3 mg/mL tRNA, 0.66
mM spermidine, 1 mM UTP, 1 mM CTP, 1 mM ATP, 1 mM GTP. Then the
measurement of fluorescence intensity was obtained from microplate reader at 30°C.
The timescale is 12 h. mRNA measurement After the reaction, total RNA was first isolated from CFPS reactions using RNAprep Pure Cell/Bacteria Kit (TIANGEN) according to the manufacturer’s instructions. The quantity and quality (A260/A280 value) of RNA samples were determined using a Uv-Vs spectrophotometer Q5000 (Quawell). For cDNA synthesis, about 200 ng of total RNA was used to a reverse
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transcription reaction using the FastKing RT Kit (TIANGEN) according to the manufacturer’s instructions. For qPCR analysis, cDNA samples were diluted 10 times with nuclease-free water. 1 µL of the diluted sample was combined with 10 µL of 2x SuperReal PreMix Plus (with SYBR Green
),
0.5
µL
of
10
µM
eGFP
gene
specific
forward
primer
(5'-
GAGCTTTTCACTGGCGTTG-3'), 0.5 µL of 10 µM eGFP gene specific reverse primer (5'TGGTGCAGATGAACTTCAGG-3') and 8 µL RNase-free water. A no reverse transcription control (RNA) was prepared for each sample. All samples were conducted in 96-well plate (Roche) and reactions were run on a LightCycler 480 (Roche). The amplification cycling conditions consisted of an initial incubation at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 20 s followed by annealing at 55°C for 20 s, and then extended at 72°C for 20 s. The standard curve was prepared from serial dilutions of the purified eGFP PCR products. Viscosity measurements The viscosity measurements were carried out on an AR-G2 rheometer (TA Instruments). Experiments were performed in the parallel plate using 400 µL saccharide solutions. The temperature is 25°C and the time scale is 1 min.
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Results and discussion
Control 3% Glucose 3% Fructose 3% Mannose 3% Galactose 3% Sorbose
b) 12
10 8 6 4 2 0
eGFP ( 104 a.u.)
eGFP ( 104 a.u.)
a) 12 10 8 6 4 2 0
0 2 4 6 8 10 12 Time (h) c)
Control 3% Maltosemonohydrate 3% Trehalose 3% Melibiose 3% Sucrose 3% Cellobiose
0 2 4 6 8 10 12 Time (h) d)
6
6
5
5
eGFP ( 104 a.u.)
eGFP ( 104 a.u.)
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4 3 2 1 0
Saccharides
4 3 2 1 0
Saccharides
Figure 1. Effect of 3% five monosaccharides (a, c) and five disaccharides (b, d) on
eGFP amount in CFPS. The dots in figure c and d represented eGFP amounts at 12 h.
We chose five typical monosaccharides (glucose, mannose, fructose, galactose
and sorbose) and five disaccharides (maltosemonohydrate, melibiose, trehalose,
sucrose and cellobiose) as crowder candidates and added to CFPS. In CFPS, cell
lysates contained basic protein synthesis machineries, energy regeneration substrates,
amino acids, nucleotides and cofactors21-23. The process of gene expression in CFPS is
shown in Figure S1a: DNA is transcribed into mRNA by RNA polymerase, and
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afterwards mRNA is translated to polypeptides which are then fold into mature proteins.
mRNA and protein amounts in the system are dynamic. In this study, we focused on
studying the crowding effect of saccharides on the mRNA and protein amounts in
CFPS. Unless stated otherwise, the reaction volume was kept at 50 J and the reaction
time was set at 12 h. The experimental group without addition of saccharides was set as
the benchmark. Enhanced green fluorescent protein (eGFP) was used as the reporter
protein. The plasmid profile of pRset-eGFP was presented in Figure S2. The
concentrations of 1%, 2% and 3% (w/v) of saccharides were tested in order to screen
out saccharides which have no obvious inhibitory effect on protein expression at low
concentrations. Unexpectedly, we found that protein amounts presented two different
trends in different saccharides (Figure S3and Figure S4). For example, with 3%
saccharides, protein amounts were partially inhibited in fructose, mannose, trehalose
and melibiose; were inhibited completely in glucose and maltosemonohydrate.
However, in galactose, sorbose, sucrose and cellobiose, no inhibitory effect was
observed, which suggested they could be used as crowders in CFPS (Figure 1). We
further examined the effects of glucose, mannose, fructose, sorbose and galactose on
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pure eGFP and found that there was no obvious difference in these five saccharides
systems (Figure S5). The inhibitions of some saccharides to protein expression still
remained unclear and we speculated it was related to enzyme activity involved in
protein synthesis. We therefore employed saccharides which had no inhibitory effect on
protein expression for the following studies, including galactose, sorbose, sucrose and
cellobiose. The chemical structures are shown in Figure S1b.
Next, we varied the concentration range of sorbose, galactose and sucrose from
4% to 20% and cellobiose from 2% to 10% and measured mRNA and protein amounts
in these crowding environments. It was noted that the cellobiose concentration was
lower than other saccharides, because cellobiose was less soluble than the other three
saccharides.
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Figure 2. Effect of sorbose (a), galactose (b), sucrose (c) and cellbiose (d) on eGFP
mRNA amounts in CFPS. Error bars represent standard deviations from three
measurements.
We first tested the effect of different concentrations of sorbose, galactose, sucrose
and cellobiose on mRNA amount in CFPS. The results show that eGFP mRNA amount
presented an increasing trend first and then declining in the presence of saccharides
(Figure2). In detail, at low concentrations of sorbose, such as 4% and 8%, mRNA
amount was higher than that of the control group (without sorbose); when the
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concentrations exceeded 8%, mRNA amount turned to decline (Figure 2a). In galactose
and sucrose systems, when the concentrations are less than 12%, mRNA amount
increased with the increment of saccharides concentration; when the concentrations
exceeded 12%, mRNA amount turned to decrease (Figure 2b and Figure 2c). Among
them, the increase of mRNA amount resulting from galactose was more obvious than
sucrose. mRNA amount was higher than the control group at 4% cellobiose; when the
concentrations exceeded 4%, mRNA amount turned to descend (Figure 2d). On the
whole, mRNA amount reached highest at 12% galactose, and copy numbers of mRNA increased from 3.3( × 106CJ ( to 16 ( × 106CJ (2 The optimal concentration of four saccharides for mRNA output was different which could be caused by the structure
difference of saccharides24. Basing on the above results, we speculated that at low
concentration of saccharides, crowding effect facilitated mRNA production due to the
increased effective concentrations of T7 RNA polymerase and reactants; however,
when the concentration reached a higher level, mRNA amount decreased because that
the diffusions of T7 RNA polymerase and other reactants were restricted in the relatively
high concentration of saccharides.
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Figure 3. Effect of sorbose (a), galactose (b), sucrose (c) and cellobiose (d) on eGFP
amount in CFPS. Error bars represent standard deviations from three measurements.
We next investigated the effect of different concentrations of sorbose, galactose,
sucrose and cellobiose on eGFP amount in CFPS. Similar tendency of protein amount
was observed as compared to mRNA amount. At low concentrations of sorbose, such
as 4% and 8%, protein amount was higher than that of the control group (without
sorbose); when the concentrations exceeded 8%, protein amount turned to decrease
(Figure 3a). When the concentrations of galactose and sucrose were less than 12%,
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protein amount increased with the increment of saccharides concentration; when the
concentrations exceeded 12%, protein amount turned to descend (Figure 3b and Figure
3c). Besides, protein amount was higher than that of the control group at 2% cellobiose;
when the concentrations exceeded 2%, protein amount turned to descend (Figure 3d).
Meanwhile, it was worth noting that eGFP expression and mRNA yield were not
consistent, completely. For example, in cellobiose system, mRNA amount reached
highest at 4% concentration of cellobiose, but eGFP expression reached highest at 2%
concentration of cellobiose (Figure 2d and Figure 3d). This inconsistency demonstrated
that the crowding effect created by saccharides also influenced the translation process
rather than just the transcription process.
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b) 4.0 3.2 2.4 1.6 0.8 0
4
c)
8 12 16 20 Csorbose(%)
4.0
4
sucrose 3.2 2.4 1.6 0.8 0.0
0
4
8 12 16 20 Csucrose(%)
4.0
galactose 3.2 2.4 1.6 0.8 0.0
0
d)
4 8 12 16 20 Cgalactose(%)
4.0
cellobiose
4
0.0
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4
sorbose
Initial expression rate ( 10 a.u./h)
4
Initial expression rate ( 10 a.u./h)
a)
Initial expression rate ( 10 a.u./h)
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Initial expression rate ( 10 a.u./h)
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3.2 2.4 1.6 0.8 0.0
0
2 4 6 8 Ccellobiose(%)
10
Figure 4. Effect of sorbose (a), galactose (b), sucrose (c), cellobiose (d) on initial protein
expression rates. Error bars represent standard deviations from three measurements.
Meanwhile, we calculated the initial reaction rates of protein production by the
following formula:
Initial reaction rates (a.u./h) = (I-I0)/t The initial reaction rates were defined the change of fluorescence intensity in initial unit time (t represented the first 1 h). The fluorescence intensity of protein in our study increased linearly in the first one hour, as presented in Figure S6. I was the eGFP
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fluorescence intensity at 1 h. I0 was the initial eGFP fluorescence intensity. We found that in sorbose and sucrose systems, initial expression rates of protein increased when
the concentration of saccharides increased from 4% to 12%; when the concentrations
exceeded 12%, initial protein expression rates turned to decrease (Figure 4a and Figure
4c). At 4% galactose, initial expression rate was higher than that of the control group;
when the concentrations exceeded 4%, initial expression rates turned to decrease
(Figure 4b). In cellobiose system, when the concentration was 2%, the initial expression
rate was higher than that of the control group (without cellobiose); when the
concentrations exceeded 2%, initial protein expression rates turned to descend (Figure
4d). Basing on the results of protein amount and initial expression rates of protien, we
proposed that when the concentration of saccharides was relatively low, crowding effect
promoted protein formation; as the concentration continued to increase and exceeded a
certain value, crowding effect inhibited protein production.
We next studied the mechanisms for the crowding effect of saccharides. We
speculated that the viscosity of saccharide solutions played critical roles in regulating
diffusion-controlled reactions according to previous studies9,
18.
The effects of
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saccharides with different concentrations on mRNA and protein amounts were related to
the viscosity and diffusion coefficients of solution. Therefore, we determined the viscosity of saccharides, and calculated the diffusion coefficients (D) of solution by Einstein stokes equations17, 25:
DAB = A"CGN AJB=2.2×10—4/rAJB. Where k is the boltzmann constant, T is absolute temperature, rA is the radius of the target molecule, JB is the viscosity of solution (the average value of three measurements). At a certain temperature, diffusion coefficient is inverse relation with
the viscosity of solution for any molecule. So the variation trend of diffusion coefficient is
theoretically the same for different molecules. To illustrate more clearly, we calculated diffusion coefficients with T7 RNA polymerase as an example (rA is 5 nm). The results were shown in Figure S8.
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trend for each saccharide (Figure S7 and Figure S8). Basing on the above experimental
results, we propose that saccharides generally create two effects: increasing the
effective concentration of reactants and thus enhancing reaction rates (Effect 1); and
limiting the diffusion of the reactants and thus slowing down the reaction rates (Effect 2).
We define the initial state as the dilute solution without saccharides in CFPS (Scheme
1a). In initial state, reactants (enzymes and substrates) can diffuse and shuttle freely
and synthesize proteins. When saccharides are added in CFPS, the viscosity of solution
increases with concentration increment of saccharides. At low concentrations of
saccharides, the reactants diffusion is not greatly restricted due to the relatively low
solution viscosity, while the effective concentrations of reactants increase owing to the
coexistence of crowders and reactants in a finite volume. As a consequence, Effect 1 is
dominant as compared to Effect 2, which results in incremental amounts of m RNA and
protein (Scheme 1b). At high concentrations of saccharides, the viscosity of solution is
relatively high due to the enhanced quantity of hydrogen bonds between saccharides
and water. The diffusion of reactants is restricted and Effect 2 is dominant, leading to
down-regulation of mRNA and protein amounts (Scheme 1c). When the viscosity of
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solution reaches a critical value, Effect 1 is comparable to Effect 2, mRNA and protein
amounts would be the highest. Overall, with the increase of saccharides concentrations
in CFPS, mRNA and protein amounts first increase and then decline when the
concentration of saccharides exceeds a critical value. Our experimental results are in
full agreement with proposed mechanism and model.
Figure 5. Crowding effect of sorbose (a), galactose (b), sucrose (c) and cellobiose (d)
on YFP amount. At low concentration range of saccharides, YFP amount presented an
upward trend with the increment concentration of saccharides; when the concentration
exceeded the critical level, YFP amount turned to descend. Error bars represent
standard deviations from three measurements.
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To verify the crowding effect of saccharides on CFPS, we further employed yellow
fluorescent protein (YFP) as the reporter protein and studied protein amount in four
saccharide systems (Figure 5). Trend of YFP amount was similar to that of eGFP in
saccharide solutions. At 4% sorbose, YFP amount was higher than that of the control
group (without sorbose); when the concentrations exceeded 4%, YFP amount turned to
decline (Figure 5a). YFP amount gradually increased as the concentration of galactose
increased from 4% to 12%; when the concentrations exceeded 12%, YFP amount
turned to decrease (Figure 5b). In sucrose system, YFP amount reached the highest
level when the concentration was 16% (Figure 5c). When the concentration of
cellobiose was less than 6%, YFP amount increased with the increased concentration of
cellobiose; when the concentrations exceeded 6%, YFP amount turned to descend
(Figure 5d).
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Conclusion
In conclusion, we have demonstrated that saccharides could create crowding
microenvironment in CFPS and studied the crowding effect of saccharides on mRNA
and protein amounts. At low concentration range of saccharides, both the mRNA
amount and protein amount present an upward trend with the concentration increment
of saccharides; when the concentrations exceed a critical value, mRNA and protein
amounts turn to decrease. Basing on the experimental results, we proposed a
mechanism that at low concentration of saccharides, the effective concentrations of
reactants increase due to the coexistence of crowders and reactants in a finite volume;
when the concentrations exceed the critical value, the molecular diffusion of reactants is
dominantly restricted due to the increased viscosity. We envision that our work offers
new insights that substances with low-molecular-weight in cells would produce crowding
effect on biochemical reactions in living systems.
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ASSOCIATED CONTENT
The supporting information is available free of charge on the ACS Publications website.
Supplementary materials and methods, Figure S1-S4 (PDF).
AUTHOR INFORMATION
Corresponding author *E-mail:
[email protected];
[email protected] Author Contributions
Lihui bai and Xiaocui guo contributed equally to this work.
Notes The authors declare no competing interests.
ACKNOWLEDGMENT
This work was in part supported by National Natural Science Foundation of China (grant
no. 21621004, 21575101 and 21622404). We thank Ms. Yang Liu, Mr. Yi Jiao, Ms. Liyi
Xu and Ms. Qing Sun at Tianjin University for their kind help on experiments and
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discussion. We are highly grateful to Dr. Xiaohui Li at Tianjin University for viscosity test
and Dr. Shaolan Zou at Tianjin University for qPCR measurement.
ABBREVIATIONS PEG, polyethylene glycol; CFPS, cell-free protein synthesis; GFP, green fluorescent
protein; YFP, yellow fluorescent protein.
REFERENCES (1) Ellis, R J; Minton, A P, Cell biology: join the crowd. Nature 2003, 425 (6953), 27-28. (2) Zhou, H X; Rivas, G; Minton, A P, Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Ann. Rev. Biophys. 2007, 37 (37), 375-397. (3) Sokolova, E, Effects of macromolecular crowding on gene expression studied in protocell models. Physical Organic Chemistry 2015. (4) Zimmerman, S B; Trach, S O, Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 1991, 222 (3), 599-620. (5) Ellis, R J, Macromolecular crowding: obvious but underappreciated. Trends Biochem. Sci 2001, 26 (10), 597-604. (6) Minton, A P, The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J. Biol. Chem. 2001, 276 (14), 1057710580. (7) Sasaki, Y; Miyoshi, D; Sugimoto, N, Effect of molecular crowding on DNA polymerase activity. Biotech. J. 2006, 1 (4), 440-446. (8) Sasaki, Y; Miyoshi, D; Sugimoto, N, Regulation of DNA nucleases by molecular crowding. Nucleic Acids Res. 2007, 35 (12), 4086-4093. (9) Ellis, R J, Macromolecular crowding: an important but neglected aspect of the intracellular environment. Curr. Opin. Struct. Biol. 2001, 11 (1), 114-119. (10) Klumpp, S; Scott, M; Pedersen, S; Hwa, T, Molecular crowding limits translation and cell growth. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (42), 16754-16759. (11) Echevera, C; Tucci, K; Kapral, R, Diffusion and reaction in crowded environments. Journal of Physics Condensed Matter 2007, 19 (19), 65146-65112. (12) Jing, Q; Chen, A; Zhao, N, A new scaling for rotational diffusion of molecular probes in polymer solutions. PCCP 2017, 19 (48), 32687-32697. (13) Carlson, E D; Gan, R; Hodgman, C E; Jewett, M C, Cell-Free Protein Synthesis: Applications Come of Age. Biotechnol. Adv. 2012, 30 (5), 1185-1194. (14) Shimizu, Y; Inoue, A; Tomari, Y; Suzuki, T; Yokogawa, T; Nishikawa, K; Ueda, T, Cell-
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free translation reconstituted with purified components. Nat. Biotechnol. 2001, 19 (8), 751755. (15) Oza, J P; Aerni, H R; Pirman, N L; Barber, K W; Haar, C M T; Rogulina, S; Amrofell, M B; Isaacs, F J; Rinehart, J; Jewett, M C, Robust production of recombinant phosphoproteins using cell-free protein synthesis. Nat. Commun. 2015, 6, 8168. (16) Nakano, H; Tanaka, T; Kawarasaki, Y; Yamane, T, Highly productive cell-free protein synthesis system using condensed wheat-germ extract. J. Biotechnol. 1996, 46 (3), 275-282. (17) Hansen, M M K; Meijer, L H H; Spruijt, E; Maas, R J M; Rosquelles, M V; Groen, J; Heus, H A; Huck, W T S, Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets. Nat. Nanotechnol. 2016, 11 (2), 191-197. (18) Ge, X; Luo, D; Xu, J, Cell-Free Protein Expression under Macromolecular Crowding Conditions. PLoS One 2011, 6 (12), e28707. (19) Sharp, K A, Analysis of the size dependence of macromolecular crowding shows that smaller is better. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (26), 7990-7995. (20) Bai, J; Liu, M; Pielak, G J; Li, C, Macromolecular and Small Molecular Crowding Have Similar Effects on L ) Structure. Chemphyschem 2016, 18 (1), 55-58. (21) Murray, C J; Baliga, R, Cell-free translation of peptides and proteins:from high throughput screening to clinical production. Curr. Opin. Chem. Biol. 2013, 17 (3), 420-426. (22) Jewett, M C; Calhoun, K A; Voloshin, A; Wuu, J J; Swartz, J R, An integrated cell-free metabolic platform for protein production and synthetic biology. Mol. Syst. Biol. 2008, 4 (1), 220-220. (23) Pedersen, A; Hellberg, K; Enberg, J; Karlsson, B G, Rational improvement of cell-free protein synthesis. New Biotechnol. 2011, 28 (3), 218-224. (24) Lee, J K; Pachtman, E A; Frumin, A M, Structural configuration of sugars and their ability to inhibit fava bean hemagglutination. Ann. N. Y. Acad. Sci. 2010, 234 (1), 161-169. (25) Nenninger, A; Mastroianni, G; Mullineaux, C W, Size dependence of protein diffusion in the cytoplasm of Escherichia coli. J. Bacteriol. 2010, 192 (18), 4535-4540.
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Langmuir
TOF Figure
1 0 0
Diluted solution
Low-viscosity solution
High-viscosity solution
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