Microcompartmentalized cell-free protein synthesis in semipermeable microcapsules composed

Microcompartmentalized cell-free protein synthesis in semipermeable

microcapsules composed of polyethylenimine-coated alginate

Daisuke Saeki,1,2Shinji Sugiura,3Toshiyuki Kanamori,3Seigo Sato,1and Sosaku Ichikawa 1,*

Faculty of Life and Environmental Sciences,University of Tsukuba,1-1-1Tennodai,Tsukuba,Ibaraki 305-8572,Japan,1Department of Chemical Science and Engineering,Kobe

University,1-1Rokkodai,Nada,Kobe,Hyogo 657-8501,Japan,2and National Institute of Advanced Industrial Science and Technology (AIST),1-1-1Higashi,Tsukuba,

Ibaraki 305-8565,Japan 3

Received 23October 2013;accepted 27January 2014

Available online 27February 2014

We describe microcompartmentalized cell-free protein synthesis in semipermeable microcapsules prepared from water-in-oil-in-water droplets by a rupture-induced encapsulation method.An aqueous solution of template DNA coding for green ?uorescent protein and enzymes for the cell-free protein synthesis was aliquoted into water-in-oil droplets using a micro ?uidic device,and the droplets were transformed into semipermeable microcapsules.Substrates for protein synthesis diffused into the microcapsules through their semipermeable polyion complex membranes composed of polyethylenimine-coated alginate.Cell-free protein synthesis was con ?rmed by detection of the ?uores-cence of the synthesized green ?uorescence protein in the microcapsules.We also used this microcompartmentalized system to synthesize protein from a single molecule of template DNA encapsulated by limiting dilution.

[Key words:Cell-free protein synthesis;Microcompartmentalization;Semipermeable microcapsule;Micro ?uidics;Rupture-induced encapsulation]

In order to realize ef ?cient selection of a target from a large population of cells,DNA,protein or small-molecules,a high-throughput screening method is essential.Such method should be economical and involve minimal consumption of expensive and rare biological reagents.The droplet-based micro-compartmentalized reaction,which was originally reported by Taw ?k and Grif ?ths (1),meets these requirements.These in-vestigators developed high-throughput systems for screening enzyme libraries in water-in-oil-in-water (W/O/W)droplets (2e 4)and used ?ow cytometry to screen the enzymes that formed in the droplets.Similar approaches using water-in-oil (W/O)droplets and lipid vesicles have been reported (5e 9).Although screening of biomolecules in microcompartments has been successfully demonstrated,improvements in the compartment size uniformity,compartment stability,droplet handling,and substrate supply to the compartments are required to make compartmentalized pro-tein screening practical.

Micro ?uidic droplet formation is a useful method for preparing uniform droplets,microbeads and microcapsules (10e 16).This droplet micro ?uidics technology provides ef ?cient encapsulation and microcompartmentalization.Recently,the use of droplet micro ?uidics for microcompartmentalized reactions has attracted attention because uniformly sized compartments can be generated,reaction conditions can be controlled,and substrates can be sup-plied to the compartments (17e 21);therefore,droplet

micro ?uidics is expected to be useful for many kinds of biochemical reactions.For example,Agresti et al.(18)used droplet micro ?uidics to prepare W/O droplets encapsulating mutated cells and then applied the technology to high-throughput screening of enzymes by means of directed evolution.However,the utility of cell encapsulation method for the synthesis of cytotoxic proteins and proteins requiring long-term maturation is restricted because of the limited cultivation time of the encapsulated cells due to lack of the nutrient supply.In addition,the utility of this method for enzymatic reaction is also restricted due to lack of the substrate supply.

Cell-free protein synthesis is a remarkable method for produc-ing and screening native and mutant proteins in the absence of living cells.The method relies on cell lysates containing the essential components for protein synthesis,such as enzymes,substrates,and template DNA.A screening system involving single-molecule polymerase chain reaction (PCR)and cell-free protein synthesis was developed by Koga et al.(22,23)to screen recombi-nant proteins by means of directed evolution.A DNA library solu-tion was aliquoted onto microplates at the concentration corresponding to a single DNA molecule per well by means of limiting dilution,and the aliquoted DNA molecules were ampli ?ed by means of PCR.The ampli ?ed DNA was translated to protein by means of cell-free protein synthesis,and then a protein library with linked genotypes and phenotypes was constructed.Koga et al.(22,23)used this method to screen protein with high enzymatic activity by adding a solution of reaction substrate to the microplate containing the enzyme library.

To number up the library size is protein screening by cell-free protein synthesis,the use of a microfabricated reactor array was proposed (24).In addition,Fallah-Araghi et al.(21)applied droplet

*Corresponding author.Tel.:t81298534627;fax:t81298534605.

E-mail addresses:shinji.sugiura@aist.go.jp (S.Sugiura),ichikawa.sosaku.fn@u.tsukuba.ac.jp (S.

Ichikawa).

https://www.360docs.net/doc/483860405.html,/locate/jbiosc

Journal of Bioscience and Bioengineering

VOL.118No.2,199e 204,

2014

micro ?uidics to directed evolution by combining single-molecule PCR and cell-free protein synthesis.Although this two-step system works,it comprises many micro ?uidic processes,including droplet formation,fusion,thermal cycling,incubation,detection,and sep-aration.Ideally,this complicated system would be replaced by a single-step reaction system,that is,direct protein synthesis from a single DNA molecule in small microcompartments.However,the amount of protein that can be synthesized from a single DNA molecule in a cell-free process is small,and detection of such a small amount of protein is dif ?cult,even in small microcompartments.

In contrast,it is known in the macroscopic experiment that the use of semipermeable membranes for continuous cell-free protein synthesis allows for long-duration synthetic reactions and high protein yields (25).Substrates are continuously sup-plied and low-molecular-weight byproducts are continuously removed through the semipermeable membranes,and proteins can be synthesized at concentrations as high as milligrams of protein per milliliter of solution,which is much higher than the concentrations that can be achieved with batch-mode processes (26e 28).

In this paper,we describe microcompartmentalized cell-free protein synthesis in uniformly sized semipermeable microcap-sules composed of alginate coated with polyethylenimine (PEI).The semipermeable microcapsules were prepared by a previously reported rupture-induced encapsulation method using a micro-?uidic device (Fig.1A)(29).The microcapsules containing tem-plate DNA and enzymes were incubated in the presence of substrates,which diffused into the microcapsules through the semipermeable membranes.We also demonstrated direct cell-free protein synthesis from a single DNA molecule encapsulated in these

microcapsules.

FIG.1.Microcompartmentalized cell-free protein synthesis in semipermeable microcapsules.(A)Schematic of the experimental procedure.(B)Schematic of the experimental apparatus for preparation of the semipermeable microcapsules.(C)Microscopy image of the formation of W/O/W droplets containing sodium alginate and new coccine dye.(D)Schematic of the formation of the PIC membrane on the W/O/W droplet surface.(B,C)Partially reproduced from Saeki et al.(29)with permission of the Royal Society of Chemistry.

200SAEKI ET AL.J.B IOSCI .B IOENG .,

MATERIALS AND METHODS

Materials Poly(dimethylsiloxane)was obtained from Dow Corning(Sylgard 184;Midland,MI,USA).Decane was obtained from Wako Pure Chemical Industries (Osaka,Japan)and used as the oil https://www.360docs.net/doc/483860405.html,li-Q water was used as the internal and external aqueous phase.Sodium alginate was obtained from Kimica Co.,Tokyo, Japan(SI-L)and added at1wt%to the internal aqueous phase.Tetraglycerin-condensed ricinoleic acid ester was obtained from Sakamoto Yakuhin Kogyo Co., Osaka,Japan(CR-310)and added at5wt%to the oil phase to stabilize the W/O droplets.Dodecyl betaine was obtained from Kao Co.,Tokyo,Japan(Amphitol20BS) and added at1wt%to the external aqueous phase to stabilize the W/O/W droplets. Sodium chloride,calcium chloride and PEI(50e100kDa)were obtained from Wako Pure Chemical Industries.Fluorescein isothiocyanate e dextran(FITC e dextran; 10kDa)was obtained from Sigma e Aldrich Co.(St.Louis,MO,USA).New coccine dye was obtained from Kanto Chemical(Tokyo,Japan).

Formation of semipermeable microcapsules We prepared semipermeable microcapsules by a modi?cation of a rupture-induced encapsulation method using our previously reported micro?uidic device(Fig.1B)(29).All?uids were injected into the microchannels at constant?ow rates controlled by syringe pumps(Pico Plus;Harvard Apparatus,Holliston,MA,USA).First,the micro?uidic device was ?lled with the external aqueous phase solution(Milli-Q water containing1wt% dodecyl betaine),and then the oil phase and the internal aqueous phase were injected.The?ow rates of the internal aqueous phase,oil phase,and external aqueous phase were0.05,0.10,and 4.68mL/h.The internal aqueous phase contained1.0wt%sodium alginate,and its osmolality was adjusted to300mOsm with0.6wt%sodium chloride.The W/O/W droplets were formed at the conjunction point of microchannels(Fig.1C),then the recovered W/O/W emulsion was placed in contact with a cross-linking solution containing0.1mol/L calcium chloride and0.1wt%PEI on a glass slide,at which point the W/O/W droplets ruptured,owing to the osmotic pressure difference(29);simultaneously,calcium alginate beads with a polyion complex(PIC)membrane were formed by means of cross-linking(Fig.1D).

To investigate the encapsulation ef?ciency and permeability of the resulting microcapsules,we premixed0.1wt%FITC e dextran in the internal aqueous phase and then carried out droplet formation using this premixed solution as the internal aqueous phase.

Cell-free protein synthesis Cell-free protein synthesis was carried out using the enzymes and substrates in an RTS100Escherichia coli HY Kit(5PRIME,Hamburg, Germany).DNA coding for green?uorescent protein(GFP,27kDa)in the kit was used as a model template(1kbp,620kDa).To investigate cell-free protein synthesis on the microplate,we mixed template DNA,enzymes for transcription and trans-lation,and substrates such as nucleic acids and amino acids;we incubated the mixture for6h at30 C for protein synthesis and for24h at5 C for protein maturation.

Cell-free protein synthesis in semipermeable microcapsules W/O/W droplets containing sodium alginate,template DNA and enzymes for cell-free pro-tein synthesis in the internal aqueous phase were prepared using the micro?uidic device.The prepared W/O/W droplets were placed in contact with the cross-linking solution to form semipermeable microcapsules.The resulting semipermeable mi-crocapsules were collected by centrifugation.The aqueous solution containing the substrates for protein synthesis was added to the suspension of microcapsules.The substrate molecules diffused into the microcapsules through the semipermeable membrane.The suspension of microcapsules was incubated for protein synthesis and maturation as described in the previous section.

Measurement and analytical methods Droplet formation was observed with an inverted microscope(DM IL LED;Leica Microsystems,Wetzlar,Germany) equipped with a CCD camera(Chameleon;Point Grey Research,Richmond,Canada). The?uorescent proteins synthesized in the microcapsules were detected with a ?uorescence microscope(DM LB2;Leica Microsystems)equipped with a cooled CCD camera(DFC300FX;Leica Microsystems)and a confocal laser scanning microscope (LSM700;Carl Zeiss Microimaging,Germany).

RESULTS AND DISCUSSION

Formation of semipermeable microcapsules FITC e dextran (10kDa)encapsulated in the microcapsules by the rupture-induced encapsulation method did not diffuse out to the external aqueous phase(Fig.2);the?uorescence intensity was sustained for at least1 week(data not shown).In contrast,new coccine dye(0.6kDa) diffused immediately during the encapsulation process(data not shown).

Calcium ions(40.1Da)quickly diffused into the internal aqueous phase and cross-linked the polyanionic alginate immediately after the droplets ruptured.This quick diffusion and isotropic gelling reaction resulted in the formation of calcium alginate microbeads retaining the size of the original droplets.Simultaneously,the polycationic PEI reacted with the outer layer of the calcium alginate microbeads,and a semipermeable PIC membrane composed of alginate and PEI was formed on the surface of the calcium alginate microbeads.The membrane was selectively permeable to various molecules,depending on their molecular weight.

The rupture-induced encapsulation method has the advantage of reduced leakage of encapsulated materials.Hydrogel-based PIC microcapsules are generally prepared by a two-step encapsulation method:preformed aqueous droplets are placed in contact with a cross-linking solution and then with a coating solution (10,11,30,31).During the?rst step,the encapsulated materials leak out because the gel structure is highly porous.Therefore,the two-step encapsulation method is generally not useful for the encap-sulation of polymers including DNA.In contrast,in the rupture-induced encapsulation method,cross-linking and coating occur simultaneously;external aqueous phase contained both of calcium ions and PEI,therefore,cross-linking reaction by calcium ions and coating reaction by PEI principally take place immediately after the rupture of the W/O/W droplet,even though the diffusion and re-action speed of calcium ion and PEI is different.These immediate cross-linking and coating reaction take place simultaneously in principle,and allow encapsulation of molecules like FITC e dextran with minimal leakage(Fig.2).In addition,the microcapsules pre-pared by the rupture-induced encapsulation method have highly uniform size distribution as described in our previous study(29). The average size and coef?cient of variation of the microcapsules prepared in this study were24.6m m and5.3%,respectively.

Cell-free protein synthesis in the semipermeable microcapsules We prepared uniformly sized microcapsules containing the template DNA(2.0ng/m L)and the enzymes neces-sary for cell-free protein synthesis.The microcapsules shrank upon addition of the substrates,probably because of the osmotic pres-sure difference between the internal aqueous phases and the sub-strate solution,which contained a high concentration of buffering salts required for cell-free protein synthesis.

We carried out the cell-free synthesis of GFP in the microcap-sules by adding the substrates in an RTS100E.coli HY Kit to the external aqueous phase.The?uorescence from the synthesized GFP was detected in the microcapsules after addition of the substrates (Fig.3A and B)but was not detected in the absence of the substrates (Fig.3C and D).No GFP?uorescence was detected in microcapsules that were not coated with PEI(Fig.3E and F).The?uorescence was detected from the internal phase of the microcapsules by means of confocal laser scanning microscopy(Fig.3G).The?uorescence was still detectable even after the microcapsules had been stored for several months at4 C(data not shown).

These results indicate that GFP was synthesized in the micro-capsules,and that the PIC membranes composed of alginate and PEI were permeable to the small substrates(less than0.6kDa)but not to the large molecules more than10kDa,including template DNA (670kDa),enzymes(larger than10kDa),and GFP(27kDa)(32). These?ndings are also supported by the results described above,

in FIG. 2.Microscope images of the semipermeable microcapsules encapsulating FITC e dextran.(A)Phase-contrast image.(B)Fluorescent image.

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which the PIC membranes were permeable to small molecules like new coccine (0.6kDa)but not to large molecules like FITC e dextran (10kDa)(Fig.2).

Effect of reaction environment on cell-free protein synthesis We also evaluated the effects of the concentrations of template DNA and sodium alginate on cell-free protein synthesis in Eppendorf microtube (Fig.4).Fluorescence intensity was detected with a microplate reader.No ?uorescent intensity was detected without templates.When the template DNA was diluted

to 0.07ng/m L,which is equivalent to a single molecule of DNA per microcapsule volume,the ?uorescence intensity was still detectable and was 1/5that when the template DNA concentration was 2.0ng/m L.Addition of sodium alginate,which is the main component of the microcapsules,barely affected the cell-free protein synthesis.In the previous study,Kwon et al.(30)reported the cell-free protein synthesis in the millimeter-sized calcium alginate gel beads.These results suggested that we might be able to carry out cell-free protein synthesis from a single molecule of template DNA in the

microcapsules.

FIG.3.Microcompartmentalized cell-free protein synthesis in the semipermeable microcapsules.The concentration of the template DNA was 2.0ng/m l,which is equivalent to 30molecules of template DNA per microcapsule.Microscope images after incubation with substrates (A,B),in the absence of substrates (C,D),and with substrates in the absence of PIC membranes (E,F).(A,C,E)Bright-?eld images;(B,D,F)?uorescent images.(A –F)Bars:30m m.(G)Confocal laser scanning microscopy images of cell-free protein synthesis in the microcapsules.Each photo is a cross-sectional image snapped at a different depth;the focus point of each image was changed by 2m m from that of the neighboring images.

202SAEKI ET AL.J.B IOSCI .B IOENG .,

Compartmentalized protein synthesis from a single molecule of DNA in the semipermeable microcapsules We demonstrated that cell-free protein synthesis could in fact be car-ried out with a single molecule of template DNA in the semi-permeable microcapsules (Fig.5).Most of the microcapsules exhibited ?uorescence when GFP was synthesized at a DNA template concentration of 2.0ng/m L,which is equivalent to 30molecules of template DNA per microcapsule (Fig.3B).In contrast,only a few microcapsules exhibited ?uorescence when the DNA template concentration was 0.07ng/m L,which is equivalent to a single molecule of template DNA per microcapsule (Fig.5B).Although the percentage of ?uorescent microcapsules at a DNA template concentration of 0.07ng/m L was low (<10%),our results provided evidence that GFP could be synthesized from a single molecule of template DNA in the semipermeable microcapsules.

The percentage of ?uorescent microcapsules (<10%)was sub-stantially lower than 63%,which is the theoretically calculated percentage assuming random encapsulation of the template DNA with a Poisson distribution at the concentration of a single DNA molecule per microcapsule.When the DNA template concentration was 0.07ng/m L,some of the encapsulated template DNA could not work,probably because the template DNA potentially adsorb on the PIC membrane,which contained polycationic PEI,via electrostatic interaction during the encapsulation process.However,quantita-tive discussion on the number of the encapsulated DNA molecule

could be complicated because the number of the DNA molecule is sensitive to purity and stability of the template,and encapsulation yield.In addition,the percentage of the ?uorescent microbeads could be affected by the number of the encapsulated template DNA molecule and interaction between PEI and DNA.Future detailed experiment is necessary to reveal the number of encapsulated DNA molecule and reaction ef ?ciency or the encapsulated template DNA molecule.

The method described herein for cell-free protein synthesis has several advantages over droplet microcompartmentalization methods.Although the amount of synthesized protein product was extremely low compared with the bulk reaction in a microplate because of the small volume of the microcapsules,the protein was retained in the small volume of the microcapsules,whereas the small-molecule byproducts were excreted through the semi-permeable membrane.The continuous exchange of substrates and byproducts through the membrane may have favored the contin-uous production of protein in the microcapsules because some byproducts inhibit protein synthesis in a concentration dependent manner (26e 28).The reaction volume of microcapsules was on the order of picoliters,6orders of magnitude smaller than the reaction volume of conventional microplates (microliters)(22,23).In addi-tion,our microcapsules permitted prolonged encapsulation of GFP (for months).Prolonged storage of protein is dif ?cult to achieve with W/O or W/O/W droplets (1,2,4).All these advantages make our system potentially useful for ultra-high-throughput formation of protein libraries.

Furthermore,protein synthesis from a single molecule of DNA in the semipermeable microcapsules can be expected to enable direct preparation of protein libraries from a solution containing DNA molecules with a variety of mutations.Such a protein library can be prepared in a single step,which makes this system more conve-nient than a two-step system involving PCR and protein synthesis (21).In addition,substrates can be supplied to the resulting protein library in the microcapsules via their semipermeable membranes,which will allow the screening of functional proteins including enzymes.

In conclusion,we developed a microcompartmentalized reac-tion system for cell-free protein synthesis in uniformly sized PEI-coated alginate semipermeable microcapsule containing enzymes and template DNA.The microcapsules were prepared by means of a micro ?uidic device.Substrates for cell-free protein synthesis diffused into the microcapsules through the PIC membranes composed of alginate and PEI.We used the developed method to synthesize GFP,which was detected by means of ?uorescence mi-croscopy and was retained in the microcapsules for months.We also synthesized GFP from a single molecule of template DNA encapsulated in the microcapsules by means of a limiting dilution method.The developed system can be expected to useful for high-throughput screening based on enzymatic activities.

ACKNOWLEDGMENTS

A part of this work was conducted at the AIST Nano-Processing Facility supported by the Nanotechnology Network Japan project of the Ministry of Education,Culture,Sports,Science and Technology (MEXT),Japan and was supported by a Grant-in-Aid for JSPS fellows from MEXT.References

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FIG.4.Effects of template DNA and sodium alginate concentrations on cell-free protein synthesis.Blank condition indicates the background ?uorescence intensity for cell-free protein synthesis without a template

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