A cell-free protein-producing gel

A cell-free protein-producing gel

(Parte 1 de 3)

ARTICLES PUBLISHEDONLINE:29MARCH2009 | DOI:10.1038/NMAT2419

A cell-free protein-producing gel

Nokyoung Park*, Soong Ho Um*†, Hisakage Funabashi, Jianfeng Xu† and Dan Luo‡

Proteins are important biomaterials and are generally produced in living cells. Here, we show a novel DNA hydrogel that is capable of producing functional proteins without any living cells. This protein-producing gel (termed ‘the P-gel system’ or ‘P-gel’) consists of genes as part of the gel scaffolding. This is the first time that a hydrogel has been used to produce proteins. The efficiency was about 300 times higher than current, solution-based systems. In terms of volumetric yield, the P-gel produced up to 5mgml−1 of functional proteins. The mechanisms behind the high efficiency and yield include improved gene stability,higherlocalconcentrationandafasterenzymeturnoverrateduetoacloserproximityofgenes.Wehavetestedatotal of16differentP-gelsandhavesuccessfullyproducedall16proteinsincludingmembraneandtoxicproteins,demonstratingthat the P-gel system can serve as a general protein production technology.

Hydrogels produced from biomolecules1–7 as well as synthetic molecules8–16 have many applications in drug delivery, tissue engineering and microfabrication. Recently, our group reported an enzyme-catalysed DNA hydrogel17 of which the scaffolding was composed entirely of branched DNA (refs 18–20). InspiredbyandonthebasisofourDNAhydrogels,weconstructeda hydrogel using similar X-shaped DNA (X-DNA) as crosslinkers but with actual genes as monomers. By deliberately incorporating the genes as part of the gel scaffolding, we created a protein-producing hydrogel (P-gel). This is the first time that a hydrogel has been used to produce proteins.

To fabricate the P-gel, we ligated X-DNA and linear plasmids (seeSupplementaryFig.S1)withinapolydimethylsiloxane(PDMS) micromould (Fig. 1a,c). Subsequently, protein was expressed simply by incubating the P-gel micropads with cell lysates for a specific time period (Fig. 1b). We have successfully used several different, commercially available cell-free systems, including lysates made from E. coli, wheat germ and rabbit reticulocyte (see Supplementary Table S1), suggesting that the P-gel format is compatible with different systems. Here, we focused on Renilla luciferase (Rluc) as the model protein and wheat germ lysates from Roche as the model, cell-free system. In this system, the reaction compartment is separated from the feeding buffer by a membrane (see Supplementary Fig. S2)21. Here, we define ‘expression efficiency’ as the amount of protein produced per unit of plasmid (gene) and ‘expression yield’ as the amount of protein produced per unit of reaction volume. Unless stated otherwise, the reactionvolumewaskeptat50µlandthereactiontimeat24h.

Current cell-free protein expression systems developed over the past 40years have led to an increased volumetric yield in the micrograms per millilitre range but seldom reaching the milligrams per millilitre level22–3. Almost all cell-free systems are solution phase systems (SPS), in which the gene templates are dispersed in solution. Here, we used SPS as the ‘benchmark’ to evaluate the productivity (efficiency and yield) of P-gel. In preliminary experiments, we produced Rluc protein with the P-gel using the same conditions as those for the SPS. Our initial results indicated that not only was functional Rluc produced from the P-gel, but the productivity of this system was significantly

Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853-5701, USA. *These authors contributed equally to this work. †Present address: Department of Materials Science and Engineering, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA (S.H.U.); Arkansas Bioscience Institute, Arkansas State University, State University, Arkansas 72467, USA (J.X.). ‡e-mail:dan.luo@cornell.edu.

higher than that of the SPS. Encouraged by this outcome, we investigated and later optimized the parameters governing protein production that were specific to P-gels. These parameters included the number of P-gel micropads, the concentration of Rluc plasmid in the P-gel scaffolding and the molar ratio between the X-DNA and the Rluc gene.

We first varied the number of P-gel micropads used in the reaction but fixed the plasmid (gene) amount at 0.99ng for each micropad.Weused100,200,400and800pads,whichcorresponded to P-gel volumes of 2, 4, 8 and 16µl, and plasmid amounts of 9.2, 198, 397 and 793 ng, respectively. Thus, through this design, we changed both the gel volume and gene amount in each reaction but fixed the P-gel gene concentration. As a control, the same amount of plasmid was used in the SPS. The protein expression results (Fig. 2a) demonstrate that, compared with the SPS control, the P-gel exhibited higher efficiency and better yield under each condition. In particular, the P-gel consisting of 400 micropads (about 397ng of genes) produced close to 100µg of luciferase in a 50µl reaction volume within 24h, equivalent to an expression efficiency of 250µg of protein per microgram of plasmid and an expression yield of 2.0mgml−1. This represents a 93.5-fold enhancement in both yield and efficiency over the SPS control. In terms of amplification ability, for each copy of the gene under this condition, the P-gel produced about 19,0 copies of the protein molecules. Figure 2a also shows that protein production from the P-gel is not linearly proportional to the number of P-gel micropads inthereaction,suggestingthattherearemorefactorsinvolvedother than P-gel volume and gene amount.

To investigate the effect of the total gene amount, we fixed the number of P-gel micropads at 400 (thus, maintaining a preset gel volume at 8µl) and varied the plasmid concentration of each micropad from 0.9 to 9ngµl−1. As shown in Fig. 2b, the Rluc expression reached a plateau with 400ng of plasmids (equal to a concentration of 50ngµl−1 per micropad). As a comparison, SPS becamesaturatedwhentheplasmidamountincreasedto4µg(equal to a concentration of 80ngµl−1, Supplementary Fig. S3).

To further explore the mechanism of the P-gel system, we varied the X-DNA/gene ratio from 1,0:1 to 6,0:1 by changing the X-DNA amount while keeping the number of micropads and the

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Glass slide

APTESP-gel precursor drop PDMS mouldP-gel padsCover with a PDMS mouldPeel off the mould

Step 1Step 2

X-DNA Plasmid

In vitro transcription and translation

P-gel pads mRNA Protein

Transcription

TranslationP-gel Ligation

1 m × 1 m × 20 µm

P-gel pads Lysate

Polymerase Amino acids ATP and so on a b

Figure 1 | Fabrication of P-gel micropads. a, A schematic diagram illustrating the formation of P-gel micropads (side view). The P-gel precursor drop, which contains X-DNA, genes and T4 DNA ligase, was confined within a PDMS mould with precisely defined dimensions: 1.0mm×1.0mm×20µm (20nl) to control and adjust the exact gene amount within the P-gel scaffolding and also to obtain a higher surface-to-volume ratio of P-gel on an APTES-coated glass slide. Extra solution was able to flow out owing to the surface modification (Step 1). After gelation, the PDMS mould was peeled off from the substrate (Step 2), and P-gel micropads were formed using the pre-designed dimensions. b, A schematic diagram of the gelation process through enzymatic crosslinking and cell-free expression with P-gel pads. c, A fluorescent image of the P-gel pads after staining with SYBR I. Each micropad was separated by 100µm spacers (scale bar: 500µm).

gene concentration constant. Figure 2c indicates that the optimal X-DNA/gene ratio was 2,0:1 for Rluc expression. When the X-DNA/gene ratio reached higher than 4,0:1, the Rluc expression decreased markedly. These results suggest that P-gel can tolerate a wide range of crosslinker/gene ratios but that too high a ratio of X-DNA/gene (>4,0:1) adversely affects P-gel expression, probably owing to changes in the properties of the gel (for example, extremely large amounts of X-DNA make the gels stiffer and more similar to a solid rather than a gel). Under these optimized conditions (plasmid density and ratio), P-gel also showed the highest transcription level (see Supplementary Fig. S4), suggesting that an increase in transcription efficiency may have a role in P-gel’s enhanced protein production.

Next, we monitored protein expression as a function of reaction time to gain insights into kinetic behaviour. Rluc expression increased linearly with reaction time (Fig. 2d). This was also true for SPS (Fig. 2d, inset), but there were two obvious differences between these two systems. The first was that the time span of the linear region for the P-gel was much longer. The P-gel continuously produced proteins for more than 36h, whereas SPS reached a plateau after only 12h. The second difference was that the speed (slope) of gene expression using the P-gel was much faster—about 73-fold faster than that of the SPS in the first 12 h (Fig. 2d, inset). Thus, the P-gelshowedgreatlyimprovedkineticbehaviourovertheSPS.

We also investigated the effect of X-DNA sequences on protein production. Three different X-DNA sequences (see Supplementary

Table S2) were tested under the same conditions. The amounts of protein produced from each P-gel were similar, suggesting that the sequences of X-DNA did not affect the amount of protein production.

From the above results, we have seen that P-gel can produce a model protein (Rluc here) with a much higher efficiency and yield compared with the SPS control. An important question is whether or not P-gel can produce other proteins in a similar fashion. To investigate the potential of the P-gel as a universal protein production system, we tested a total of 16 P-gels with 16 different genes coding for reporter proteins, membrane proteins, kinases and highly repetitive, toxic proteins (see Supplementary Table S3). The molecular weights ranged from 16 to 110kDa. All 16 proteins were successfully produced from the P-gel system with about 1–7mgml−1 total protein yields (see Supplementary Fig. S5). Notably, the total Rluc production (both active and inactive forms) from P-gel was 134 times higher than that of SPS. In addition to the total protein yield, we also measured the amount of active proteins produced from P-gel when functional assays were available (see Table 1 and Supplementary Table S3). Compared with the SPS controls, P-gels produced far more active proteins. Figure 3a,b shows visual comparisons of two proteins, (Rluc and Aequorea coerulescens green fluorescent protein (AcGFP)), produced from both P-gels and SPS. These images visibly demonstrate that the P-gel has produced significantly more functional proteins than SPS.

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ARTICLES NATUREMATERIALSDOI:10.1038/NMAT2419

(100 pads)198 ng (200 pads)

Functional Rluc e xpr ession

(mg ml

) (lines ) ease (bar

Functional Rluc e xpr ession

Functional Rluc e xpr ession

(mg ml

SPS P-gel a b xpr ession

(mg ml

Figure 2 | Functional Rluc expression from P-gel with different parameters. a, Effect of the total gene amounts on expression, determined by varying the number of P-gel micropads in the reaction (filled squares). The same amounts of the plasmid were used in SPS control reactions (open squares). The bars indicate the fold increase in functional protein expression efficiency of P-gels over SPS controls. b, Effect of the monomer (plasmid) concentrations of P-gel on Rluc expression. The line was fitted using a second-order polynomial regression. c, Effect of the crosslinker (X-DNA) and plasmid ratio of P-gel on functional Rluc expression. d, Time course of functional Rluc expressions of P-gel (open diamonds) and SPS (filled squares) and plotted in different y-axis scales (inset, the arrows indicate their corresponding axes). Error bars represent standard deviations from three replicates.

Table 1|Reporter protein production yield and efficiency.

P-gel production SPS production Fold increase (P-gel over SPS)

Protein Total (mgml−1)

Total production

Active protein

*Specific activities were calculated for the total amount of proteins produced. Activities of standard proteins: Rluc, 7.5×10 RLUmg (RLU: relative luminescence unit); AcGFP, 2.1×10 RFUmg (RFU: relative fluorescence unit); chloramphenicol acetyltransferase (CAT), 1.5×10 unitmg .

Indeed, it was surprising to see that by simply converting the gene from a solution format to a crosslinked hydrogel format, the P-gel system was capable of producing proteins with productivity well above that of the already optimized SPS. To understand P-gel’s better performance relative to SPS, we further studied the underlying mechanisms by investigating transcription and translation separately. At the transcription level, the messenger RNA amounts of the reaction solution were followed using northern blotting (Fig. 4a, lanes 1-3). The P-gel produced 53 times more mRNA than the SPS control (Fig. 4a, lane 4 versus lane 5). This strongly suggests that the transcription from P-gel was significantly enhanced. Many factors may have contributed to this enhanced transcription that leads to higher protein expressions.

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