ARTICLES
PUBLISHED ONLINE: 29 MARCH 2009 | 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 5 mg ml−1of functional proteins. The mechanisms behind the high efficiency and yield include improved gene
stability, higher local concentrationand a faster enzyme turnover rate due to a closer proximity of genes. Wehave tested a total
of 16 different P-gels and have successfullyproduced all 16 proteins including membrane and toxic proteins, demonstrating that
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).
Inspired by and on the basis of our DNA hydrogels, we constructed a
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
(see Supplementary Fig. S1) within a polydimethylsiloxane (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
reaction volume was kept at 50 µl and the reaction time at 24 h.
Current cell-free protein expression systems developed over
the past 40 years have led to an increased volumetric yield in
the micrograms per millilitre range but seldom reaching the
milligrams per millilitre level22–33. 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, ArkansasState University, State University, Arkansas 72467,
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 0.99 ng for each
micropad. We used 100, 200, 400 and 800 pads, which corresponded
to P-gel volumes of 2, 4, 8 and 16 µl, and plasmid amounts of 99.2,
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 397 ng of genes) produced close to 100µg of luciferase in
a 50 µl reaction volume within 24 h, equivalent to an expression
efficiency of 250 µg of protein per microgram of plasmid and
an expression yield of 2.0 mg ml−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 19,000 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
in the reaction, suggesting that there are more factors involved other
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 8 µl) and varied the plasmid concentration of each
micropad from 0.99 to 99 ngµl−1. As shown in Fig. 2b, the Rluc
expression reached a plateau with 400 ng of plasmids (equal to a
concentration of 50 ng µl−1per micropad). As a comparison, SPS
became saturated when the plasmid amount increased to 4 µg (equal
to a concentration of 80 ng µl−1, Supplementary Fig. S3).
To further explore the mechanism of the P-gel system, we varied
the X-DNA/gene ratio from 1,000:1 to 6,000:1 by changing the
X-DNA amount while keeping the number of micropads and the
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