A cell-free protein-producing gel

A cell-free protein-producing gel

(Parte 2 de 3)

One of the obvious factors was gene protection. Table 2 summarizes the results of our investigations on the effect of gene protection. We started with the SPS control and then sequentially added the same amount of free linear double-stranded DNA

(dsDNA) or free X-DNA (but without enzymatic crosslinking, that is, no gel formation). With a simple addition of free dsDNA (40bp long, which corresponds to the total length of two arms of X-DNA), we observed a 5.5 times higher expression compared with the SPS control. By adding free X-DNA to the SPS, we observed an expression that was 18 times higher than the SPS control. These studies suggested that dsDNA and X-DNA might have protected genes. The protection effect of X-DNA was further studied by carrying out enzymatic digestion of plasmid mixed with X-DNA or plasmid itself. As shown in Supplementary Fig. S6a, X-DNA protects the plasmid DNA from DNase digestion (as a control, X-DNA does not protect mRNA from RNase digestion (see Supplementary Fig. S6b)). However, the expression from the above mixture of plasmid with X-DNA was still 5 times lower than that from the P-gel, suggesting that the gel format is necessary to reach the highest expression level. When we made a gel by entrapping plasmids within a DNA hydrogel without physically crosslinking the gene

434 NATURE MATERIALS | VOL 8 | MAY 2009 | w.nature.com/naturematerials

NATUREMATERIALSDOI:10.1038/NMAT2419 ARTICLES

Lane number

(kDa) 98

Rluc

P-gel SPS AcGFP Blank SPS P-gel a b

Figure 3 | Comparison of the proteins expressed from P-gel and SPS. a, Total protein comparison. SDS–polyacrylamide gel electrophoresis assays of two expressed proteins: Rluc and AcGFP. Lane 1 corresponds to the pre-stained protein standards (SeeBlue Plus2, Invitrogen). The red arrows indicate the Rluc (34kDa, lane 2) and AcGFP (27kDa, lane 4) expressed from P-gels. Lanes 3 and 5 are the lysates from SPS controls for Rluc and AcGFP, respectively. Lysate control (blank control) is in lane 6. The lanes were reorganized from the same gel without any image manipulation. b, Functional protein comparison. Left: bioluminescence from Rluc; right: green fluorescence from AcGFP.

Functional Rluc e xpr ession (mg ml

Figure 4 | Mechanism studies of P-gel expression. a, A northern blot analysis of the Rluc mRNA from P-gel and the SPS control. Lane 1, 2 and 3: standard mRNA at 25ng, 2.5ng and 0.25ng, respectively. Lane 4, 5 and 6: mRNA extracted from the same volume of P-gel (16ng mRNA), SPS (0.3ng mRNA) and blank (0ng mRNA) lysates, respectively. The P-gel produced 53 times more mRNA than the SPS control. The mRNA amounts from P-gel and SPS were calculated using ImageJ (http://rsb.info.nih.gov/ij/) based on the mRNA standard. b, Rluc expression with a variation of surface-tovolume ratio. Different PDMS micromoulds were microfabricated with the following dimensions: width and length at 200µm and 400µm, respectively, with depths of 10µm, 30µm or 70µm. By manipulating the appropriate number of P-gel micropads, the total volume as well as the gene amounts of P-gel was kept constant, while varying the overall surface areas. Error bars represent standard deviations from three replicates.

to the gel scaffold, the expression level was 62 times higher than SPS but was still about 6% of that of the P-gel system, suggesting that physically crosslinking the gene within the gel scaffold is another important step. We further explored the effect of linear linking between X-DNA and plasmid in the format of X-plasmid-X (that is, capping the plasmid with two X-DNAs but, again, without crosslinking and thus without gel formation, Supplementary Table S4). We observed a twofold enhancement, suggesting that the X-DNA capping may have protected the plasmid. Putting together these observations, our results (see Table 2 and Supplementary Table S4) indicate that (1) conditions that protect DNA (for example, adding free dsDNA, free X-DNA, capping the gene with X-DNA or encapsulating the gene inside a gel) will enhance the cell-free protein expression; and (2) more importantly, physically crosslinking the genes into the DNA hydrogel scaffold provides a novel format with extra enhancement for cell-free protein production.

In addition to the gene protection, transcription efficiency itself is another key factor for protein expression. Although the kinetics of transcription are rather complicated in the P-gel system owing to the coupled transcription and translation processes, we can attribute the enhanced transcription efficiency to two important parameters that are unique to the P-gels: (1) a higher overall gene concentration due to the compressed volume of the gel—a condition that cannot be realized in the solution phase because of the extra volume of the solvent and the fixed solubility of DNA, and (2) a faster enzyme turnover rate due to the closer proximity of genes. Detailed calculations are discussed in Supplementary Discussion S1.

Aftertheabovestudiesatthetranscriptionlevel,wesubsequently examined the translation process. It is reasonable to assume that transcription occurred mostly on or close to the surface of the P-gel whereas translation occurred mainly outside (in the bulk solution) the P-gel micropads for the following reasons: (1) the P-gel micropads were extremely thin (only 20µm) and relatively large in surface (millimetres in length and width); (2) P-gels were incubated with lysates after, not before, the gelling process, and thus no encapsulation could occur; and (3) the translation machinery is composed of protein complexes including ribosomes that are very large in size, and thus would have difficulty diffusing within the P-gel pores. To validate this surface-dominant hypothesis, we fixed the volume of total P-gel micropads, but varied the total surface area. Indeed, the P-gel expression of Rluc increased with an increase of surface area (Fig. 4b), strongly suggesting that the surface area, not the total volume, had an important role in the P-gel system. Figure 2c also revealed that when the ratios of X-DNA and plasmid varied from 1,0 to 4,0 (thus, changed the internal pore sizes, see Supplementary Fig. S7), protein expression levels were very similar, suggesting that pore sizes had no direct effects on the P-gel expression.

NATURE MATERIALS | VOL 8 | MAY 2009 | w.nature.com/naturematerials 435

ARTICLES NATUREMATERIALSDOI:10.1038/NMAT2419

Table 2|Effects of gene template conditions on protein production.

Condition Schematic diagram Rluc yield (mgml−1, based on activity) Fold increase

SPS (plasmid alone) Lysate solution

SPS with dsDNA (plasmid+free dsDNA)

Lysate solution

SPS with X-DNA (plasmid+free X-DNA)

Lysate solution

Plasmid-entrapped gel (plasmid inside a gel)

Lysate solution

P-gel (plasmid covalently crosslinked to gel matrix)

Lysate solution

By simply crosslinking free genes with X-DNA, we have, for the first time, produced a variety of different proteins with a high efficiency and yield from a hydrogel. Some of these proteins are important for complex materials synthesis but until now have been impossible to produce from a living cell (for example, a ‘glue protein’ from mollusc mussels). In addition, protein production from the P-gel can be further enhanced through optimization of reaction conditions (see Supplementary Discussion S2). More importantly, compared with cell-based systems, non-physiological perturbations, such as adding detergents or changing temperatures, can be applied to improve P-gel protein production (for example, expressing membrane proteins) without worrying about affecting other bioprocesses or killing cells, as long as the cell-free systems used will not be inactivated. We expect that other formats, such as those that are column based or microfluidic-channel based, will be easily adapted to the P-gel system for massive parallel and/or totally automatic protein production. We further predict that the tremendous protein production potential of the P-gel system will make high-throughput protein engineering and onsite protein production a reality in the near future. This novel protein production system, less limited by biology but more similar to a cell by virtue of the gel format, will have an immediate and important impact on materials science and engineering including catalyses, biomaterials, tissue engineering, drug discovery and drug delivery.

Methods

Synthesis of X-DNA and linear plasmids. All oligonucleotides were commercially synthesized by Integrated DNA Technology. The X-DNA sequences (see Supplementary Table S2) were designed and the X-DNAs were prepared and characterized following the same procedures as described in our previous publications . The Rluc gene was PCR-amplified from the pRL-Null vector (Promega) using two primers: ATG CCA TGG CTT CGA AAG T ATG ATC CAG and TAC C G TTA TTG TTC ATT T GAG AAC TCG C. After amplification, the Rluc gene was inserted into the Nco I and Sma I sites of the expression vector pIVEX1.3WG (Roche Applied Science) to generate pIVEX1.3RL (for wheat germ lysate). All other genes (except AcGFP) were cloned into the same cloning sites. The AcGFP gene was first cut out from the plasmid pAcGFP1 (Clontech) using Nco I and Stu I and then was subcloned into the pIVEX1.3WG vector at the Nco I and Sma I sites to generate pIVEX1.3AcGFP. All of the plasmids were linearized with Apa I before being used to construct the P-gels for expression. The size of the entire luciferase plasmid is 4,134 bp, and the size of the luciferase geneis936bp(or1,331bpwiththeT7promoter,5’UTRand3’UTR).

Construction of P-gel micropads. X-DNA and linear plasmids were first mixed at a pre-determined molar ratio in the presence of T4 DNA ligase (Promega) to form the P-gel precursor. For an X-DNA/plasmid ratio of 2,0:1, we used 35.0µm of X-DNA and 17.5nM of plasmid. P-gel micropads were then created by confining the precursor in a PDMS mould fabricated with precisely defined dimensions using standard photolithography and soft lithography processes (Fig. 1). Briefly, a

436 NATURE MATERIALS | VOL 8 | MAY 2009 | w.nature.com/naturematerials

NATUREMATERIALSDOI:10.1038/NMAT2419 ARTICLES

10µl drop of the P-gel precursor was added onto an 3-aminopropyltriethoxysilane (APTES)-modified glass slide and covered with the PDMS mould. After gelation for at least 4h at room temperature with gentle shaking, the PDMS mould was peeled off, leaving the P-gel micropads on the glass substrate ready for use.

Expressions of proteins. Coupled transcription and translation kits (Rapid Translation System) were purchased from Roche, and reactions were carried out by following the procedures suggested by the manufacturer. For protein expression, the P-gel micropads containing specific amounts of plasmids were first transferred from the glass slide to the reaction solution. In the SPS controls, the linear plasmid was added directly to the reaction solution. For the SPS experiment in the presence of non-coded DNA, 23.5µg of 40bp free dsDNA and 80bp of blunt-ended free X-DNA were respectively added to the SPS control. Unless stated otherwise, the reaction volume was kept at 50µl and the feed solution at 1ml. The protein expressions were conducted in the Proteomaster (Roche) at 24 C with 900r.p.m. of shaking. After incubation for a specific time, the reaction solutions were stored at −80 C for various assays.

Protein assays. The detailed methods for protein assays are described in Supplementary Method S1. Briefly, the amount of total expressed proteins was quantified based on SDS–polyacrylamide gel electrophoresis assays, western blotting and/or enzyme-linked immunosorbent assays when antibodies were available. The active protein amount was evaluated based on functional assays such as measuring luminescence or fluorescence.

Northern blotting. A digoxigenin (DIG)-labelled DNA probe was synthesized by random primed labelling with a DIG-High Prime Labeling and Detection kit (Roche) using the Rluc gene (Xba I–Nhe I fragment of Promega’s pRL-null vector) as a template. The positive control, mRNA encoding luciferase, was used as a quantitative standard and was synthesized with the Riboprobe In Vitro Transcription System (Promega) using the Apa I linearized plasmid pIVEX1.3 RL as a template. The products were further purified following DNase treatment. Before electrophoresis, total RNA was isolated from the experimental cell-free lysates with a QIAamp Viral RNA Isolation Mini Kit (Qiagen). The RNA was finally eluted in water and treated with DNase. Five microlitres of each eluate was then electrophoresed on a 1% agarose/formaldehyde gel. The transfer to a positively charged nylon membrane, pre-hybridization, hybridization with a DIG-labelled probe, low stringency washing as well as high stringency washing were all carried out following the NorthernMax protocol (Ambion). The DIG label was then detected bychemiluminescenceaccordingtothemanufacturer’smanual(Roche).

Received 24 April 2008; accepted 23 February 2009; published online 29 March 2009

References

1. Kang, H. W., Tabata, Y. & Ikada, Y. Fabrication of porous gelatin scaffolds for tissue engineering. Biomaterials 20, 1339–1344 (1999). 2. Holmes, T. C. et al. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc. Natl Acad. Sci. USA 97, 6728–6733 (2000). 3. Kisiday, J. et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: Implications for cartilage tissue repair. Proc. Natl Acad. Sci. USA 9, 996–10001 (2002). 4. Lutolf, M. P. et al. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nature Biotech. 21, 513–518 (2003). 5. Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Mater. 3, 249–253 (2004). 6. Kong, H. J., Kaigler, D., Kim, K. & Mooney, D. J. Controlling rigidity and degradation of alginate hydrogels via molecular weight distribution. Biomacromolecules 5, 1720–1727 (2004). 7. Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotech. 23, 47–5 (2005). 8. Jeong, B., Bae, Y. H., Lee, D. S. & Kim, S. W. Biodegradable block copolymers as injectable drug-delivery systems. Nature 388, 860–862 (1997). 9. Kiser, P. F., Wilson, G. & Needham, D. A synthetic mimic of the secretory granule for drug delivery. Nature 394, 459–462 (1998). 10. Miyata, T., Asami, N. & Uragami, T. A reversibly antigen-responsive hydrogel.

Nature 399, 766–769 (1999). 1. Beebe, D. J. et al. Functional hydrogel structures for autonomous flow control inside microfluidic channels. Nature 404, 588–590 (2000). 12. Halberstadt, C. et al. A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep. Tissue Eng. 8, 309–319 (2002).

13. Lin, D. C., Yurke, B. & Langrana, N. A. Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel. J. Biomech. Eng. 126, 104–110 (2004). 14. Li, J. et al. Self-assembled supramolecular hydrogels formed by biodegradable

PEO-PHB-PEO triblock copolymers and alpha-cyclodextrin for controlled drug delivery. Biomaterials 27, 4132–4140 (2006). 15. Dong, L., Agarwal, A. K., Beebe, D. J. & Jiang, H. Adaptive liquid microlenses activated by stimuli-responsive hydrogels. Nature 442, 551–554 (2006). 16. Peppas, N. A., Hilt, J. Z., Khademhosseini, A. & Langer, R. Hydrogels in biology and medicine: From molecular principles to bionanotechnology. Adv. Mater. 18, 1345–1360 (2006). 17. Um, S. H. et al. Enzyme-catalysed assembly of DNA hydrogel. Nature Mater. 5, 797–801 (2006). 18. Li, Y. G. et al. Controlled assembly of dendrimer-like DNA. Nature Mater. 3, 38–42 (2004). 19. Li, Y. G., Cu, Y. T. H. & Luo, D. Multiplexed detection of pathogen DNA with

DNA-based fluorescence nanobarcodes. Nature Biotech. 23, 885–889 (2005). 20. Um, S. H., Lee, J. B., Kwon, S. Y., Li, Y. & Luo, D. Dendrimer-like DNA-based fluorescence nanobarcodes. Nature Protoc. 1, 995–1000 (2006). 21. Betton, J. M. Rapid translation system (RTS): A promising alternative for recombinant protein production. Curr. Protein Peptide Sci. 4, 73–80 (2003). 2. DeVries, J. K. & Zubay, G. DNA-directed peptide synthesis. I. The synthesis of the alpha-fragment of the enzyme beta-galactosidase. Proc. Natl Acad. Sci. USA 57, 1010–1012 (1967). 23. Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y. & Alakhov, Y. B.

A continuous cell-free translation system capable of producing polypeptides in high-yield. Science 242, 1162–1164 (1988). 24. Kim, D. M. & Choi, C. Y. A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotech. Prog. 12, 645–649 (1996). 25. Kim, D. M. & Swartz, J. R. Prolonging cell-free protein synthesis with a novel

ATP regeneration system. Biotech. Bioeng. 6, 180–188 (1999). 26. Kim, D. M. & Swartz, J. R. Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotech. Bioeng. 74, 309–316 (2001). 27. Kim, D. M. & Swartz, J. R. Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotech. Bioeng. 85, 122–129 (2004). 28. DiTursi, M. K., Cha, J., Newman, M. R. & Dordick, J. S. Simultaneous in vitro protein synthesis using solid-phase DNA template. Biotech. Prog. 20, 1705–1709 (2004). 29. Ghosh, D. et al. Transcription of T7 DNA immobilised on latex beads and Langmuir-Blodgett film. J. Biochem. Biophys. Methods 62, 51–62 (2005). 30. Yang, J. H. et al. Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system. Biotech. Bioeng. 89, 503–511 (2005). 31. Calhoun, K. A. & Swartz, J. R. Total amino acid stabilization during cell-free protein synthesis reactions. J. Biotech. 123, 193–203 (2006). 32. Rege, K. et al. In vitro transcription and protein translation from carbon nanotube-DNA assemblies. Small 2, 718–722 (2006). 3. Mei,Q.,Fredrickson,C. K.,Simon,A.,Khnouf,R.&Fan,Z. H.Cell-freeprotein synthesis in microfluidic array devices. Biotech. Prog. 23, 1305–1311 (2007).

Acknowledgements

The work is partially supported by NYSTAR Faculty Development Program Award, NYSTAR CAT grant, US National Science Foundation’s CAREER award (grant number: 0547330) and a USDA NRI grant. We wish to acknowledge L. Ding, M.R. Hartman, S. Y. Kwon, S. Tan and C. Umbach for technical support and helpful discussions. The authors also thank M. J. Campolongo, J. C. March, J. Kahn, E. J. Rice and T. Tran for proofreading this manuscript.

Author contributions

N.P., S.H.U. and D.L. conceived and designed the experiments. N.P., S.H.U., H.F. and J.X. carried out the experiments. N.P., S.H.U. and D.L. analysed the data. N.P., S.H.U., H.F., J.X. and D.L. co-wrote the paper. All authors discussed the results and commented on the manuscript.

(Parte 2 de 3)

Comentários