WO2015144859A9

WO2015144859A9 - Cell-free protein synthesis with semi-continuous or continuous supply of rna template

Info

Publication number: WO2015144859A9
Application number: PCT/EP2015/056662
Authority: WO
Inventors: Leopold Georgi; Stefan Kubick; Victoria SCHULDT
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.
Priority date: 2014-03-26
Filing date: 2015-03-26
Publication date: 2016-04-28
Also published as: WO2015144859A3; WO2015144859A2

Abstract

The invention relates to a method for semi-continuously or continuously supplying freshly synthesized RNA from a continuously proceeding in vitro transcription formulation to an in vitro translation formulation for cell-free protein synthesis. The method contains the following steps: immobilizing the freshly synthesized RNA from the transcription and using said RNA for the in vitro translation. Either the RNA can be transferred to the translation formulation or the translation formulation can be transferred to the RNA. In any case, RNA is semi-continuously or continuously fed to the translation and the transcription continues to proceed even after RNA removal. The invention further relates to the ability to automate said method.

Description

Automate cell-free protein synthesis with semicontinuous or continuous delivery of freshly synthesized RNA template

The invention relates to a method for carrying out the reaction and for automating in vitro protein expression. The method involves the immobilization of an RNA template during continuous in vitro transcription and the semicontinuous or continuous transfer of this RNA to an in vitro translation compartment for cell-free protein synthesis. Alternatively, the translation approach to RNA can be transported.

Prior art and background of the invention

In the recent past, cell-free protein synthesis has emerged as a simple and efficient alternative to protein expression in cell-based systems [1, 2, 3]. Cell-free protein synthesis is based on cell extracts (lysates) whose cellular machinery is used for translation. For lysate production, prokaryotic cells such as Escherichia coli or eukaryotic cells from wheat germ, reticulocytes and insect cells can be used. The absence of cell walls results in an open system, allowing observation of the system and specific adaptation of reaction conditions. Complex proteins, such as pharmaceutically relevant proteins, can be synthesized, generating completely new biochemical properties even with the help of noncanonical amino acids [4, 5]. The entire process of cell-free protein synthesis has been optimized in the past, especially at the biochemical level to obtain higher yields and active proteins. However, new reaction concepts have also been developed at the level of reaction: there are basically two different ways of carrying out the reaction: the coupled system, in which transcription and translation take place simultaneously in the same compartment, and the linked system, in which transcription and transcription take place Translation into succession and spatially separated, and the "linked" system allowed optimal adaptation of the reaction conditions for both reactions - transcription and translation. Either the transcription mix is added directly to the translation or the synthesized RNA is purified, for example, by gel filtration and then added for translation. Furthermore, there are various formats for in vitro translation: The batch format in which all components are present in one approach. It has a simple structure, but results in only short protein synthesis times. This is due to the depletion of energy sources, eg ATP and GTP, as well as the accumulation of reaction products such as inorganic phosphate, AMP and GMP, which inhibit the reaction. The more complicated continuous formats allow longer reaction times and thus higher yields: In the continuous flow cell-free (CFCF) system, sources of energy are permanently supplied and the reaction products are removed by ultrafiltration. [6] The dialysis version of the "continous exchange cell-free "System (CECF System) permanently removes the low molecular weight inhibitory reaction products by dialysis, which simplifies the design and is therefore more cost effective [7]. Another limiting factor - especially for the long-lived formats - is the degradation of RNA templates by nucleases.

Brief description of the invention

The present invention relates to a method for the cell-free synthesis of polypeptides, comprising the following steps:

1) immobilization of the RNA;

2) translation of the RNA;

The RNA is repeatedly immobilized during a semicontinuous or continuous in vitro RNA synthesis, which does not terminate transcription; and this RNA is repeatedly combined with the translation approach and translated.

Description of the invention

The invention is based on the technical problem that complex proteins such as membrane proteins or proteins with post-translational modifications can be produced in a functionally active manner only in eukaryotic systems. For optimal protein yield, the transcriptional and translational responses are separated ("linked" system) to optimize both reactions individually, but the mRNA that is synthesized during transcription becomes a template for protein synthesis in translation Therefore, an active mRNA transfer from the transcriptional to the translational compartment is needed, furthermore, the mRNA is degraded relatively rapidly by nucleases and therefore needs to be replenished continuously The proposed method makes it possible to automatically transfer mRNA without interfering with transcription and translation, whereby always freshly synthesized mRNA can be replenished. An automation of a method for the transfer of fresh mRNA saves time and personnel costs.

Current efforts for cell-free synthesis of proteins include coupled systems and linked systems, whereas in the former transcription and translation take place simultaneously in a reaction compartment, which does not require the transfer of mRNA, but the individual reactions can not be optimized individually is essential for eukaryotic cell-free systems in order to obtain higher protein yields, whereas in the linked system the reactions take place sequentially. The transcription solution is completely translated or completely purified, and only the RNA is translated. The transcription then stops running and the entire mRNA-containing volume is given once for translation. The mRNA is degraded by nucleases, which leads, especially in long-lived formats such as CFCF (Continuous Flow Seif reporting protein synthesis) and CECF (Continuous Exchange Seif reporting Protein Synthesis) over time, that insufficient mRNA template is available for effective translation stands. In order to reduce this problem it was tried to stabilize the mRNA. This can be done for example by immobilization, but this protects only the terminal mRNA regions from degradation and so far leads to lower yields in translation.

The invention relates to a comparatively easily automatable method for cell-free protein synthesis, in which mRNA is immobilized during a continuously running transcription and actively transferred to translation.

A method is performed for cell-free synthesis of polypeptides in which the RNA is repeatedly immobilized during a continuous in vitro RNA synthesis, which does not terminate transcription, and this RNA is then repeatedly combined with the translation approach and translated, with transcription and translation takes place in separate compartment.

1) immobilization of the RNA;

2) translation of the RNA; The RNA is repeatedly immobilized during a semicontinuous or continuous in vitro RNA synthesis, which does not terminate transcription; and this RNA is repeatedly combined with the translation approach and translated.

Continuous in vitro RNA synthesis: RNA is continuously produced in the reaction vessel without interruption

Semicontinuous in vitro RNA synthesis: RNA is produced in the reaction vessel, whereby the RNA synthesis is briefly interrupted and resumed. This can e.g. be achieved by regular temperature cycles between 4 ° C and 37 ° C.

One embodiment of the invention is that the RNA is transferred after the immobilization for translation.

Another embodiment of the invention is that the translation approach is transferred to the RNA.

Another embodiment of the invention is that the method is realized in a microfluidic platform. Another embodiment of the invention is that the immobilized RNA is translated.

Another embodiment of the invention is that the immobilized RNA is eluted and then the free RNA is translated.

Another embodiment of the invention is that the immobilized RNA is temperature-controlled or ion-controlled or eluted by elimination or otherwise eluted and then the free RNA is translated.

Another embodiment of the invention is that the RNA is covalently or non-covalently immobilized.

Another embodiment of the invention is that the RNA is spatially removed by a spacer from the Immobilisierungsmatrix defined.

Another embodiment of the invention is that the RNA is immobilized via oligonucleotides and the oligonucleotides in turn are covalently or non-covalently immobilized via a biotin-streptavidin interaction. The oligonucleotides can be spatially removed from the immobilization matrix by a spacer or immobilized directly on the matrix. The immobilization is carried out a) by initially free oligonucleotides which hybridize with the RNA and subsequent immobilization of the RNA oligonucleotide hybrid, or b) by immobilized oligonucleotides, which in turn immobilize RNA by hybridization.

Another embodiment of the invention is that the RNA is immobilized via oligonucleotides and the oligonucleotides in turn are covalently or non-covalently immobilized.

Another embodiment of the invention lies in the fact that the oligonucleotides are spatially removed in a defined manner by a spacer from the immobilization matrix.

Another embodiment of the invention is that the RNA or oligonucleotides is immobilized via biotin-streptavidin interaction.

Another embodiment of the invention is that natural or synthetic RNA templates are synthesized.

Another embodiment of the invention is that the RNA is transcribed from PCR products, plasmid DNA, genomic DNA, cloned DNA fragments, cDNA libraries, or synthetic oligonucleotides.

Another embodiment of the invention is that the cell-free system is based on wheat germs, reticulocytes, yeast, insect cells, CHO cells, cultured human cells or other eukaryotic and prokaryotic origins or is synthetic.

Another embodiment of the invention is that the RNA is immobilized on non-magnetic, magnetic or magnetizable particles such as nano- or microparticles or other solid phases such as smooth surfaces or hydrogels or column material. The wall of the incubation vessel itself could also be used for immobilization.

Another embodiment of the invention is that the solid phase a) is reused or b) is discarded.

Another embodiment of the invention is that magnetizable particles with immobilized mRNA are moved and / or separated with permanent magnets.

Another embodiment of the invention is that magnetisiebare particles with immobilized mRNA with electromagnets / coils are moved and / or separated. Another embodiment of the invention is that the process fluids in the system, such as transcription solution, translation solution or washing solution, a) are moved or b) are stationary.

Another embodiment of the invention is that s i c h between the process fluids, separating fluids, such as air, oil or others, are located. These release fluids can a) be permanently there or b) be removed temporarily.

Another embodiment of the invention is that the transcription is extra slow - for example, by the use of a slower RNA polymerase - which ensures that always freshly synthesized mRNA is transferred.

Another embodiment of the invention is that the transcription is interrupted - for example by cooling - thereby ensuring that always freshly synthesized mRNA is transferred.

Another embodiment of the invention is that the RNA is intermediately stored at low / no nuclease activity (for example, by cooling) and is then first delivered semicontinuously or continuously for translation.

Another embodiment of the invention is that smaller or larger particles with RNA immobilization function are immobilized in a column.

Another embodiment of the invention is that non-magnetic, magnetic or magnetizable particles such as nanoparticles or microparticles or other solid phases such as smooth surfaces or hydrogels for RNA immobilization are used and the automation is done via pipetting robot.

Important advantages of the procedure consist in the fact

(i) that transcription continues after mRNA removal to synthesize mRNA.

(ii) that the mRNA is continuously or semicontinuously translated and that the rate of mRNA addition for translation can be varied.

(iii) that freshly synthesized mRNA can be fed on and thus compensate for mRNA losses by nuclease degradation.

(iv) the method is compatible with the CECF and CFCF formats.

(v) that the method is relatively easy to automate. In general, freshly synthesized mRNA is transferred semicontinuously or continuously from the transcription reaction to the translation reaction, without terminating the RNA synthesis.

The method can also be used for very small reaction volumes in the range below 10 μΙ. In addition, the RNA immobilization matrix is reusable.

An appropriate structure of the reaction in a production plant for proteins is easily recognizable.

The method for carrying out the reaction for cell-free protein synthesis described here is relevant in all biochemical and biotechnological disciplines dealing with recombinant protein expression. Especially in the field of production of functional proteins with post-translational modifications and the expression of membrane proteins for the elucidation of protein structures or for pharmaceutical research and protein production, this method could be of great importance. The method is automatable and forms the basis of an automated reactor for cell-free protein synthesis with separately controlled and controllable compartments. The method can be implemented in small systems in the form of microfluidic platforms or even on a large scale.

Another embodiment of the present invention relates to apparatus for

Execution, observation and control of a cell-free protein synthesis in a microfluidic system, in which RNA is synthesized, repeatedly immobilized and translated.

The immobilization of the RNA takes place on magnetic particles and these are moved by the microfluidic system by means of the device.

The device comprising a support for the microfluidic system consisting of a polymer plate having structured channels (e.g., made by milling or stamping). The dimensions of the microfluidics are preferably about 127 mm x 85 mm and a thickness of about 1, 5 mm. Another component of the device is a 3-axis movable multifunction head with holders for a permanent magnet and a camera for process control. Furthermore, the system includes an electronically controlled

Temperature control unit and thermocouples for generating various

Temperature zones on the microfluidic system. Finally, the fixed microfluidics is connected to a pump system for fluid movement. The multifunctional head can be positioned, for example, by stacking two linear stages and a pneumatic lifting cylinder or by a combination of spindle drives in 3-dimensional space.

The permanent magnet is attached to the multifunction head and the magnetic field is focused by an attached metal cone onto a microfluidic channel. If the metal cone is guided with the focused magnetic field to a channel containing magnetic particles dissolved in liquid, then the particles collect in the area of this field. Is the metal cylinder now by moving the multifunctional head along a

moved fluid-filled fluidic channel, then follow the particles of this movement.

The multifunctional head is equipped with a camera which tracks the movement of the particles.

Necessary pumping steps are accomplished by a syringe pumping array of e.g. 8 independently controllable syringe pumps controlled.

Software controls the movement of the syringe pumps, the 3-axis system and the temperature control.

One embodiment of the present invention provides a microfluidic TRITT

In the separate compartments / areas for the transcription and translation different biochemical conditions can be adjusted and thus the respective reactions independently optimized as well as the continuous synthesis of the biomolecules can be guaranteed.

Exemplary modifications of the Reaktiosnbedinqunqen:

Specific RNA templates were stabilized with 5 'and 3' hairpin structures against degradation by nucleases, immobilized via oligonucleotides during in vitro transcription, and then coupled to magnetic particles. Furthermore, on-chip mRNA transfer was performed by magnetic particles, which are TRITTED

Platform was used for the production of the cytotoxic protein pierisin. At the same time, the non-canonical amino acid was incorporated for fluorescence labeling. The system according to the invention avoids the degradation of the mRNA, since these are continuously in the translation area is transported. The TRITT platform is a modular microreactor that can be adapted to different reaction conditions. Optimizations of the reaction conditions are easy to make due to the separate reaction areas / compartments.

Description of the figures

illustration 1

The illustrated microfluidic TRITT platform is suitable for carrying out the method according to the invention. TRITT stands for "Transcription-RNA Immobilization & Transfer - Translation".

Transcribed RNA is automatically transferred from the transcription area to the transcription area of the device. Automatic washing steps are integrated. The different areas can be used in parallel. The TRITT concept should be compatible with all reaction formats described above.

Figure 2

Magnetizable streptavidin-loaded particles were functionalized with biotinylated oligonucleotides (Hyb3A-15T) that are complementary to the constant part of the mRNA (N ₀ -eYFP-C ₀ ). eYFP-encoding mRNA was transcribed from a PCR template. During continuous transcription mRNA was repeatedly immobilized on the functionalized particles. Again and again, new particles were used, or the particles were reused after elution of the mRNA. In both cases the same amount of mRNA was immobilized for equal times. By extending the incubation time from 30 min to 45 min, more mRNA could be immobilized. The amount of immobilized mRNA can be adjusted by incubation time and particle amount. Transcription is not disturbed by the immobilization of the mRNA.

Figure 3

Magnetizable streptavidin-loaded particles (C1 beads) were functionalized with biotinylated oligonucleotides (Hyb5A-15T or Hyb3B) that are complementary to the constant part of the mRNA. eYFP-encoding mRNA was transcribed from a PCR template (N ₀ - eYFP-Co). The mRNA was immobilized on the functionalized particles.

On the one hand, the mRNA was again eluted from the particles and placed for translation. Fluorescence intensities of the synthesized eYFP were detected, which are in the range of reference translation with conventionally purified by gel filtration mRNA lie. The translation with the previously immobilized and re-eluted mRNA is thus possible without inhibiting the reaction.

On the other hand, the immobilized mRNA was directly translated. As a reference, the translation of free mRNA, which was conventionally purified by gel filtration, was carried out in the presence of the unfunctionalized particles. The translation is inhibited. The translation of immobilized mRNA on these particles leads to similar fluorescence intensities as the reference translation of free mRNA in the presence of unfunctionalized particles. The translation is not or hardly additionally inhibited by immobilized mRNA. Thus, with an alternative solid phase for mRNA immobilization that does not inhibit translation, effective translation should be possible.

Figure 4

Magnetizable streptavidin-loaded particles were functionalized with biotinylated oligonucleotides that are complementary to the constant part of the mRNA. mRNA was transcribed from linear PCR templates and from circular expression vectors as well as linearized expression vectors. Furthermore, mRNA encoding eYFP and luciferase was synthesized. In each case, the same amount of mRNA could be immobilized. For quantification, on the one hand, the amount of mRNA was compared before and after the immobilization process and, on the other hand, the immobilized mRNA was eluted again after a washing process. Both methods led to the same result.

Figure 5

Magnetizable streptavidin-loaded particles (C1 beads) were functionalized with biotinylated oligonucleotides that are complementary to the constant part of the mRNA. eYFP-encoding mRNA was transcribed from a PCR template. The mRNA was immobilized on the functionalized particles. The mRNA-loaded particles were washed on the one hand in a reaction vessel and eluted. In parallel, the mRNA-loaded particles were washed and eluted in a specially designed microfluidic system with magnetically-activated particle transfer. In both ways, the same amount of mRNA was eluted. This demonstrates the automatability of the washing and elution step in a micro-fluidic system. Figure 6

Magnetizable streptavidin-loaded particles (C1 beads) were functionalized with biotinylated oligonucleotides that are complementary to the constant part of the mRNA. The transcription of the eYFP-encoding mRNA was carried out by a PCR template, The immobilization of this mRNA to magnetizable particles, the magnetically actuated separation of the particles, the washing process and the elution of the immobilized mRNA were in a reaction vessel and parallel in a specially designed microfluidic System performed. In both ways, the same amount of mRNA could be immobilized, transported and eluted. This shows the automation of the entire process in a microfluidic system.

Figure 7

Construction of the microfluidic system TRITT for cell-free protein synthesis (CFPS). (A) Exploded drawing of the chip structure. A COC substrate (Cyclic Olefin Copolymer Substrate) with microfluidic structures has been permanently bonded to a cover plate by thermal bonding. Temperature sensors and thermal actuators connected to a heat spreader and the microfluidic chip control the process temperatures. A mobile magnetic device transfers magnetizable particles and binds the mRNA in the microfluidic structures. (B) Top view of the microfluidic structure. The transcription (TK) compartment or the TK space is divided into three chambers, which serve for the stepwise immobilization of the synthesized mRNA to functionalized magnetizable particles. Following transfer of the mRNA loaded particles, the particles were washed to completely remove the TK mixture and the mRNA was removed by temperature as well as low salt elution. The solution containing mRNA was stirred in agitation mixture, with the mRNA serving as a template for protein synthesis. The particles reused for immobilization. (C) An integrated heating resistor and temperature sensor for temperature control. A printed circuit board (PCB) with a heating resistor and a temperature sensor are connected to or mounted on a heat spreader and connected to the compartment of the microfluidic system via thermal grease. (D) Photo of the microfluidic system. The TRITT chip is placed on a magnetic movement machine (xy (-) mechanical stage). Magnetizable particles in the microfluidic structure follow the magnetic field. Figure 8

Transcription 4 is carried out on a solid phase 6 in a transcriptional compartment 1. Translation 5, in which polypeptides 7 are produced, takes place in translation compartment 2 (FIG. 8A).

, FIG. 8C shows the implementation of the inventive method in reaction vessels (reaction vessel one 8, reaction vessel two 9), in which the translation batch is transferred to the RNA. Fig. 8D shows an embodiment of the invention in which the immobilized RNA is eluted and then the free RNA is translated. The immobilized RNA is temperature-controlled or ion-controlled or eluted by cleavage or otherwise, and then the free RNA is translated. FIG. 8E schematically shows an embodiment variant of the inventive method in which immobilized RNA is translated.

Figure 9

Figure 9 shows various immobilization possibilities of RNA for use in the inventive method. The RNA 3 bound to a solid phase 6 can be immobilized by covalent or noncovalent bonds 12 (FIG. 9A). Fig. 98 illustrates an embodiment in which the RNA is spatially removed by a spacer 10 defined by the immobilization matrix. In the embodiment shown in FIG. 9C, the RNA is immobilized via oligonucleotides 11, wherein the oligonucleotides themselves are covalently or non-covalently immobilized. FIG. 9D shows an embodiment in which the oligonucleotides are spatially removed in a defined manner from the immobilization matrix via a spacer. Fig. 9E shows a performance form in which RNA or oligonucleotides are immobilized via biotin-streptavidin interaction. Working Example

1) In Vitro Cell-Free Synthesis of Polypeptides eYFP- or pierisin-encoding mRNA was transcribed continuously from a PCR template, a plasmid, or a linearized plasmid using T7 polymerase at 37 ° C (300 μg DNA in the transcriptional assay). The transcription time was between 30 minutes and 20 hours.

The transcribed mRNA was immobilized during transcription by using biotinylated oligonucleotides that are complementary to the constant part of the mRNA, the oligonucleotides in turn bound to magnetizable streptavidin-loaded particles (eg streptavidin coated superparamagnetic dynabeads of the firmnavitrogen) and where no spacer or a Oligo (dT) ₁₅ spacer was used to spatially remove the oligonucleotides from the immobilization matrix.

The mRNA-loaded particles were removed from transcription after 10-30 min (depending on the RNA concentration in the transcription, which increases over time) at room temperature (RT) with 5-30 μM mM HEPES buffer at 0.5-2.5 mM Mg ²⁺ (MgCl ₂ or CH ₃ COO) ₂ mg) and / or 50-100 mM KCl with 2-3 repetitions of the washing process and the RNA was washed at 25 ° C-70 ° C with 0.5-2μΙ_ water, pure 30 mM HEPES buffer or 30 mM HEPES buffer with MgCl ₂ or CH ₃ COO) ₂ Mg or KCl eluted from the particles. The free mRNA-containing eluate was supplied to the translational mix after cooling to 27 ° C and the free RNA was translated. This process was repeated several times to deliver semicontinuously RNA for translation.

The translation took place in a volume of 20-50 μΙ_ at 27 ° C in Sf21 insect cell lysate system. Depending on the system, translation can take 90 minutes to at least 24 hours.

The number of repetitions for the immobilization and the transfer of the RNA from the transcription to the translation depends on the translation volume (the more translational volume, the more RNA and thus repetitions are needed) and the amount of particles (the more particles, the fewer repetitions). For the initial deployments of RNA for 20 μΙ translational volume using about 0.2 mg beads, for example, 6 repeats are needed. Subsequently, only degraded RNA must be tracked, which can be carried out for example every 60-120 min. The particles were reused or discarded.

For the particle separation from the transcription, the transfer of the particles, the washing and elution step as well as the tempering of the individual reaction steps (transcription, elution, translation) a microfluidic system was used. The microfluidic system consisted of the material cyclo-olefin copolymer (COC) and had approximately the dimensions of a microtiter plate (127 mm x 85 mm) and a thickness of about 1, 5 mm. The microfluidic structures had a depth of 0.3-1 mm and the channels a width of 0.3-1 mm and were made by milling or injection molding. For sealing the channels with COC film (e.g., 135 μηη), a thermal or adhesive-based joining process was used. Three chambers with volumes of 10-30 μΙ_, e.g. 12.5 μΙ_ per chamber used as reaction vessels at 37 ° C for transcription. In these chambers also the immobilization of the RNA to the functionalized magnetisable particles took place. The particles were then transported through the microfluidic structures by means of a NdFeB permanent magnet (1 cm x 1 cm x 1 cm, Br = 13000 Gauss) and a matched steel cone (eg 1 cm height, at 1 cm base diameter), in a 2-10 μΙ_ Washed at room temperature (30-100 μΙ_ wash buffer) and then eluted at 25 ° C to 80 ° C (elution: detach the RNA from the particles in a small buffer volume - 0.5-5 μΙ_). This droplet of dissolved RNA can be fluidly transported to the translation system (operated, for example, at 27 ° C). The various temperature zones on the microfluidic chip were adjusted by electronic heating elements and by feedback control via a temperature sensor. The process fluids (transcription / translation solutions, washing / elution solutions) were moved in the microfluidic system, but at any time separated from one another by the separating fluid air.

2) Design of the microfluidic system, microfluidic assembly, microfluidic chips and its manufacture

7 gives an overview of the microfluidic system for cell-free protein synthesis. The microfluidic structure (Figure 2 AB) was micromilled into a 1.55 mm amorphous thermoplastic COC substrate (Cyclic Olefin Copolymer COC (TOPAS 6013-S04)). Access holes allow connection to a liquid flow. Around To obtain a liquid-tight microfluidic system with homogeneous surface properties, the structured substrate with the rectangular channels (500 μηη wide and deep), the reaction chambers (height 500 μηη) and access holes by thermal bonding permanently with a 135 μηι thick COC film (TOPAS 6013) than Cover plate (Fig. 7 A) connected. Steel cannulas were stuck in the access holes and silicone tubing was slipped over the cannulas to connect the fluid flow. The fluid movement was ultimately achieved by pressure differences caused by syringe pumps. Other access holes served as fill openings for pipette tips. The micro-milled COC walls were modified by a low-viscous solution of fluoro-acrylate polymer dissolved in the organic solvent hydrofluoroether (Certonal FC-742, Nordson Deutschland GmbH). Flushing with dissolved fluoro-acrylate polymer results in the deposition of a dry, oleophobic and hydrophobic film which adhere through intermolecular interaction with the walls of the microfluidic system and modify surface properties such as roughness, surface tension and surface charge. The process temperatures were controlled by the integrated temperature sensors and thermal actuators, such as heating pads and heating resistors, mounted on an aluminum heat spreader (Figure 7 A / C). Three temperature zones were realized on the chip: 37 ° C for the transition chambers / compartments, 70 ° C for the elution chamber / elution compartment, and 27 ° C for the translation chamber / translation compartment. Homogeneity of the surface heating was determined by IR thermography (IR). Cam SC6000) verified. Air vents were placed around the elution chamber to ensure thermal isolation of the various temperature zones. To manipulate the RNA-transferring magnetisable particles in the microfluidic system, a permanent magnet with a tapered attachment (Figure 7A) was mechanically moved through a motorized xy (-z) stage. The cone-shaped attachment was necessary to focus the magnetic field of a 10 mm ³ cubic NdFeB permanent magnet. In order to optimize the geometry of the attachment, the magnetic field density around the magnet and attachment assembly was calculated using the Finite Element Method (FEM) with the ANSYS software (version 14.5). references

[1] M.G. Casteleijn, A. Urtt, and S. Sarkhel, "Expression without boundaries: Cell-free protein synthesis in pharmaceutical research," International Journal of Pharmaceutics, Vol. 440, No. 1, pp. 39-47, 2013.

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[3] ED Carlson, R. as described above, as described above, Gan, CE Hodgman, and MC Je, "Cell-free protein synthesis: applications come of age," Biotechnology Advances, Vol. 30, No. 5, pp. 1 185 -1 194, 2012.

[4] CJ Noren, SJ Anthony-Cahill, MC Griffith, and PG Schultz, "A general method for site-specific incorporation of unnatural amino acids into proteins," Science, Vol. 244, No. 4901, pp. 182-188, 1989th

[5] I. Hirao, T. Ohtsuki, T. Fujiwara, T as described above. Mitsui, T. Yokogawa, T. Okuni, H. Nakayama, K. Takio, T. Yabuki, T. Kigawa, K. Kodama, T. Yokogawa, K.

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[6] AS Spirin, VI Baranov, La Ryabova, SY Ovodov, and YB Alakhov, "A continuous cell-free translation system capable of producing polypeptides in high yield," Science, Vol. 242, Item 4882, pp. 1 164, 1988.

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Claims

claims

1 . A method for cell-free synthesis of polypeptides, the following steps

includes:

1) immobilization of the RNA;

2) translation of the RNA;

2. The method of claim 1, comprising the following sections:

1) Transcription of DNA to mRNA

2) Immobilization of the mRNA to magnetizable particles

3) Transfer of magnetizable particles

4) elution of the mRNA

5) translation of the RNA; wherein transcription and translation take place in separate compartments.

The method of claim 1, wherein the immobilized RNA is for translation

is transferred and / or the translation mixture is transferred to the RNA.

4. The method according to claims 1 and 2, wherein the mRNA is transferred on a chip by means of magnetic forces.

5. The method of claim 1 and 4, wherein the method is performed in a microfluidic platform and / or by a pipetting robot.

6. The method of claims 1 to 5, wherein either the immobilized RNA is translated or eluted and then the free RNA is translated.

7. The method according to any one of claims 1 to 6, wherein the immobilized RNA

temperature-controlled or ion-driven or by elimination or otherwise eluted and then free RNA is translated.

8. The method according to any one of claims 1 to 7, wherein the RNA is immobilized either covalently or non-covalently via a biotin-streptavidin interaction.

9. The method according to any one of claims 1 to 8, wherein the RNA by a

Spacer defined by the immobilization matrix is spatially separated.

10. The method according to any one of claims 1 to 9, wherein the RNA via

Oligonucleotide is immobilized and the oligonucleotides are in turn covalently or non-covalently immobilized via a biotin-streptavidin interaction. The oligonucleotides can be spatially removed from the immobilization matrix by a spacer or immobilized directly on the matrix.

1 1. The method according to any one of claims 1 to 10, wherein natural or

Synthetic RNA template can be synthesized, and wherein the natural RNA using PCR products, plasmid DNA, genomic DNA, cloned DNA

Fragments, cDNA libraries, or synthetic oligonucleotides is transcribed.

12. The method of any one of claims 1 to 11, wherein the cell-free system is based on wheat germ, reticulocytes, yeast, insect cells, CHO cells, cultured human cells or other eukaryotic and prokaryotic origins or is synthetic.

13. The method according to any one of claims 1 to 12, wherein the RNA to not

magnetic, magnetic or magnetizable particles.

14. The method according to any one of claims 1 to 13, wherein magnetizable particles with immobilized mRNA with permanent magnets or electromagnets / coils are moved and / or separated / immobilized.

15. The method according to any one of claims 1 to 14, wherein the process fluids in

System, such as transcription solution or washing solution, either moved or stationary.

The process of any one of claims 1 to 15, wherein between the process fluids, separation fluids such as air, oil or others are. These release fluids may be permanently there or temporarily removed.

17. Apparatus for carrying out the method according to claims 1 to 16.

18. The apparatus of claim 17, comprising a support for a microfluidic system, which consists of a polymer plate with structured channels. Another component of the device is a 3-axis movable multifunction head with holders for a permanent magnet and a camera for process control. Furthermore, the system includes an electronically controlled temperature control unit and thermocouples for generating various temperature zones on the microfluidic system. The fixed microfluidics is connected to a pump system for fluid movement.

19. The apparatus of claim 18, wherein the multifunctional head is stacked

two linear tables and a pneumatic lifting cylinder or by a

Combination of spindle drives in 3-dimensional space is positioned.

20. The apparatus of claim 18, wherein the permanent magnet is attached to the multifunction head and the magnetic field is focused by an attached metal cone onto a microfluidic channel.

21. The apparatus of claims 18 to 20, wherein a camera is mounted on the multi-function head (Description: which tracks the movement of the particles)

The apparatus of any one of claims 18 to 21, wherein necessary pumping steps are controlled by a syringe pumping array (described in, for example, 8 independently controllable syringe pumps).

23. The apparatus according to any one of claims 18 to 22, wherein software controls the movement of the syringe pumps and the 3-axis system and takes over the temperature control.