WO2015144859A2 - Automatisation de la synthèse protéique acellulaire avec alimentation semi-continue ou continue d'arn matrice fraîchement synthétisé - Google Patents

Automatisation de la synthèse protéique acellulaire avec alimentation semi-continue ou continue d'arn matrice fraîchement synthétisé Download PDF

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WO2015144859A2
WO2015144859A2 PCT/EP2015/056662 EP2015056662W WO2015144859A2 WO 2015144859 A2 WO2015144859 A2 WO 2015144859A2 EP 2015056662 W EP2015056662 W EP 2015056662W WO 2015144859 A2 WO2015144859 A2 WO 2015144859A2
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rna
translation
immobilized
mrna
transcription
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PCT/EP2015/056662
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German (de)
English (en)
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WO2015144859A9 (fr
WO2015144859A3 (fr
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Leopold Georgi
Stefan Kubick
Victoria SCHULDT
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Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.
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Publication of WO2015144859A2 publication Critical patent/WO2015144859A2/fr
Publication of WO2015144859A3 publication Critical patent/WO2015144859A3/fr
Publication of WO2015144859A9 publication Critical patent/WO2015144859A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • 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.
  • 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.
  • 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 present invention relates to a method for the cell-free synthesis of polypeptides, comprising the following steps:
  • 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.
  • 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.
  • 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.
  • 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.
  • CFCF Continuous Flow Seif reporting protein synthesis
  • CECF Continuous Exchange Seif reporting Protein Synthesis
  • 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.
  • the present invention relates to a method for the cell-free synthesis of polypeptides, comprising the following steps:
  • 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.
  • RNA is continuously produced in the reaction vessel without interruption
  • 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.
  • RNA is covalently or non-covalently immobilized.
  • RNA is spatially removed by a spacer from the Immobilmaschinesmatrix defined.
  • 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.
  • 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.
  • RNA or oligonucleotides is immobilized via biotin-streptavidin interaction.
  • Another embodiment of the invention is that natural or synthetic RNA templates are synthesized.
  • RNA is transcribed from PCR products, plasmid DNA, genomic DNA, cloned DNA fragments, cDNA libraries, or synthetic oligonucleotides.
  • 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.
  • 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.
  • 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.
  • magnetisiebare particles with immobilized mRNA with electromagnets / coils are moved and / or separated.
  • 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.
  • transcription is interrupted - for example by cooling - thereby ensuring that always freshly synthesized mRNA is transferred.
  • 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.
  • 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.
  • the method is compatible with the CECF and CFCF formats.
  • the method can also be used for very small reaction volumes in the range below 10 ⁇ .
  • the RNA immobilization matrix is reusable.
  • 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.
  • 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.
  • the system includes an electronically controlled
  • 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
  • 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.
  • 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
  • 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.
  • RNA is repeatedly immobilized during transcription, which does not terminate the transcription. This RNA is repeatedly combined with the translation mixture and translated. The RNA is either removed again from the immobilization matrix or remains at the translation on the solid phase.
  • 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.
  • RNA is covalently or non-covalently immobilized.
  • RNA is spatially removed by a spacer defined by the immobilization matrix.
  • RNA is immobilized via oligonucleotides and the oligonucleotides are in turn covalently or non-covalently immobilized.
  • RNA or oligonucleotides are immobilized via biotin-streptavidin interaction.
  • RNA is immobilized via oligonucleotides.
  • the immobilization is carried out by initially free oligonucleotides which hybridize with the RNA with subsequent immobilization of the RNA oligonucleotide hybrid.
  • RNA is immobilized via oligonucleotides.
  • the immobilization is carried out by immobilized oligonucleotides, which in turn immobilize RNA by hybridization.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the immobilized mRNA was directly translated.
  • 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.
  • an alternative solid phase for mRNA immobilization that does not inhibit translation, effective translation should be possible.
  • 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.
  • Magnetizable streptavidin-loaded particles 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.
  • Magnetizable streptavidin-loaded particles were functionalized with biotinylated oligonucleotides that are complementary to the constant part of the mRNA.
  • the transcription of the eYFP-encoding mRNA was performed by a PCR template.
  • the immobilization of this mRNA to magnetizable particles, the magnetically activated separation of the particles, the washing process and the elution of the immobilized mRNA were carried out in a reaction vessel and in parallel in a specially designed microfluidic system. 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.
  • 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.
  • magnetizable streptavidin-loaded particles eg streptavidin coated superparamagnetic dynabeads of the firmnavitrogen
  • dT Oligo
  • 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 ⁇ M 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 0.5-2 ⁇ _ 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 ⁇ _ 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).
  • the translation volume the more translational volume, the more RNA and thus repetitions are needed
  • the amount of particles the more particles, the fewer repetitions.
  • microfluidic system 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.
  • COC film e.g., 135 ⁇
  • 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
  • FIG. 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.
  • the structured substrate with the rectangular channels (500 ⁇ wide and deep), the reaction chambers (height 500 ⁇ ) and access holes by thermal bonding permanently with a 135 ⁇ thick COC film (TOPAS 6013) than Cover plate (Fig. 7 A) connected.
  • 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.
  • IR thermography IR thermography
  • RNA-transferring magnetisable particles in the microfluidic system 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).

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Abstract

La présente invention concerne un procédé d'alimentation semi-continue ou continue d'ARN fraîchement synthétisé à partir d'un mélange transcriptionnel in vitro s'écoulant en continu dans un mélange traductionnel in vitro pour la synthèse protéique acellulaire. Le procédé comprend les étapes suivantes : immobilisation de l'ARN fraîchement synthétisé à partir de la transcription et utilisation de cet ARN pour la traduction in vitro. Pour cela, on peut soit transférer l'ARN dans le mélange traductionnel, soit transférer le mélange traductionnel dans l'ARN. Dans chaque cas, on alimente la traduction en ARN de manière semi-continue ou continue et la transcription se poursuit également après le prélèvement d'ARN. L'invention concerne en outre la possibilité d'automatiser ce procédé.
PCT/EP2015/056662 2014-03-26 2015-03-26 Automatisation de la synthèse protéique acellulaire avec alimentation semi-continue ou continue d'arn matrice fraîchement synthétisé WO2015144859A2 (fr)

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EP14161671.4 2014-03-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019236761A1 (fr) * 2018-06-06 2019-12-12 Syngulon Sa Ingénierie de peptides antimicrobiens

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* Cited by examiner, † Cited by third party
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US20080214783A1 (en) * 2004-11-12 2008-09-04 Japan Science And Technology Agency Method of Synthesizing Protein, mRna Immobilized on Solid Phase and Apparatus for Synthesizing Protein

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019236761A1 (fr) * 2018-06-06 2019-12-12 Syngulon Sa Ingénierie de peptides antimicrobiens

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