Apparatus for automatically preparing cell-free proteins and method for preparing proteins using same

A protein and cell-free technology, applied in biochemical equipment and methods, peptide preparation methods, chemical instruments and methods, etc., can solve the problem of low protein expression efficiency

Active Publication Date: 2015-09-02

BIONEER

11 Cites 3 Cited by

AI-Extracted Technical Summary

Problems solved by technology

[0018] The present inventors have worked hard to develop a system and kit that can overcome the problems of the automated protein synthesizer ExiProgen TM The disadvantage of low protein expression efficiency, and whi...

Method used

[0074] The dialysis tubing 110 preferably includes an elastic tube 114 disposed around the outer portion of the dialysis membrane 112, the elastic tube 114 being in contact with the top structure 111 and the bottom structure 113 to improve adhesion to both.

[0107] The present invention attempts to overcome this problem by adding a small amount of small molecule expression solution and replacing it in multiple steps.

[0217] Pipette the small molecule expression solution of the multi-well plate kit into the small molecule expression solution wells of the reaction vessel of the protein expression reaction unit. Here, the volume of the small molecule expression solution wells corresponds to about 2.5-3 times the volume of each protein expression mixture. Heat (26-38°C) the reaction temperature control unit located below the protein expression reaction unit and periodically mix the solution by inserting a pipette into the small molecule expression solution. In this process, the energy source in the small molecule expression solution, etc., is introduced into the mixture through the dialysis membrane of the dialysis tube containing the protein expression mixture, and the protein expression inhibitor in the mixture is discharged...

Abstract

The present invention relates to an apparatus for automatically preparing cell-free proteins and a method for preparing proteins using the same, and more particularly, to an apparatus for automatically preparing cell-free proteins and a method for manufacturing proteins using the same, the apparatus comprising: a protein expression reaction unit including a reaction vessel comprising a plurality of dialysis tubes provided with a dialysis membrane by an open superstructure; a reaction temperature control unit for heating or cooling the protein expression reaction unit; a pipet array which includes a plurality of pipets and is capable of suctioning or discharging a solution by using the pipets; a pipet array movement unit capable of moving solutions by moving the pipet array up and down, back and forth, or left and right; a protein purification unit provided with a magnetic field application apparatus; and a multi-well plate provision unit capable providing a multi-well plate kit which supplies a solution used in the preparation of proteins. In addition, the present invention relates to the apparatus for automatically preparing cell-free proteins and the preparation method therefor, which has a pure target protein synthesizing function in which an affinity tag is removed, excluding a target protein in which an affinity tag is coupled, and a function for rapidly and automatically exchanging, by a desired elution solution, through multi-step dialysis, a leaching solution in which the resulting purified proteins are dissolved, with the desired elution solution for containing proteins such that a plurality of proteins can be simply and automatically prepared in mass. Moreover, the present invention relates to a reaction kit for automatically preparing cell-free proteins, comprising: a multi-well plate kit for storing a solution necessary for preparing proteins; and a dialysis tube provided with the dialysis membrane. The apparatus for automatically preparing cell-free proteins according to the present invention can automatically express and purify target proteins, and dialyze the proteins in the buffer solution for containing proteins thereby enabling the immediate use of the resulting purified proteins. Furthermore, the method for preparing proteins according to the present invention can continuously supply energy sources necessary for cell-free protein expressions by multi-leveled exchanges of a low molecular expression solution such that a maximum of five times more target proteins can be obtained compared with obtaining target proteins by using a conventional method for preparing cell-free proteins in an arrangement form, and since the preparation of pure target proteins by removing an unnecessary affinity tag using proteases when purifying target proteins by using magnetic particles, the present invention is very useful in protein production for industrial, medicinal and research purposes wherein circular proteins can be produced in which artificial sequences exerting negative influences on the activities and structures of a protein are removed.

Application Domain

Bioreactor/fermenter combinationsBiological substance pretreatments +7

Technology Topic

Protein proteinSupply energy +17

Image

Examples

Experimental program(8)

Example Embodiment

[0159] Example 1: Preparation of template DNA

[0160] 1-1: Preparation of plasmid DNA

[0161] In the present invention, the AcGFP, CAT, RFP and TEV-AcGFP genes are used for cell-free protein production. These genes are used to confirm whether a protein is produced by a cell-free protein production system. It is obvious to those skilled in the art that the present invention can be applied to any protein produced by a cell-free protein production method.

[0162] Each gene was synthesized by a gene synthesis method (NBiochem. Biophys. Res. Commun. 1998, 248, 200-203). Each synthesized gene was treated with restriction enzymes, and then cloned into a histidine-tagged cell-free expression vector pBIVT (Bioneer, Korea). At the same time, TEV-AcGFP was constructed to include the sequence (Glu-Asn-Leu-Tyr-Phe-Gln↓Gly) between the histidine tag and the target protein, which was cleaved by TEV.

[0163] Examples of expression vectors that can be used in the present invention include, but are not limited to, pBIVT (Bioneer, Korea), pIVEX (Roche, Germany), pET (Novagen, Germany), pK7, pQE, and the like.

[0164] Specifically, restriction enzyme treatment and cloning were performed in the following manner.

[0165] For the gene product synthesized by restriction enzyme treatment, put 1μl of NdeI (Bioneer, Korea), 1μl of SalI (Bioneer, Korea), and 2μl of 10X AccuCut in each tube TM Buffer (Bioneer, Korea), 10 μl of synthetic gene product and 6 μl of sterile distilled water were mixed and then incubated at 37°C for 3 hours. For the restriction enzyme treatment of the expression vector, put 1μl of NdeI (Bioneer, Korea), 1μl of SalI (Bioneer, Korea), and 2μl of 10X AccuCut in each tube TM Buffer (Bioneer, South Korea), 10 μl of expression vector and 6 μl of sterile distilled water were mixed and then incubated at 37°C for 3 hours. From each reaction product treated with restriction enzymes, DNA was purified using Accuprep Gel Extraction Kit (Bioneer, Korea).

[0166] Next, put 5μl of 2X Quick Ligation Buffer (Promega, USA), 1μl of T4DNA Ligase (Promega, USA), 3μl of restriction enzyme-treated synthesized gene product and 1μl of restriction in each tube. The endonuclease-treated vector was mixed, and then incubated at 16°C for 1 hour. Next, add 10 μl of the incubated reaction solution to 100 μl of E. coli competent cells, place on ice for 30 minutes, then incubate at 42° C. for 90 seconds, and place on ice for 5 minutes. The reaction solution was inoculated to an LB plate containing kanamycin, and then incubated at 37°C for 16 hours.

[0167] The white colonies were collected and incubated in 10 μl of LB liquid medium for about 16 hours. After centrifugation, the supernatant was removed and the plasmid DNA was purified from the precipitate using AccuPrep Plasmid DNA Preparation Kit (Bioneer, South Korea). The purified DNA is sequenced to confirm whether the gene synthesized by each gene synthesis method is correct. In order to obtain DNA for protein synthesis, the corresponding colonies were incubated, and plasmid DNA was purified in the same manner as described above. Measured by UV spectrophotometer (Shimadzu, Japan), the purity of plasmid DNA (A 260/280 ) Is 1.8-2.0.

[0168] 1-2: Preparation of PCR products

[0169] Use ExiProgen TM ProXpress PCR Template Kit (Bioneer, Korea) constructs PCR products.

[0170] Specifically, in order to amplify the target gene, a set of primers with overlapping sequences at the 5'and 3'ends are prepared, and the sample with the target gene is used as a template (genomic DNA, T vector, etc.) to perform primary PCR. In the PCR reaction, use the AccuPowerProFi Taq PCR Premix provided in the kit. The PCR reaction conditions were as follows: initial denaturation at 94°C for 5 min, then 30 cycles, each cycle consisting of 30 sec at 94°C, 30 sec at 58°C, and 1 min at 72°C, followed by a final extension at 72°C for 5 min. The primary PCR product was purified using AccuPrep Gel Extraction kit (Bioneer, Korea).

[0171] Using the primary PCR product as a template, a secondary overlap PCR was performed to synthesize PCR fragments for protein synthesis. The PCR reaction was carried out by adding the cassette set and primer pair provided in the kit. The PCR reaction conditions were as follows: denaturation at 94°C for 5 min, then 30 cycles, each cycle from 94°C for 1 min, 48°C Composed at 1 min and 72°C for 1 min, and then finally extended at 72°C for 5 min. The secondary PCR product was purified using AccuPrep Gel Extraction Kit (Bioneer, Korea).

Example Embodiment

[0172] Example 2: Preparation of cell lysate

[0173] First, E. coli cells (BL21(DE3), Novagen, USA) were cultured in a 350l fermentor (2×YT medium) at 37°C. In the absorbance (OD 600 ) Is 0.6, add 1mM IPTG to express T7RNA polymerase, and in the absorbance (OD 600 When) is 3.0-3.5, stop the culture, harvest the cells by centrifugation and store at -50°C.

[0174] Use 500ml washing buffer (10mM Tris(oAc) pH8.2, 14mM Mg(oAc)) with 100g harvested E.coli cells 2 , 60mM K (OAc), 1mM DTT (dithiothreitol), 0.05% (v/v) 2-mercaptoethanol (2-ME)) wash, and then centrifuge (3000RPM, 30min). Repeat this step 4 times.

[0175] After washing, the E.coli cells were weighed and added to a buffer (10mM Tris(oAc)pH 8.2, 14mM Mg(oAc)2, 60mM K(OAc), 1mM DTT (disulfide) with a volume equivalent to 1.1 times its weight. Threitol)) in and dispersed. Then, the cells were lysed using a cell homogenizer (Pressure Cell Homogeniser, Stansted Fluid Power) under constant pressure (280 psi).

[0176] The cell lysate was centrifuged at 16,000 RPM at 4°C for 30 minutes, and the supernatant was collected. Then, each 10ml cell lysate contains 3ml buffer (10mM Tris(oAc)pH 8.2, 14mM Mg(oAc) 2 , 60mM K (OAc), 1mM DTT (dithiothreitol)) buffer was added to the supernatant, while adding 26.7g/ml creatine kinase to the supernatant. Then, the solution was incubated at 37°C for 80 minutes, thereby obtaining a pre-cultured solution. Next, the pre-cultured solution was centrifuged (11,000RPM, 20min, 4°C), thereby obtaining a cell lysate. Store the cell lysate at -70°C until use.

Example Embodiment

[0177] Example 3: Preparation of expression solution and small molecule expression solution

[0178] By adding 114mM HEPES-KOH (pH 7.5), 2.4mM ATP, each 1.7mM CTP, GTP and UTP, 2mM DTT, 90mM K(Glu), 80mM NH 4 (OAc), 12mM Mg(OAc), 68g/ml folinic acid (L-5-formyl-5,6,7,8-tetrahydrofolate), 2.5mM amino acid mixture, 2% PEG 8000 and 67mM creatine phosphate Mix each other to prepare an expression solution for cell-free protein expression. In addition, by mixing the expression solution, DEPC DW and 5% NaN in a ratio of 46.7:52.3:1 3 To prepare small molecule expression solutions.

[0179] Store the expression solution and small molecule expression solution at -20°C until use.

PUM

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