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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
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  • Abstract
  • Description
  • Claims
  • Application Information

AI 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 while eliminating the need to increase the size of the system caused by using a small molecule expression solution with an expression solution volume 10 times or more, it improves the expression level of the target protein

Method used

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

Examples

Experimental program
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Embodiment 1

[0159] Embodiment 1: the preparation of template DNA

[0160] 1-1: Preparation of plasmid DNA

[0161] In the present invention, AcGFP, CAT, RFP and TEV-AcGFP genes are used for cell-free protein production. These genes are used to confirm whether the protein is produced by the cell-free protein production system, and it is obvious to those skilled in the art that the present invention is applicable to any protein produced by the 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 the histidine-tagged cell-free expression vector pBIVT (Bioneer, Korea). Meanwhile, TEV-AcGFP was constructed to include a sequence (Glu-Asn-Leu-Tyr-Phe-Gln↓Gly) located between the histidine tag and the target protein, which is cleaved by TEV.

[0163] Examples of expression vectors that can be used in the present inve...

Embodiment 2

[0172] Embodiment 2: the preparation of cell lysate

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

[0174] Wash 100 g of harvested E.coli cells with 500 ml of washing buffer (10 mM Tris (oAc) pH 8.2, 14 mM Mg (oAc) 2 , 60mM K(OAc), 1mM DTT (dithiothreitol), 0.05% (v / v) 2-mercaptoethanol (2-ME)), and then centrifuged (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 threitol)) 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 lysates were centr...

Embodiment 3

[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 of 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 mix, 2% PEG 8000 and 67mM creatine phosphate Mix with each other to prepare expression solution for cell-free protein expression. Alternatively, by mixing expression solution, DEPC DW, and 5% NaN at a ratio of 46.7:52.3:1 3 To prepare small molecule expression solution.

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

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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.

Description

technical field [0001] The present invention relates to an automated cell-free protein production system and a method of producing protein using the system. More specifically, the present invention relates to an automated cell-free protein production system comprising: a protein expression reaction unit comprising a reaction vessel comprising a plurality of dialysis tubes each comprising a dialysis membrane and open at the top; A reaction temperature control unit for heating or cooling a reaction vessel; a pipette array, including a plurality of pipettes and using the pipette to suck or discharge a solution; a pipette array moving unit, in the up-down direction, front-rear direction, or left-right direction moving the pipette array in a direction to move the solution; a protein purification unit comprising a magnetic field applying device; and a multiwell plate installation unit in which a multiwell plate kit is installed, the multiwell plate installation unit is supplied with...

Claims

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Application Information

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IPC IPC(8): C12M1/38C07K1/22
CPCC07K1/36C12M23/08C12M23/12C07K1/22C12M23/44C12M41/12C12M41/48C12M47/12C07K1/02C07K1/047C12M23/38C12M29/00C12M29/04C12M33/04C12P21/00
Inventor 朴翰语金钟甲韩指元赵俞相金敏贞金荷娜李洋源金楠镒
Owner BIONEER
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