KR101644906B1 - Method for analyzing interaction between target cell and transcript or protein synthesized in single container - Google Patents

Abstract

The present invention relates to a method for analyzing interaction between a target cell and transcriptome or protein, comprising the following steps of: expressing transcriptome or protein by adding template genes to a single container including an alive target cell; and a reaction mixture for biosynthesis of ex vivo transcriptome or protein, and inducing a reaction between the expressed transcriptome or protein and the target cell; and checking the degree of the reaction between the transcriptome or protein and the target cell. The present invention enables an ex vivo expression of the transcriptome or protein and interaction between the expressed transcriptome or protein and the target cell to be performed at once in a single container, thereby enabling easy and quick analysis. Therefore, the present invention can be used for screening for selecting valid transcriptome, protein, or the like.

Description

[0001] The present invention relates to a method for analyzing the interaction between target cells and a transcript or protein synthesized in a single container,

The present invention relates to a method for producing a target cell; And a reaction mixture for extracellular ( ex vivo ) transcription or protein biosynthesis, wherein a template gene is added in a single container to express the transcript or protein, and the reaction of the expressed transcript or protein with the target cell Inducing; And a step of confirming the degree of the reaction between the transcript or the protein and the target cell, and a method for analyzing the interaction between the target cell and the transcript or protein.

The genetic information entered into the DNA is transcribed into RNA and then translated into protein to implement its function or to perform its function at the transcribed RNA level. In general gene recombination technology, the expression of RNA or protein is usually made from the DNA introduced into the cell. In some cases, however, transcription or translation of the gene may be caused by enzymes and synthesis machinery Enzymes, ribosomes, and translation factors), which can be used for in vitro transcription and cell-free translation (cell-free protein synthesis, in vitro protein synthesis and in vitro translation ).

As such, siRNA, a gene product expressed from DNA, or a peptide penetrating into cells have been used for the purpose of infiltrating into specific cells or binding to the cell surface to regulate the activity of the cells, and the effect of the expressed gene product on cells In order to analyze the gene product, the gene product was expressed using gene recombinant techniques, and then the cells were analyzed by separating and purifying the cells. Thus, the process of expressing transcripts or proteins, which are gene products, and the analysis process proceed separately. For the analysis of function or activity, there is a need to separate or purify after expression.

Generally, in the synthesis reaction of proteins in cells, first, the information is transferred from the DNA having genetic information to the mRNA, and the ribosome then synthesizes the protein by translating the information of the mRNA.

In vitro transcription is the synthesis of RNA in vitro using RNA polymerase and cofactor, substrate NTP, and template DNA, and the synthesis of proteins outside of the cell, such as in vitro, is called cell-free protein synthesis), and the cell-free protein synthesis is an intracellular protein synthesis mechanism involved in the production of proteins in the cell and extracts only its factors, so that the physiological control mechanism of cells outside the cell is excluded, As a technique for mass production of a target protein in a short period of time, a desired protein biosynthesis mechanism, that is, a ribosome, an initiation factor, a renal factor, a termination factor, an aminoacyl tRNA synthetase, (Yoshihiro Shimizu et al., 2001, Nature Biotechnology, 19 (8): 751-755; Tae-W an Kim et al., 2006, Journal of Biotechnology, 126 (4): 554-561). The conventional cell-free protein synthesis system maintains high performance comparable to protein synthesis in the rate of polymer synthesis reaction and accuracy of translation reaction, and is known as a useful method for obtaining a target protein without performing a complicated purification process. Several inventions have been disclosed for improving synthesis efficiency and economics for more useful industrial applications.

 Korean Patent No. 0892889 discloses a method for producing a recombinant protein and a fusion protein is disclosed in Korean Patent No. 0749053, and Korean Patent No. 1476953 discloses a method for producing a cell-permeable protein Although disclosed for this enhanced hepcidin-targeted novel peptide and its use, a method for analyzing the synthesis of an extracellular transcript or protein and interaction with a cell in a container containing living cells of the present invention has been disclosed none.

BRIEF SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, And extracellular ( ex vivo transcription or protein biosynthesis of a transgene or a protein; and expressing the transgene or protein by inducing a reaction between the expressed transgene or protein and the target cell; And determining the degree of the reaction between the transcription product or the protein and the target cell, and a method for analyzing the interaction between the transcription product or the target cell and the transcript or protein according to the present invention, And confirming the interaction between the gene product obtained through extracellular transcription and protein synthesis in a container containing living cells and the target cell contained in the container, thereby completing the present invention Respectively.

In order to achieve the above object, And extracellular ( ex vivo transcription or protein biosynthesis of a transgene or a protein; and expressing the transgene or protein by inducing a reaction of the transgene or protein with the target cell; And confirming the degree of the reaction between the transcription product or the protein and the target cell, and analyzing the interaction between the transcription product or the protein and the target cell.

The present invention relates to a method for producing a target cell; And extracellular ( ex vivo transcription or protein biosynthesis of a transgene or a protein; and expressing the transgene or protein by inducing a reaction of the transgene or protein with the target cell; And determining the degree of the reaction between the transcription product or the protein and the target cell. The present invention relates to a method for analyzing the interaction between a transcription product or a protein and a target cell, Since the interaction can be analyzed by one-stop analysis, it is possible to perform a simple and rapid analysis, so that it can be very useful for screening for screening effective transcripts or proteins.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram of a method for analyzing the interaction of a target cell with a transcript or protein synthesized in a single container. FIG.
FIG. 2 is a schematic diagram of a construct (A) containing the phoA signal sequence used to produce the TMab4 scFv antibody of the present invention and a construct (B) containing the ubiquitin and factor Xa cleavage site sequences.
FIG. 3 shows the results of analysis of cell permeability of an antibody (TMab4 scFv) produced by cell-free protein synthesis in the presence of a target cell (HeLa).
4 shows the results of confirming inhibition of growth of Escherichia coli cells by an antimicrobial peptide (Pexiganan) produced by cell-free protein synthesis in the presence of E. coli cells.

(A) a living target cell; And a reaction mixture for extracellular ( ex vivo ) transcription or protein biosynthesis, wherein a template gene is added in a single container to express the transcript or protein, and the reaction of the expressed transcript or protein with the target cell Inducing; And

(b) determining the degree of the reaction between the transcript or the protein and the target cell, and a method for analyzing the interaction between the target cell and the transcript or protein.

The extracellular ( ex vivo transcript or protein biosynthesis is preferably but not limited to in vitro transcription or cell free protein synthesis.

The in vitro transcription ( in vitro transcription) further preferably comprises, but is not limited to, a substrate NTP, an RNA polymerase and a cofactor.

Preferably, the interaction is for confirming whether the transcript or the protein has permeability or antimicrobial activity to the target cell, and more preferably, the influx into the cytoplasm of the antibody or the like produced using the cell-free protein synthesis method , Or a method of easily analyzing the antibacterial activity of an antimicrobial peptide produced using a cell-free protein synthesis method, but the present invention is not limited thereto.

The method for easily analyzing the influx into the cytoplasm of the antibody produced using the cell-free protein synthesis method comprises (a) introducing into the cell-free protein synthesis reaction solution containing Factor Xa a promoter, ubiquitin, Factor Xa cleavage site, Producing an antibody by adding a sequence operably linked to a target antibody, GFP ₁₁ and a streptavidin binding peptide; And

(b) treating the produced antibody with an animal cell expressing GFP _{1 -10} to detect GFP fluorescence in the cytoplasm of the animal cell, but is not limited thereto.

Preferably, the transcript is siRNA or miRNA, and the protein is any one selected from an antibody, an enzyme, and an antimicrobial peptide, but is not limited thereto.

The transcription product or protein may be introduced with a reporter region for confirming interaction with the target cell, and the introduction of the reporter is characterized in that the transcription product or the protein does not affect the activity in interaction with the target cell .

The target cells are preferably E. coli, yeast, fungi, plant cells or animal cells, but are not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

[Materials and Methods]

1. Materials

HeLa cells were purchased from the American Type Culture Collection (ATCC) and stored in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS, GE Healthcare, Logan, UT).

A mixture of ATP, GTP, UTP, CTP, creatine phosphate, creatine kinase and E. coli total tRNA was purchased from Roche Applied Science and E. coli-derived BL21 Star (DE3) Were purchased from Rosen. All other chemical reagents were purchased from Sigma-Aldrich and used without further purification.

The S12 cell extract prepared from E. coli-derived BL21 Star (DE3) (Invitrogen, Carlsbad, Calif.) Was used as a raw material for protein synthesis.

The antimicrobial peptide gene was purchased from Bioneer and cloned into pK7 vector with Ndel / Sall.

2. Cell-free protein synthesis

1) Cell-free protein synthesis of TMab4 scFv antibody for cell permeability confirmation

5 mM HEPES-KOH (pH 8.2), 1.2 mM ATP, 0.85 mM each of GTP, UTP and CTP, 80 mM ammonium acetate, 8-tetrahydrofolic acid), 1.0mM each of 20 amino acids, 2% PEG (8000), 3.2 U / ㎖ of creatine kinase, creatine phosphate 67mM, L- of 0.01mM [U- ¹⁴ C] leucine (11.1 GBq / (GSSG), 3 mM of reduced glutathione (GSH), 27% (v / v) of S12 cell extract and 26.7 / / ml of PCR-amplified template gene were subjected to a standard reaction for cell-free protein synthesis The mixture (4 ml) was used and the reaction mixture was incubated at 30 ° C for 1 hour.

2) Cell-free protein synthesis of antimicrobial peptide (PEXIGANAN)

The cells were incubated at 37 ° C in a solution containing 57 mM HEPES-KOH (pH 8.2), 1.2 mM ATP, 0.85 mM GTP, UTP and CTP, 2 mM DTT, 0.17 mg / ml E. coli total tRNA mixture, 0.64 mM cAMP, 90 mM potassium glutamate, Ammonium acetate, 12 mM magnesium acetate, 5-formyl-5,6,7,8-tetrahydrofolic acid (34 μg / ml), 20 amino acids of 1.0 mM each, 2% PEG (8000), 3.2 μg / ml creatine kinase (CK), 1067 mM creatine phosphate, 10 μg / ml DNA and 24% (v / v) S12 cell extract were used, For 3 hours.

Example  1. In a container with living cells TMab4 scFv  Antibody Acellular cell  Protein synthesis and cell permeability  Confirm

The DNA encoding the heavy and light chain variable regions of the cytotransmab TMab4 (Kim et al., 2009., Biochem Biophys Res Commun, 379 (2), 314-318) was amplified by PCR and the scFv version (TMab4 (G4S) ₃ linker sequence to generate the < RTI ID = 0.0 > scFv). < / RTI > TMab4 GFP peptide in ₁₁ scFv sequence (Cabantous et al., 2005., Nat Biotechnol, 23 (1), 102-107) and a speed-streptavidin binding peptide (SBP2) (Barrette-Ng et al., 2013., Acta Crystallogr D Biol Crystallogr, 69 (Pt 5), 879-887). The resulting sequence (TMab4 scFv-GFP11-SBP2) was been cloned in plasmid pIg20m, named pIg20m TMab4 scFv-GFP -SBP2 ₁₁ (FIG. 2A).

The gene of TMab4 scFv cloned into plasmid pIg20m was amplified by PCR using primers of T7 promoter and T7 terminator sequence. The ubiquitin sequence was added before the heavy chain variable region (VH) using overlap-extension PCR (OE-PCR) (Fig. 2B) to enhance expression levels and solubility of TMab4 scFv. To remove the ubiquitin sequence from the translation product, a Factor Xa cleavage site was introduced between the ubiquitin and VH sequences.

(HeLa-SA-GFP _{1 -10} ) expressing the fusion construct of streptavidin and GFP _{1 -10} fragments were incubated in a container containing the reporter cells with 57 mM HEPES-KOH (pH 8.2), 1.2 mM ATP, Each of 0.85 mM GTP, UTP and CTP, 80 mM ammonium acetate, 34 μg / ml of 1-5-formyl-5,6,7,8-tetrahydrofolic acid, 20 amino acids of each 1.0 mM, 2 (PEG) (8000), 3.2 U / ml creatine kinase, 67 mM creatine phosphate, 0.01 mM L- [U- ¹⁴ C] leucine (11.1 GBq / mmol), 2 mM oxidized glutathione (GSSG), 3 mM reduced glutathione (G), 27% (v / v) S12 cell extract and 26.7 μg / ml PCR-amplified template gene were incubated for 1 hour at 30 ° C. in a standard reaction mixture (4 ml) for cell-free protein synthesis.

The TMab4 scFv antibody produced by the cell-free protein synthesis was analyzed based on the -GFP complementarity isolated in the reporter cells (Kim et al., 2015., Biochem Biophys Res Commun, 467 (4), 771-777).

As described in Figure 2, the TMab4 scFv construct contains the SBP2 sequence after the GFP ₁₁ fragment, so that the interaction between streptavidin and SBP2 brings the GFP _{1 -10} and GFP ₁₁ sequences in close proximity, May appear. Thus, the TMab4 scFv antibody produced by the cell-free protein synthesis shows GFP fluorescence by permeating HeLa-SA-GFP _{1 -10} cells in the container.

After the protein synthesis, the cells were incubated at 37 ° C and 5% CO ₂ for 12 hours, and GFP fluorescence was observed using a confocal microscope. As shown in FIG. 3, in the presence of the TMab4 scFv antibody gene GFP fluorescence. From these results, it was possible to synthesize cell-free protein in the presence of living cells and to confirm the interaction with the cells by confirming that the synthesized antibody permeates into the cells.

Example  2. Live Escherichia coli ( ATCC  2223) Acellular cell  Identification of antimicrobial activity of protein synthesis and synthesized antimicrobial peptide

Escherichia coli (ATCC 2223) was added to a reaction mixture for synthesis of 30 μl of acellular protein containing an antimicrobial peptide gene. Escherichia coli was grown from OD ₆₀₀ to 1.0, followed by addition of 1 μl. Cell-free protein synthesis was then carried out at 30 ° C for 3 hours to synthesize an antimicrobial peptide, which was spread on a plate without antibiotics, allowed to stand overnight and colony observed.

Cell-free protein synthesis was performed at 30 ° C for 3 hours under the condition that no antimicrobial peptide gene was used as a control. The cells were spread overnight on an antibiotic-free plate, and left overnight to observe the number of colonies.

As a result, when the gene was contained, the number of E. coli was reduced by the synthesized antimicrobial peptide, and when the gene was absent, the antimicrobial peptide was not synthesized and the number of E. coli was relatively large (FIG. From these results, it was confirmed that cell-free protein synthesis was possible in the presence of living cells, and interaction between the synthesized protein or peptide and the cells could be analyzed.

Claims (7)

(a) living target cells; And a reaction mixture for extracellular ( ex vivo ) transcription or protein biosynthesis, wherein a template gene is added in a single container to express the transcript or protein, and the reaction of the expressed transcript or protein with the target cell Inducing; And
(b) determining the degree of the reaction between the transcript or the protein and the target cell, and analyzing the interaction between the target cell and the transcript or protein. The method of claim 1, wherein the outer (ex The cells vivo transcripts or protein biosynthesis can be detected by in vitro transcription ( in vitro transcription, or cell free protein synthesis. < Desc / Clms Page number 20 > 3. The method of claim 2, wherein the in vitro transcription ( in vitro transcription) further comprises a substrate NTP, an RNA polymerase and a cofactor. A method for analyzing the interaction of a transcript or protein with a target cell. 2. The method of claim 1, wherein the interaction is for verifying whether the transcript or protein has permeability or antimicrobial activity to the target cell. The method according to claim 1, wherein the transcript is siRNA or miRNA, and the protein is any one selected from an antibody, an enzyme, and an antibacterial peptide. The method according to claim 1, wherein the transcript or protein is introduced with a reporter region for confirming interaction with the target cell. The method according to claim 1, wherein the target cell is an E. coli, a yeast, a fungus, a plant cell or an animal cell.

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