KR101667023B1 - Method for simply analyzing uptake into cytoplasm of antibody produced by cell-free protein synthesis - Google Patents

Abstract

The present invention relates to a method for easily analyzing the influx of an antibody produced by a cell-free protein synthesis method into the cytoplasm, and is useful for the development of novel antibodies targeting intracellular proteins and their interaction have. In addition, based on the cleaved GFP complementation cleaved through the present invention, it is possible to shorten the time required for confirming antibody delivery into the cytoplasm without time-consuming multistage dyeing procedures and to easily and rapidly analyze the inflow into the cytoplasm In addition, there is an advantage that antibodies produced by cell-free protein synthesis can be directly used for analysis without purification.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0002] The present invention relates to a method for easily analyzing the inflow of an antibody produced by a cell-free protein synthesis method into the cytoplasm,

The present invention relates to a method for easily analyzing influx of an antibody produced by a cell-free protein synthesis method into the cytoplasm.

Generally, the synthesis reaction of a protein in a cell is firstly transferred from the DNA having the genetic information to the mRNA, and then the ribosome translates the mRNA information to synthesize the protein. In this way, cell-free protein synthesis, which performs protein synthesis in cells in vitro in vitro, extracts only the intracellular protein synthesis machinery involved in protein production in cells and their factors, Is a technology for mass production of a target protein in a short period of time by artificially repeating only the synthesis process of the protein in a state in which the physiological regulatory mechanism of the protein is excluded. Aminoacyl tRNA synthetase can be used either separately or in combination with those contained in the cell extract (Yoshihiro Shimizu et al., 2001, Nature Biotechnology, 19 (8): 751-755; Tae-Wan 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.

On the other hand, antibodies having unique target specificity and strong binding affinity have been used in various fields ranging from molecular sensors to diagnostic and therapeutic agents. In particular, the therapeutic antibody development field has continued to grow since the commercialization of orthoclone OKT3 in 1986. 47 monoclonal antibody products have been approved in the US and Europe since 2014 and the therapeutic antibody market is expected to reach $ 125 billion by 2020. Although antibodies are the fastest growing segment of modern therapeutics, antibodies have been limited to surface-exposed proteins or secreted proteins for therapeutic antibody intervention, since antibodies generally can not pass through cell membranes. Thus, given that 20-30% of the cellular proteins are distributed within the cytoplasm, developing a method of delivering antibodies across the cell membrane will provide an opportunity to explore the unexplored regions of the novel antibody target. A number of methods have been evaluated for antibody delivery into cells including microinjection, electroporation and conjugation with the protein delivery domain (PTD). However, these methods have a number of barriers to be addressed such as cytotoxicity, loss of stability, lysosomal degradation, endosome reuse and low membrane permeability of the delivered antibody.

Korean Patent No. 0892889 discloses a recombinant protein production method and fusion protein, Korean Patent No. 0749053 discloses a method for synthesizing cell-free protein, Korean Patent No. 1476953 discloses a method for enhancing cell permeability Lt; RTI ID = 0.0 > novel < / RTI > peptides and uses thereof. However, a method for easily analyzing the inflow into the cytoplasm of an antibody produced using the cell-free protein synthesis method of the present invention has not yet been disclosed.

The present invention is derived by the request as described above, the present inventors have found that a promoter, ubiquitin, Factor Xa cleavage site, the heavy chain variable region and a cell membrane-permeable light chain variable region comprising the light chain variable region is introduced scFv antibody, GFP ₁₁ and streptavidin A gene construct which is operably linked to a sequence coding for a streptavidin binding peptide was prepared and a single chain variable fragment (scFv) of a cytotransmab having a cell membrane permeability activity by synthesizing a cell-free protein using the gene ) Antibody. Further, the present inventors have completed the present invention by confirming that the scFv antibody can be easily analyzed for infiltration into the cytoplasm based on the cleaved GFP-complementation (GFP-complementation).

In order to achieve the above object, the present invention relates to a method for producing a cell-free protein synthesis reaction mixture comprising (a) a step of introducing a promoter, ubiquitin, Factor Xa cleavage site, target antibody, GFP ₁₁ and streptavidin Adding a sequence operably linked to the peptide to produce an antibody; 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. And the like.

The present invention also relates to a method for introducing a promoter, ubiquitin, Factor Xa cleavage site, 6xHis tag, heavy chain variable region and cell membrane permeable light chain variable region in 5 'to 3' direction into a cell-free protein synthesis reaction solution containing Factor Xa A scFv antibody consisting of a light chain variable region, a sequence encoding GFP ₁₁ and a streptavidin binding peptide, and a gene in which a terminator is operably linked is added to produce an antibody by a cell-free protein synthesis method.

The present invention also provides an antibody produced by the above method and consisting of a scFv antibody, GFP ₁₁ and a streptavidin binding peptide consisting of a light chain variable region in which a 6xHis tag, a heavy chain variable region and a cell membrane permeable light chain variable region are introduced.

The present invention relates to a method for easily analyzing the influx of an antibody produced by a cell-free protein synthesis method into the cytoplasm, and is useful for the development of novel antibodies targeting intracellular proteins and their interaction have. In addition, based on the cleaved GFP complementation cleaved through the present invention, it is possible to shorten the time required for confirming antibody delivery into the cytoplasm without time-consuming multistage dyeing procedures and to easily and rapidly analyze the inflow into the cytoplasm In addition, there is an advantage that antibodies produced by cell-free protein synthesis can be directly used for analysis without purification.

Figure 1 shows the construct (A) containing the phoA signal sequence used to produce the TMab4 scFv antibody of the present invention, the construct (B) containing no phoA signal sequence, and the ubiquitin and factor Xa cleavage site sequences (C). ≪ / RTI >
Fig. 2 shows the results of cell-free protein synthesis of TMab4 scFv antibody according to presence or absence of phoA signal sequence. The filled bar represents the total protein and the empty bar represents the soluble protein.
Figure 3 shows the transmembrane activity of the TMab4 scFv antibody produced from Escherichia coli transformed with the construct containing the phoA signal sequence.
Fig. 4 shows the results of cell-free protein synthesis of the TMab4 scFv antibody according to the presence of the ubiquitin tag sequence. The filled bar represents the total protein and the empty bar represents the soluble protein.
FIG. 5 is a graph showing the activity of the < RTI ID = 0.0 > inubiquitin < / RTI > tag in- situ cutting process.
Figure 6 is a ubiquitin tag of ubi-TMab4 scFv antibody according to the factor-Xa enzyme concentration in Western blot results confirmed situ cleavage. Lane 1 did not process Factor Xa; Lane 2 was treated with Factor Xa at 20 占 퐂 / mL; Lane 3 was treated with 40 占 퐂 / mL Factor Xa; Lane 4 was treated with Factor Xa at 60 占 퐂 / mL; Lane 5 was treated with Factor Xa at 80 占 퐂 / mL; Lane 6 shows the response of 100 ug / mL of Factor Xa treated.
FIG. 7 shows the cell membrane permeability of the TMab4 scFv antibody produced by the cell-free protein synthesis method.

The present invention

(a) a cell-free protein synthesis reaction solution containing Factor Xa is operably linked with a sequence encoding a promoter, ubiquitin, Factor Xa cleavage site, target antibody, GFP ₁₁ and streptavidin binding peptide as a synthetic template Adding a gene to produce an antibody; 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; and It provides a simple way to analyze the influx.

In the method according to an embodiment of the present invention, the factor Xa cleaves the factor Xa cleavage site and acts on the factor Xa cleavage site fused after the ubiquitin sequence to remove the ubiquitin, thereby cleaving the native amino acid sequence of TMab4 scFv Thereby producing an antibody.

In the process according to one embodiment of the invention, the GFP fluorescence streptavidin HeLa cell line _{(HeLa-SA-GFP 1 -10} ) to avidin and expressing the fusion constructs of GFP and GFP fragments _1-10 and ₁₁ streptavidin ( Streptavidin-binding peptide 2 (SBP2) interacts with the cleaved GFP ₁₁ and GFP _{1 -10} (SEQ ID NO: 2) during the culture for 12 to 15 hours by resuspending the TMab4 scFv antibody containing the streptavidin- And the fragments can be detected by fusion through complementarity. GFP _{1 -10} and GFP ₁₁ may be composed of the amino acid sequences of SEQ ID NOS: 1 and 2, respectively, but are not limited thereto.

In the method according to an embodiment of the present invention, the target antibody may be a single chain variable fragment (scFv) antibody consisting of a heavy chain variable region and a light chain variable region, but is not limited thereto.

In the method according to an embodiment of the present invention, the light chain variable region may be introduced with a transmembrane light chain variable region, but is not limited thereto. The transmembrane transmissible light chain variable region may comprise the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

In a method according to an embodiment of the present invention, the gene is an scFv antibody consisting of a light chain variable region in which a promoter, ubiquitin, Factor Xa cleavage site, heavy chain variable region and transmembrane light chain variable region are introduced in 5 'to 3' But are not limited to, those comprising a sequence and a terminator encoding GFP ₁₁ and a streptavidin binding peptide (SBP2). The promoter and terminator may be, but are not limited to, a T7 promoter and a T7 terminator, respectively.

In the method according to an embodiment of the present invention, the animal cell may be, but is not limited to, a HeLa cell expressing GFP _{1- 10} .

In the method according to an embodiment of the present invention, the gene may further include, but is not limited to, a 6xHis tag coding sequence. The 6xHis tag is designed to facilitate the purification of the antibodies produced, in general, it can be purified by Ni ^{+ 2} affinity chromatography.

In the method according to an embodiment of the present invention, the concentration of Factor Xa in the cell-free protein synthesis reaction solution may be 40 μg / mL or more, preferably 40 to 100 μg / mL, but is not limited thereto.

In the method according to one embodiment of the present invention, the produced antibody of step (b) may be purified or untreated, and preferably purified, but is not limited thereto.

The present invention also relates to a method for introducing a promoter, ubiquitin, Factor Xa cleavage site, 6xHis tag, heavy chain variable region and cell membrane permeable light chain variable region in 5 'to 3' direction into a cell-free protein synthesis reaction solution containing Factor Xa A scFv antibody consisting of a light chain variable region, a sequence encoding GFP ₁₁ and a streptavidin binding peptide, and a gene in which a terminator is operably linked is added to produce an antibody by a cell-free protein synthesis method.

The present invention also provides an antibody produced by the above method and consisting of a scFv antibody, GFP ₁₁ and a streptavidin binding peptide consisting of a light chain variable region in which a 6xHis tag, a heavy chain variable region and a cell membrane permeable light chain variable region are introduced. In addition, the antibody may be an antibody having no 6xHis tag.

The method for producing the antibody may be to produce an antibody by adding a gene having no 6xHis tag among the genes, and the antibody thus produced may be an antibody having no 6xHis tag in the produced antibody.

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). IgG sepharose resin and streptavidin resin were purchased from GE Healthcare and Sigma-Aldrich, respectively. ATP, GTP, UTP, CTP, creatine phosphate and creatine kinase were purchased from Roche Applied Science and L- [U- ¹⁴ C] leucine was purchased from Perkin Elmer, Lt; / RTI > 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. According to the experiment, the BL21 Star (DE3) strain transformed with the plasmid pTUM4 was used to prepare S12 cell extracts after being induced to overexpress four kinds of foldases DsbA, DsbC, FkbA and SurA.

2. In E. coli TMab4 scFv  Expression of antibodies

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 cloned into the plasmid pIg20m and named pIg20m TMab4 scFv-GFP11-SBP2 (Figure 1A).

BL21 (DE3) plysE derived from Escherichia coli transformed with plasmid pIg20m TMab4 scFv-GFP11-SBP2 was inoculated into 2 x TYA medium (18 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 100 g / L ampicillin and pH 7.4) at 37 ° C. When the OD ₆₀₀ value reached 0.8, the cells were induced with 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and cultured at 30 ° C. for 20 hours. Since the TMab4 scFv-GFP11-SBP2 was cloned in frame to the phoA signal sequence, the produced antibodies were purified from the culture medium after centrifugation (9,000 RCF, 4 캜 and 40 min). The purified cell culture was loaded onto a 2 mL IgG Sepharose resin column equilibrated with TBS (Tris-buffered saline) at pH 7.4. After washing with 50 mL of TBS, the protein was eluted with 10 mL of 0.1 M acetic acid (pH 3.0). The eluate was neutralized with 2M Tris (pH 9.5) in 0.25mL, salts of 14mL removed (desalting) buffer _{(137mM NaCl, 8.4mM KH 2 PO} 4, 3.6mM NaHPO 4, 2.7mM KCl, 10% glycerol and pH 6.5) (Spin-X, Corning, Lowell, Mass.) After which the final concentration of purified TMab4 scFv was determined by BCA (bicinchoninic acid assay kit (ThermoFisher Scientific, Waltham, Mass.).

3. TMab4 scFv  Antibody  Acellular cell  synthesis

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. 1C) to enhance the expression level 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.

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 / mL 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 standard reaction for cell-free protein synthesis The mixture (4 mL) was used and the reaction mixture was incubated at 30 DEG C for 1 hour. During protein synthesis, the ubiquitin sequence in For situ cleavage, 40 / / mL of Factor Xa enzyme was added to the reaction mixture. The TMab4 scFv antibody produced by the cell-free protein synthesis method was quantified by measuring TCA-insoluble radioactivity using a Tri-Carb 2810TR liquid scintillation counter (PerkinElmer, Waltham, Mass.). Cell-free synthetic proteins were analyzed by 12% tricine gel stained with coomassie blue and Western blot.

After completion of the cell-free synthesis reaction, the centrifuged (20,000 RCF and 10 minutes) supernatant was mixed with 1.2 mL streptavidin agarose beads equilibrated with PBS (phosphate-buffered saline). After holding at 4 ° C for 1 hour, the resin was washed three times with 10 mL of PBS and the binding protein was eluted with 1.6 mL of 5 mM desthiobiotin.

4. Separated- GFP  Complementarity split - GFP complementation ) For cell membrane-permeation analysis

The TMab4 scFv antibody produced from transformed E. coli or produced by cell-free protein synthesis was analyzed based on the -GFP complementarity isolated in reporter cells (Kim et al., 2015., Biochem Biophys Res Commun, 467 ), 771-777), streptavidin and avidin _{1 -10} GFP HeLa cell line _{(HeLa-SA-GFP 1 -10} ) expressing the fusion construct of the fragment was used as a reporter cells. As described in Figure 1, 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. HeLa-SA-GFP _1-10 cells were inoculated into 6-well plates at 3 × 10 ⁴ cells per well and incubated for 12 hours at 37 ° C. and 5% CO ₂ to confirm permeation of the TMab4 scFv antibody into the cytoplasm Lt; / RTI > The medium was removed and the cells were washed with PBS to resuspend the purified TMab4 scFv antibody and fresh medium in a 1: 3 ratio mixture. The re-suspended cells per well was transferred to a 3 × 10 ⁴ 6-well plates at a cell density. After further incubation for 12 hours, the cells were washed three times with PBS and fixed on the plate surface. Cell activity was then measured using MTT assay, fixed with 4% paraformaldehyde for confocal microscopy and stained with Hoechst 33342.

Example  One. TMab4 scFv  Antibody Acellular cell  Expression and analysis

Cell-free protein synthesis uses a translation mechanism that is separate from the cells for protein production. Compared with the cell-based expression method of Comparative Example 1 below, acellular-protein synthesis provides a faster and more flexible option for gene expression, allowing rapid analysis of the expression of various gene sequences. In addition, the reaction conditions for cell-free synthesis can be precisely adjusted according to the physicochemical properties of the target protein. In this Example 1, a TMab4 scFv antibody was produced using a cell-free protein synthesis method as a method for producing a TMab4 scFv antibody. As a result, in the initial experiment in which the PCR amplified gene construct of TMab4 scFv antibody was expressed in the standard reaction mixture as shown in Fig. 2, about 220 / / ml of the TMab4 scFv antibody was synthesized, but only a part of the synthesized protein was present in the soluble fraction (About 18%). These results suggest that the translation product may contain the phoA signal sequence, which may interfere with the proper folding of the TMab4 scFv antibody, due to the absence of the transmembrane structure of E. coli. Thus, constructs without phoA signal sequence were constructed, but removal of this sequence did not improve the solubility of the TMab4 scFv antibody and reduced the total amount of synthesized protein to 75 μg / mL. To increase the production of soluble enhanced scFv antibodies comprising two disulfide (SS) linkages, the reaction mixture was supplemented with a redox buffer (2 mM oxidized glutathione and 3 mM reductase (Oh et al., 2006., Biotechnol Prog, 22 (4), 1225-1228). In addition, S12 cell extracts were prepared after overexpression of four foldases DsbA, DsbC, FkbA and SurA in E. coli strains. However, the application of these oxidation conditions did not significantly increase the solubility of the TMab4 scFv antibody in nature.

It is well known that the amino acid sequence of the complementarity determining regions (CDRs) significantly affects the stability and solubility of the antibody. The TMab4 scFv antibody has a transmembrane hT4 VL with multiple cation residues in CDR1, which is thought to be responsible for their low expression and stability.

Comparative Example  One. In E. coli TMab4 scFv  Expression of antibodies

Approximately 0.4 mg of TMab4 scFv antibody was obtained from 3 L of transformed E. coli culture. GFP fluorescence was not observed in the cytoplasm of the reporter cells up to 7 μM (295 μg / mL) when the TMab4 scFv antibody purified at various concentrations was added to 200 μl of the HeLa-SA-GFP _{1 -10} cell culture medium ). These results indicate that the antibody produced through E. coli has lower cell membrane-permeability or stability because TMab4 is in the scFv form. The addition of large quantities of the TMab4 scFv antibody would produce a detectable fluorescence signal. Cell-based expression methods have limited throughput in testing multiple gene constructs. In particular, subsequent membrane-permeation assays require large-scale cultures of E. coli to obtain purified TMab4 scFv antibodies. In addition, inefficient folding of the TMab4 scFv antibody during the expression in E. coli cells may cause a partially low delivery ratio.

Example  2. Ubiquitin  Using tags to improve availability TMab4 scFv  Production of antibodies

In Example 2, the ubiquitin sequence was fused to the N-terminus of the gene construct used for cell-free synthesis of TMab4 scFv. As shown in Figure 1C, the ubiquitin-tag sequence was added in place of the phoA sequence through an overlap-extension PCR (OE-PCR) reaction. When cultivated under disulfide conditions, the new construct (FIG. 1C) significantly increased the total amount and soluble fraction of synthesized protein (FIG. 4), as compared to the construct without the ubiquitin sequence (FIGS. 1A and 1B). For ubiquitin-TMab4 scFv, 180 [mu] g / ml of the TMab4 scFv antibody was produced, of which about 50% was in the soluble fraction. Therefore, the amount of soluble TMab4 scFv antibody synthesized in 4 mL of cell-free synthesis reaction was similar to that obtained from 3 L of E. coli cell culture.

Example  3. TMab4 scFv of Ubiquitin  Tag In situ  Cleavage and Membrane Permeation Analysis

The ubiquitin-tagged TMab4 scFv construct of the present invention comprises Factor Xa cleavage sites between the ubiquitin tag and the VH sequence. As shown in Figure 5, during the acellular synthesis, the in To produce the TMab4 scFv antibody with the native amino acid sequence through situ cleavage, various concentrations of Factor Xa enzyme were added to the reaction mixture prior to incubation. As a result, Western blot analysis of the translation product, as shown in Figure 6, showed TMab4 scFv (41.93 kDa) antibody at the expected size depending on the concentration of Factor Xa used in the reaction mixture. The ratio of soluble TMab4 scFv antibody was confirmed by comparing the ubiquitin-tagged TMab4 scFv antibody with the original TMab4 scFv antibody, and it was confirmed that the ubiquitin tag was completely removed when Factor Xa of 40 ㎍ / mL or more was treated.

The TMab4 scFv antibody synthesized from 4 mL of the reaction mixture supplemented with 40 μg / mL of Factor Xa was purified with streptavidin-coated agarose resin and dissolved in 5 mM desthiobiotin solution After elution, about 130 μg of purified protein was obtained in a total of 1.5 mL. When reporter cells were treated with purified TMab4 scFv antibody, GFP fluorescence was observed at a much lower concentration (750 nM) than when using the same antibody produced in E. coli cells (Fig. 7). Observation of GFP fluorescence indicates that the cell-free synthesized antibody is internalized into the cell and secreted into the cytoplasm from the intermediate endosomal vesicle. Compared with the antibody produced through E. coli, the successful permeation of the cell-free synthetic antibody into the cell membrane was judged to be due to the difference in cell permeability or GFP complementation efficiency in the reporter cells. In any of these cases, Will be affected by. The reaction conditions of a cell-free system containing a redox potential can thus be meticulously adjusted to specific antibodies, while E. coli cells can only provide a pre-set environment in their periplasmic space, This may not be an optimal condition for antibody folding. Thus, the flexibility of the cell-free synthesis system in terms of manipulating the molecular environment of protein synthesis would be very beneficial for the functional expression of various types of antibody molecules.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Method for simply analyzing uptake into cytoplasm of antibody          produced by cell-free protein synthesis <130> PN15447 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> GFP1-10 <400> 1 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val   1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu              20 25 30 Gly Glu Gly Asp Ala Thr Ile Gly Lys Leu Thr Leu Lys Phe Ile Cys          35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu      50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg  65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg                  85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Lys Tyr Lys Thr Arg Ala Val Val             100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Thr         115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn     130 135 140 Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Thr Val Arg His Asn Val Glu Asp Gly Ser Val                 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro             180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Val Leu Ser         195 200 205 Lys Asp Pro Asn Glu Lys Gly Thr     210 215 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> GFP11 <400> 2 Arg Asp His Met Val Leu His Glu Tyr Val Asn Ala Ala Gly Ile Thr   1 5 10 15 <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Tmab4 VL <400> 3 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr Ala Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys

Claims (10)

(a) a promoter in the 5 'to 3' direction as a synthetic template in a cell-free protein synthesis reaction solution containing Factor Xa; Ubiquitin; Factor Xa cleavage site; A scFv antibody comprising a light chain variable region and a light chain variable region into which a transmembrane transmissible light chain variable region is introduced; A sequence encoding GFP ₁₁ and a streptavidin binding peptide; And adding an operably linked gene to the terminator to produce an antibody; 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; and The method of analyzing the inflow of delete delete delete 2. The method according to claim 1, wherein the animal cell is a HeLa cell expressing GFP _{1- 10} . 2. The method of claim 1, wherein the gene further comprises a 6xHis tag coding sequence. The method according to claim 1, wherein the concentration of Factor Xa in the cell-free protein synthesis reaction solution is 40 μg / mL or more. The method according to claim 1, wherein the antibody produced in step (b) is not purified or purified. Factor Xa, a light chain variable region in which a promoter, ubiquitin, Factor Xa cleavage site, 6xHis tag, heavy chain variable region and transmembrane light chain variable region are introduced in a 5 'to 3' direction as a synthetic template in a cell-free protein synthesis reaction solution A method for producing an antibody by a cell-free protein synthesis method by adding a scFv antibody, a sequence encoding GFP ₁₁ and a streptavidin binding peptide, and a terminator-operably linked gene. 9. An antibody produced by the method of claim 9, consisting of a scFv antibody, GFP _11, and a streptavidin binding peptide, wherein the light chain variable region comprises a 6xHis tag, a heavy chain variable region and a transmembrane light chain variable region.

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