WO2006011589A1 - Procédé de préparation de population de gènes artificiels et de population de protéines artificielles par polymérisation aléatoire des séquences de motif de plusieurs types - Google Patents

Procédé de préparation de population de gènes artificiels et de population de protéines artificielles par polymérisation aléatoire des séquences de motif de plusieurs types Download PDF

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WO2006011589A1
WO2006011589A1 PCT/JP2005/013913 JP2005013913W WO2006011589A1 WO 2006011589 A1 WO2006011589 A1 WO 2006011589A1 JP 2005013913 W JP2005013913 W JP 2005013913W WO 2006011589 A1 WO2006011589 A1 WO 2006011589A1
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artificial
motif
population
sequences
sequence
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PCT/JP2005/013913
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Japanese (ja)
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Kiyotaka Shiba
Hirohide Saito
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Japan Science And Technology Agency
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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

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  • the present invention aims to create a functional artificial gene or a functional artificial protein, an artificial gene population in which a large number of motif sequences are randomly polymerized, an artificial protein population encoded by the artificial gene population, and production thereof Regarding the law.
  • evolutionary molecular engineering in vitro molecular evolution
  • a strategy to obtain a molecule with the desired function and activity by "selection" from a random sequence group prepared in advance rather than rationally designing artificial molecules is adopted.
  • evolutionary molecular engineering consists of (1) selecting a small molecule with the desired activity from a combinatorial molecular population (DNA or protein) with various sequence diversity, and (2) selecting the gene The same reaction is repeated after amplification by gene amplification reaction (PCR), and (3) an artificial molecule having the desired reaction activity is evolved.
  • the error prone PCR method is a method of preparing a mutant population by randomly introducing base substitutions into a parent gene by performing PCR amplification in the presence of manganese ions with the parent gene as a saddle type.
  • the efficiency of introducing a base substitution mutation is increased by the error prone PCR method, there is a problem that not only amino acid substitution but also the probability that a codon encoding an amino acid is replaced with a stop codon is increased.
  • unintended deletion mutations are likely to occur under conditions that increase the mutation rate (Trends Biochem. Sci. 26: 100-106, 2001). There is a problem that the coded stop codon appears.
  • error prone PCR is usually performed under conditions that introduce a low mutagenesis rate, ie, substitution of 2 to 3 bases per gene and a mutation rate of 1 residue as an amino acid. For this reason, it is considered difficult to create artificial molecules that greatly change functions and substrate affinity.
  • DNA shuffling is a method of preparing a population of mutants by homologous recombination in vitro using a gene DNA encoding a protein and one or more similar DNAs having sequence homology. Can do.
  • the DNA shirt-fling method is based on homologous recombination, recombination between relatively similar sequences is possible. It is limited to. That is, it is difficult to shuffle between DNAs with low sequence similarity.
  • a series of complicated operations such as DNA cleavage and ligation are required.
  • a method using a gene population in which a part or all of a natural gene sequence is randomized by chemical synthesis is effective for the production of functional nucleic acids such as artificial RNA enzymes (ribozymes).
  • functional artificial proteins have not been created. The reasons for this are as follows: (1) Genes with long ORFs (Open Reading Frames) are difficult because stop codons that stop protein translation appear frequently in populations with random mutations. 2) Since the sequence space composed of 20 types of proteins is vast compared to the sequence space of nucleic acids composed of 4 types of bases, it is extremely difficult to select functional proteins from random sequence populations. It is difficult.
  • a microgene serving as a repetitive unit is a motif sequence corresponding to the intended function or structure in three reading frames using the multifunctional base sequence design method invented by the present inventor (JP 2001-352990 A). Since it is designed to code, an artificial gene population with high latent ability can be prepared.
  • Patent Document 1 Japanese Patent No. 3415995.
  • Patent Document 2 JP 2001-352990 A.
  • Non-patent literature l Science, 219, 666-671, 1983.
  • Non-Patent Document 2 Science, 67,938-947,1997.
  • Non-Patent Document 3 PCR Methods Appl, 2: 28-33, 1992.
  • Non-Patent Document 4 Nature, 370: 389-391, 1994.
  • Non-Patent Document 5 Trends Biochem. Sci. 26: 100-106, 2001.
  • Non-Patent Document 6 Proc. Natl. Acad. Sci. USA, 94: 3805-3810, 1997.
  • the subject of the present invention is a functional artificial gene or!
  • Tsuji provides an artificial gene group in which multiple motif sequences are randomly polymerized, an artificial protein group encoded by the human gene group, and a method for producing the same.
  • a single strand that encodes a motif sequence at least in part can randomly polymerize DNA that is not subject to high overall sequence similarity, and avoids the occurrence of stop codons. It is an object of the present invention to provide a method for producing a combinatorial artificial gene and an artificial protein population that enables random polymerization of DNA sequences having a long translation reading frame (ORF) by randomly polymerizing DNA.
  • ORF long translation reading frame
  • the present inventors constructed at least three kinds of single-stranded DNAs containing sequences encoding arbitrary motif sequences, and the single-stranded DNA sequences.
  • a base sequence capable of forming a complementary base pair with each other in a part of the terminal sequence of the DNA mixing the various single-stranded DNAs in an arbitrary ratio, and allowing DNA polymerase to act on them.
  • ORFs long-range translational reading frames
  • the present invention constructs at least three kinds of DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and a part of the terminal sequence of the single-stranded DNA sequence is
  • a DNA polymerase is allowed to act on a reaction mixture in which various single-stranded DNAs constructed by the method are mixed at an arbitrary ratio. It consists of proceeding a random polymerization reaction between DNAs encoding, and creating an artificial gene population in which multiple motif sequences are randomly polymerized.
  • the motif sequence in the present invention it is desirable to construct a single-stranded DNA in which at least 3 types, preferably 4 or more types of motif sequences exist, respectively.
  • a part of the complementary base pair of the terminal sequence of the single-stranded DNA sequence can usually be introduced at the 3 'end, and the number of the complementary base pairs is 6 bases. It is preferable that this is the case.
  • one or more bases that are not paired with the partner base are preferably present in the range of 1 to 3, so that the efficiency of the polymerization reaction by DNA polymerase can be increased.
  • single-stranded DNA containing a sequence encoding an arbitrary motif sequence is based on a functional motif sequence, a structural motif sequence, a human peptide sequence obtained by evolutionary molecular engineering, or protein engineering knowledge. It can be a microgene constructed on the basis of the designed sequence.
  • stop codons can be excluded in advance for each translation reading frame. Furthermore, in the present invention, by adding a restriction enzyme recognition sequence to a single-stranded DNA that encodes multiple types of motif sequences, the frequency of occurrence of motif sequences used in random polymerization reactions can be directly monitored by restriction enzyme reactions. As described above, an artificial gene population can be produced.
  • the artificial gene population produced by the method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to the present invention further translates the artificial gene population and converts it into an artificial protein.
  • an artificial protein population in which a large number of motif sequences are randomly inserted can be produced.
  • an artificial gene population is produced by randomly polymerizing single-stranded DNAs encoding multiple types of motif sequences without inserting base mutations or deletions. By translating the gene population, it is possible to produce an artificial protein population in which a large number of motif sequences are randomly inserted without depending on the frame shift.
  • a single gene DNA encoding a multiple types of motif sequences is randomly polymerized while inserting base mutations and deletions to produce a human gene population, and the artificial gene population is translated into a frame.
  • an artificial protein population can be created in which many types of motif sequences are randomly inserted.
  • prepared artificial gene populations in which a large number of the motif sequences of the present invention are randomly polymerized or artificial protein populations into which various motif sequences are randomly inserted are functional groups of these populations.
  • Functional artificial genes or functional artificial proteins can be created by screening genes or functional proteins.
  • the present invention provides [1] (1) constructing at least three or more DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and (2) A complementary base sequence is introduced so that part of the terminal sequence of the single-stranded DNA sequence can form a complementary base pair with each other, and (3) various single-stranded DNAs constructed by the method are arbitrarily selected.
  • a method for producing an artificial gene population in which a large number of motif sequences according to [1] or [2] are randomly polymerized, and [4] the number of complementary base pairs is 6
  • the concentration of the double-stranded DNA varies within the range of 0.2 ⁇ to 20 ⁇ .
  • the present invention also [9] mixes various constructed single-stranded DNAs into a reaction solution and acts from the 3 ′ end to the end with the complementary base sequence of the introduced single-stranded DNA as a saddle.
  • a single-strand D ⁇ containing a sequence encoding an arbitrary motif sequence is a functional motif sequence, structural motif sequence, evolution Based on artificial peptide sequences obtained from molecular engineering, or sequences designed based on protein engineering knowledge
  • a method for preparing an artificial gene population in which a large number of motif sequences are randomly polymerized according to any one of the above [1] to [8], which is a constructed microgene, and [11] a mouth-mouth gene The multi-motif sequence described in [10] above, wherein the stop codon is excluded in each translation reading frame in the single-stranded DNA sequence to be synthesized.
  • a method for creating a gene population and [12] single-strand DNA strength containing sequences encoding arbitrary motif sequences Random polymerization reaction by adding restriction enzyme recognition sequences to single-strand DNA encoding multiple motif sequences
  • the multiple motif sequences described in any one of [1] to [11] above are randomly polymerized so that the frequency of the motif sequences used in can be directly monitored by restriction enzyme reaction Method for manufacturing an artificial gene population, consisting of [13] above [1] to [12] Artificial genes Group The fabricated by work made method of any, many species motif sequence was polymerized randomly.
  • the present invention provides [14] a beta incorporating a gene produced by a method for producing an artificial gene population in which the multiple motif sequences according to any one of [1] to [12] are randomly polymerized, A method of creating artificial protein populations that randomly insert various motif sequences characterized by introduction and expression into cells, and [15] single-stranded DNA encoding multiple motif sequences
  • a beta incorporating a gene produced by a method for producing an artificial gene population in which the multiple motif sequences according to any one of [1] to [12] are randomly polymerized A method of creating artificial protein populations that randomly insert various motif sequences characterized by introduction and expression into cells, and [15] single-stranded DNA encoding multiple motif sequences
  • an artificial gene population is produced by random polymerization without insertion, and a polymorphic molecular population is obtained without depending on frameshift by translating the artificial gene population.
  • FIG. 1 is a diagram showing an outline of production of an artificial protein population using the motif sequence of the present invention.
  • FIG. 2 is a diagram showing a design of a single-stranded DNA containing a restriction enzyme recognition sequence (top) and an electrophoretogram of a random polymerization reaction in Examples of the present invention.
  • M represents a single DN A.
  • FIG. 3 is a diagram showing the results of confirming multiple types of random DNA polymerization by restriction enzyme reaction in Examples of the present invention.
  • FIG. 4 is a diagram showing the sequence of a DNA polymerization product having a restriction enzyme recognition motif in an example of the present invention.
  • FIG. 5 is a diagram showing design (A) and design (B) of single-stranded DNA having a BH1-B H4 motif present in an apoptosis-regulating protein in an example of the present invention.
  • Design A was designed to form a complementary base pair with the 3 ends of KY-1372, 3 ends of KY-1372, KY-1375, KY-1375, and KY-1377.
  • Design B was designed to form a complementary base pair with the ends of KY-1372 and KY-1379 and the ends of KY-1374 and KY-1375.
  • FIG. 6 is an electrophoretogram of a single-stranded DNA polymerization reaction containing a BH1-BH4 motif in an example of the present invention. o It was confirmed that the four types of single-stranded DNA synthesized in Design A and Design B proceeded with the polymerization reaction.
  • FIG. 7 is a diagram showing the results of confirmation of single-stranded DNA random polymerization containing a BH1-BH4 motif by restriction enzyme reaction in an example of the present invention. Electrophoresis after the restriction enzyme reaction confirmed that each motif polymerized randomly.
  • FIG. 8 a, b and c are artificial gene populations obtained by design A in the examples of the present invention. It is a figure which shows the arrangement
  • FIG. 9 is a diagram showing the results of confirming the expression of an artificial protein in mammalian cells in an example of the present invention.
  • the gene for artificial protein A6 obtained by design A was introduced into breast cancer cell line MCF-7, and its expression was confirmed by immunostaining with Alexa-His antibody. These artificial proteins were found to be localized and expressed in mitochondria.
  • FIG. 10 is a diagram showing the sequences of the artificial gene population obtained by design B and the artificial protein population that is the translation product in the example of the present invention.
  • FIG. 11 In the examples of the present invention, the results of the design of single-stranded DNA having BH1-BH4 motif (C, D) and electrophoretic diagram of single-stranded DNA polymerization reaction present in the apoptosis-control protein are shown.
  • FIG. In design (C), four types of single-stranded DNA (KY-137 2, KY-1389, KY-1391) were mixed and polymerized in equal amounts, whereas in design (D), KY-1372: KY -1389: KY- 1390: KY-1391 2: 2: 0. 4: 0. 3 Encoding ⁇ ⁇ 4 and ⁇ 3 motifs ⁇ — 1372 and ⁇ — 1389 Increase the ratio of polymerization reaction Added to the system.
  • FIG. 12 is a diagram showing the results of confirming single-stranded DNA random polymerization containing ⁇ 1- ⁇ 4 motifs by restriction enzyme reaction in Examples of the present invention. In designs C and D, partial cleavage by three restriction enzymes was confirmed.
  • FIG. 13] a and b are diagrams showing the sequences of an artificial gene population obtained by design C and an artificial protein population which is a translation product thereof, in an example of the present invention.
  • FIG. 14 is a diagram showing the sequences of an artificial gene population obtained by design D and an artificial protein population that is a translation product thereof, in Example of the present invention.
  • an artificial protein population (C) and an artificial protein population (D) are provided in an embodiment of the present invention.
  • FIG. 4 is a diagram showing the results of comparing the appearance frequencies of BH1-BH3 motifs in FIG.
  • the protein population (D) prepared by increasing the concentration of single-stranded DNA (KY-1389) encoding the BH3 motif the frequency of appearance of the BH3 motif increased.
  • FIG. 16 is a diagram showing various localization patterns of artificial proteins in human cancer cells in Examples of the present invention.
  • FIG. 17 shows a gene encoding artificial protein D29 obtained in the examples of the present invention.
  • FIG. 6 is a diagram quantifying the number of apoptotic cells induced in breast cancer cell line MCF-7 by introduction of. The effect was equivalent to the natural apoptosis-inducing protein Bax, and about 30% of the cells were apoptosis-positive cells. In cells transfected with the control artificial protein A10 gene, we found that such apoptosis-positive cells were not confirmed.
  • FIG. 18 is a graph showing that apoptosis is effectively induced in cells expressing D29 in the examples of the present invention. Double staining by confocal microscopy (green; expressed protein, red; apoptosis-positive cells) confirmed the correlation between cells expressing D29 and apoptosis-positive cells. It was found that apoptosis was not induced in cells expressing the control artificial protein A10.
  • FIG. 19 is a diagram quantifying the inhibitory activity of the growth inhibition induced by the cervical cancer cell line HeLa by introducing the gene encoding the obtained artificial protein A10 or D16 in the examples of the present invention.
  • Natural type apoptosis-inducing protein BIM, or anticancer drug etoposide (VP-16) or staurosporine (STS) reduces the number of HeLa cells (quantified by WST-1; see control pcDNA), but introduces A10 or D16 gene As a result, the proliferation activity was partially recovered.
  • This inhibitory effect on growth inhibition was also observed in cells transfected with a plasmid encoding the gene for Bel-xL, a natural apoptosis-inhibiting protein.
  • FIG. 20 is a view showing that apoptosis induced by the obtained artificial protein A10 or D16 force STS can be suppressed in Examples of the present invention.
  • the medium was replaced with a medium containing 125 nM STS, and the cells were further cultured for 23 hours.
  • apoptosis was effectively induced in the cells transfected with the control pcDNA or D29, whereas in the cells transfected with the apoptosis-inhibiting Bel—xL gene.
  • the number of TUNEL positive cells decreased.
  • Apoptosis was partially suppressed in cells transfected with A10 and D16 genes.
  • FIG. 21 is a diagram showing that cells expressing the obtained artificial protein D16 become apoptosis-negative in the examples of the present invention.
  • the artificial protein was greened with Myc-secondary antibody and FITC secondary antibody. Apoptotic cells were double-stained with TMR-red label (red). In cells expressing D16 and Bcl— x L, apoptosis was suppressed. Cells that expressed D29 were confirmed to be positive for apoptosis.
  • the present invention aims to effectively create a functional artificial gene or functional artificial protein, an artificial gene population in which a large number of motif sequences are randomly polymerized, and an artificial protein population encoded by the artificial gene population And providing a method for producing the same.
  • the method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to the present invention consists of (1) constructing at least three types of DNA sequences of single-stranded DNA containing sequences encoding arbitrary motif sequences. And (2) introducing a complementary base sequence so that part of the terminal sequence of the single-stranded DNA sequence can form a complementary base pair with each other, and (3) various single strands constructed by the method.
  • the method for producing an artificial gene population of the present invention will be specifically described with reference to an example.
  • multiple types (multiple types) of single-stranded DNA containing the desired motif sequence are synthesized.
  • synthesis is carried out so that a part of the three rules of a plurality of types of single-stranded DNA can form complementary base pairs.
  • the number of complementary base pairs is preferably 6 bases or more.
  • it is synthesized so that there are one or more bases (preferably 1 to 3 bases) that are not paired with the partner base at both ends of the complementary base pair. By this operation, the efficiency of the polymerization reaction can be increased.
  • the reaction when the reaction is performed using four types of single-stranded DNA, the remaining three types of single-stranded DNA 3 'sequences are complementary to the 3' sequence of one type of single-stranded DNA. It may form, or two types of single-stranded DNA with homologous ⁇ -terminal sequences, while the other two types of single-stranded DNA You can design your DNA sequences so that they are evenly complementary.
  • the concentration of single-stranded DNA added to the reaction system can vary from 0.2 / z / to 20 / ⁇ M, and the concentration of single-stranded DNA to be used for the polymerization reaction can be increased. To the reaction system.
  • the frequency of motif sequences used in random polymerization reactions can be controlled.
  • the complementary regions of these multiple types of single-stranded DNA function as a trapezoid for a PCR reaction using a thermostable polymerase.
  • a thermostable DNA polymerase containing 3, ⁇ 5 exonuclease activity
  • a double-stranded DNA polymer population in which multiple types of single-stranded DNA containing a motif sequence are randomly extended and ligated is synthesized under the conditions of PCR using DNA polymerase (eg, Vent polymerase), for example 94
  • DNA polymerase eg, Vent polymerase
  • One cycle of 10 seconds at 55 ° C and 60 seconds at 55 75 ° C is performed for 7 minutes at 69 ° C until DNA polymerization can be confirmed (30 to 65 cycles).
  • the method for producing the artificial gene population of the present invention has been specifically described above with examples. However, in the method for producing the artificial gene population of the present invention, a variety of single-stranded D as described above can be used. Control the frequency of motif sequences used in random polymerization reactions by adjusting the combination of NA's complementary base pairs and the concentration of each Z or multiple single-stranded DNA Is possible.
  • a plurality of motif sequences are arbitrarily selected and randomly polymerized to produce an artificial gene population and an artificial protein population. Is constructed based on sequences such as functional motif sequences, structural motif sequences, artificial peptide sequences obtained from evolutionary molecular engineering, or sequences designed based on protein engineering knowledge. It is possible to create an artificial gene population by predicting a certain degree of function with a reasonable rationality.
  • An artificial protein population produced by the method for producing an artificial gene population of the present invention in which various motif sequences are randomly inserted is introduced into a host cell and expressed by introducing a vector incorporating the gene into the host cell. It is possible to produce artificial protein populations in which various motif sequences are randomly inserted.
  • Vectors used for the production of the artificial protein population, host cells and their gene expression methods include vectors, host cells and gene expression methods known in the art.
  • An artificial gene population in which a large number of motif sequences produced by the method of the present invention are randomly polymerized, or an artificial protein population into which various motif sequences are randomly inserted, is used for the artificial gene population or artificial protein population. By screening functional genes or functional proteins, functional artificial genes or functional artificial proteins can be created.
  • FIG. 1 shows a strategy for producing an artificial protein population using the motif sequence of the present invention.
  • This method consists of (1) design of multiple types of single-stranded DNA encoding motif sequences, (2) random polymerization reaction between multiple types of single-stranded DNA, and (3) efficiency of random polymerization reaction by restriction enzyme cleavage reaction. Study, (4) Transformation of random polymer into E. coli and sequencing, (5) Expression of artificial protein and screening of functional artificial protein in E. coli or mammalian cells.
  • an artificial gene and an artificial protein population are generally prepared by random polymerization of four types of motif sequences.
  • the number of motifs that can be used is not limited to four, (more than that is theoretically possible). Specific experimental examples are given below.
  • the fourth to ninth sequences (underlined) of KY-1354, KY-1355, KY-1356, KY-1357 are the restriction enzyme recognition sequences GAAT TC (EcoRI), AAGCTT (HindIII), TCTAGA, respectively. (Xbal) and AGATCT (Bglll).
  • the 10th to 15th 6 bases of KY-1354 (5, -GGCGGG-3,) are the 6th base of 11th to 16th of KY-1355, KY-1356, KY-1357 (5 CCCGCC -Designed to form complementary base pairs with 3 ').
  • adenine (A) was introduced at the ⁇ terminal and 10th position of KY-1355, KY-1356, and KY-1357 so that a complementary base pair of 6 bases or more could not be formed with KY-1354. Furthermore, each of the four types of single-stranded DNA has a length (16-17 bases), Tm (: ⁇ 58 ° C), and GC content (65-69%) close to each other. Strand DNA was designed.
  • This reaction solution contains 10 X ThermoPol Reaction Buffer (NEW ENGLAND BioLabs'lx ThermoPol Reaction Buffer: 20 mM 2-amino-2-hydroxymethyl-1,3, monopropanediol hydrochloride (hereinafter tris-hydrochloric acid) pH 8.8, 10 mM Vent DNA polymerases with potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X—100) 5; z L, 350 ⁇ (1 ', 3' ⁇ 5 'etasonuclease activity ( 2 units / ⁇ L, NEW EN GLAND BioLabs) 2.6 L and 4 types of single-stranded DNA (KY— 1354— KY— 135 7) was mixed in the following 6 ratios.
  • 10 X ThermoPol Reaction Buffer NW ENGLAND BioLabs'lx ThermoPol Reaction Buffer: 20 mM 2-amin
  • KY— 1354 20 pmol only (1 DNA
  • the DNA polymer was cleaved using an arbitrary restriction enzyme.
  • 50 ⁇ L of 5 kinds of reaction solutions containing different restriction enzymes were prepared as follows. 5 ⁇ L of the above DNA polymerization reaction solution, 10 ⁇ restriction enzyme buffer B (Hindlll, EcoRI), M (BglII), or H (Xbal) 5 ⁇ L, (Roche Diagnostics, Basel), restriction enzyme (Roche Diagnostic, Basel) has (1) no enzyme, (2) HindllKlOunits / ⁇ L) 2 ⁇ L, and (3) BglII (lOunits / ⁇ L) for each of the five reaction solutions.
  • Random polymerization reaction solution between the above DNA sequences is commercially available Zero Blunt TOPO PCR
  • Cloning and sequencing were performed using Cloning Kit (Invitrogen, CA).
  • the ligation reaction is performed on a 6 ⁇ L reaction scale, and this reaction solution contains 1 ⁇ L of DNA polymerization product, 1 L of Salt Solution, 1 L of TOPO vector, and 3 ⁇ L of ultrapure water.
  • 2 ⁇ L of the reaction solution was mixed with TopOlOcell (Invitrogen) and transformed.
  • Select 18 clones containing the insert purify the plasmid with QIAGEN mini kit (Qiagen), and sequence with the capillary sequencer CEQ2000XL DNA analyzer (Beckman) by dye termination method using DTCS cycle sequence reaction kit (Beckman) It was determined.
  • FIG. 4 shows three clones (EHBX1 to EHBX3: SEQ ID NOs: 6, 7, and 8) obtained from the polymer power obtained by mixing four kinds of single-stranded DNAs of KY-1356 and KY-1357. As shown in FIG. 4, an artificial gene population in which DNA containing 3 or 4 kinds of restriction enzyme recognition sequences was randomly polymerized at an arbitrary ratio could be obtained.
  • BcHd a member of the family, has been shown to be important in inhibiting apoptosis, and Noxa is important in promoting apoptosis.
  • the partial sequence of BH1, BH2, and BH4 motifs in human BcHd protein and the partial sequence of BH3 motif in hu man Noxa protein are used as motif sequences for artificial protein population creation. Tried. Part encoding the following peptide sequence Minutes were selected from each BH1-BH4 motif.
  • BH2 ENGG WDTF
  • BH3 LRRFGDKLN ⁇
  • BH4 RELWDFL.
  • KY-1372 (SEQ ID NO: 9), KY-1377 (SEQ ID NO: 10), KY-1375 (SEQ ID NO: 11), KY-1374 (SEQ ID NO: 12) are the restriction enzyme recognition sequences GTCGAC (Sail), It was designed to include AAGCTT (HindIII), AGATCT (BglII), and GAATTC (EcoRI) ( Figure 5, Design A, underlined).
  • KY— 1372 3 ′ end 7 bases (5,-GGCGGGG-3,), KY— 1377, KY— 1375, KY-1374 ⁇ end 7 bases (5 '-CCCCGCC-3') It was designed to form a complementary base pair.
  • adenine (A) and cytosine (C) were inserted so that no interaction of 7 base pairs or more was formed at the 9th base from the ⁇ terminal and ⁇ terminal of KY-1372.
  • the length (40-45), GC base content (55-65%), intramolecular hairpin formation ability, and Tm are close to each other. Designed as follows.
  • the composition of this reaction solution is 5 ⁇ L of 10 X ThermoPol Reaction Buffer (same as Example 1), 2.6 L of 350 ⁇ M dNTP ⁇ Vent DNA polymerases (2 units / ⁇ L, NEW ENGLAND BioLabs), and Four types of single-stranded DNA (KY-1372, KY-1377, KY-1375, KY-1374) were mixed at various ratios, and the same DNA random polymerization reaction as in Example 1 was performed.
  • a pretreatment for the polymerization reaction a reaction was carried out at 94 ° C for 10 minutes and at 69 ° C for 10 minutes.
  • the polymerization reaction temperature was set to 72 ° C.
  • the polymerization reaction cycle was performed at 94 ° C for 10 seconds and at 55 ° C for 60 seconds, and 40 cycles of the reaction proceeded. [0038] After completion of the polymerization reaction, an elongation termination reaction was carried out at 69 ° C for 7 minutes.
  • the DNA polymerization product was electrophoresed at 100 V for 15 minutes using 1.0% TAE agarose gel (Agarose ME Iwai Chemical Tokyo) and Mupid electrophoresis apparatus (Cosmo Bio, Tokyo) to confirm the polymerization reaction (Fig. 6). .
  • the DNA polymer was cleaved using an arbitrary restriction enzyme.
  • 50 ⁇ L of five reaction solutions containing different restriction enzymes were prepared as follows. 5 ⁇ L of DNA polymerization reaction solution, 10 ⁇ restriction enzyme buffer H (SalI, or EcoRI), or 5 ⁇ L of M (Hindm, or Bglll) (Roche Diagnostics, Basel), restriction enzyme (Rochedai (Agnostic, Basel) has (1) no enzyme, (2) Sail (40units / ⁇ L) ⁇ 2 ⁇ (3) HindIII (10units Z ⁇ L) in 2 ⁇ m for each of the five reaction solutions.
  • the DNA polymerization reaction product containing the BH1-BH4 motif was cloned and sequenced using a commercially available pcDNA3.1Directiona 1 TOPO Expression Kit (Invitrogen, CA).
  • an artificial gene was ligated into a mammalian expression vector (pcDNA3.1D / V5-His-TOPO, Invitrogen) for screening functional artificial proteins in mammalian cell systems.
  • the ligation reaction is performed on a 6 ⁇ L scale. Polymerization reaction product l / L, Salt Solution 1 L, TOPO vector 1 L, ultrapure water 3 L force S are included.
  • the reaction solution was reacted at room temperature for 30 minutes, the 2 / ⁇ reaction solution was mixed with Ding 0-10 ⁇ 11 (1 nvitrogen), allowed to stand on ice for 30 minutes, and transformed. From the screening PCR, 10 types of clones containing inserts were selected, and the plasmids were purified with QIAGEN mini kit (Qiagen). The sequence was determined with (Beckman) (SEQ ID NO: 13-42).
  • FIG. 8 The gene sequence and amino acid sequence of the obtained artificial protein are shown in Fig. 8 (SEQ ID NOs: 13-42).
  • Figure 8 showed that an artificial protein population in which artificial DNA encoding the BH1-BH4 motif was randomly inserted could be created.
  • the motif depends on the frame shift that occurs during the DNA polymerization reaction.
  • the gene was designed to be introduced (the reading frame would be shifted if no frame shift occurred) o Many reading frame arrangements different from the reading frame encoding the BH1-BH4 motif appeared as shown in Fig. 8. .
  • a library can be created using multiple motif sequences, but sequence diversity can be increased by using sequences in different translation reading frames.
  • a plasmid encoding the gene for MxA6 (see Fig. 8, a, b, c) was used as one of the representative human breast cancer cell lines, MCF-7
  • the gene was introduced into.
  • V pcDNA vector (Invitrogen) that does not encode an artificial protein was introduced into cells.
  • MCF-7 was fixed with methanol 24 hours after gene introduction, and the expression of histidine-tag and artificial protein was confirmed by immunostaining with Alexa-His-antibody (Qiagen) (FIG. 9). As shown in Fig.
  • the 3 'end 7 bases of KY-1372 or KY-1379 are complementary to the 7 end bases of KY-1375 and KY-137 4 (5' -CCCCGCC-3 ') It was designed to form a target base pair.
  • the 9th base from the 3 'end and the 3' end of KY-1372 and KY-1379 does not form an interaction of 7 base pairs or more, so that adenine (A) and cytosine (C) are added. Each inserted.
  • the DNA polymer was cleaved using an arbitrary restriction enzyme.
  • the reaction is the same as in the section on the production of artificial protein population (A), except that Xhol is carotenized as a restriction enzyme.
  • the cleavage reaction was allowed to proceed at 37 ° C for 90 minutes.
  • Transformation and sequencing were performed in the same manner as in the section on production of artificial protein population (A).
  • the gene sequence and amino acid sequence of human protein is shown in FIG. 10 (SEQ ID NO: 4453).
  • the BH1-BH4 motif is an artificial protein depending on the frame shift caused by random base deletion or insertion that occurs during the DNA polymerization reaction. Randomly inserted into the population. For this reason, the appearance frequency of the BH1—BH4 motif was relatively low (a lot of different translation reading frames appear).
  • the design of the single-stranded DNA was modified so that the BH1-BH4 motif was randomly polymerized without depending on the frame shift.
  • the other methods are basically the same as the production of the artificial protein population (A). More BH1—BH4 modules without relying on frameshift It is expected in Example 4 that the chief appears in the artificial protein population.
  • KY— 1372 Four types of single-stranded DNAs KY— 1372, KY— 1389 (SEQ ID NO: 54), KY— 1390 (SEQ ID NO: 55), KY — 1391 (SEQ ID NO: 56) was designed (FIG. 11, KY— 1372: BH4 motif, KY— 1389: BH3 motif, KY— 1390: BH1 motif, KY— 1391: BH2 motif).
  • KY-1389, KY-1390, and KY-1391 are sequences with CC attached to the ⁇ end of KY-1377, KY-1374, and KY-1375, respectively.
  • the randomly polymerized BH1-BH4 motif appears in the artificial protein population without depending on the frame shift.
  • KY— 1372, KY— 1389, KY— 1390, and KY— 1391 were designed to include the restriction enzyme recognition sequences GTCGAC (Sail), AAGCTT (Hindlll), GAATTC (EcoRI), and AGATCT (Bglll), respectively. C, underlined).
  • KY— 1372 3 ′ end 7 bases (5, -GG CGGGG-3,) force KY— 1389— KY— 1391 3 ′ end 7 bases (5,-CCCCGCC-3,) and complementary base pair Designed to form.
  • adenine (A) and cytosine (C) were inserted so that no interaction of 7 base pairs or more was formed at the ⁇ terminal of KY-1372 and the ninth base from the ⁇ terminal.
  • the single-stranded DNA random polymerization reaction solution 50 ⁇ L of the single-stranded DNA random polymerization reaction solution was prepared in the same manner as in the section on the production of artificial protein population ( ⁇ ).
  • the polymerization reaction was carried out at 72 ° C and 45 cycles, and the polymerization was confirmed by 1% agarose gel (Fig. 11).
  • Transformation and sequencing were performed in the same manner as in the section on production of artificial protein population (A).
  • the gene and amino acid sequence of the artificial protein obtained by Design C are shown in FIGS. 13a and b (SEQ ID NOs: 57-74).
  • FIGS. 13a and b SEQ ID NOs: 57-74.
  • Fig. 13 we succeeded in obtaining an artificial protein population in which four BH1-BH4 motif sequences appear frequently.
  • the proportions of KY-1 372 and KY-1389 encoding ⁇ ⁇ ⁇ 4 and ⁇ 3 motifs were increased and added to the polymerization reaction system. All the reaction conditions were the same as in the production of the artificial protein population (C) in Example 4.
  • FIG. 11 shows an electrophoretogram of the random polymer
  • FIGS. 14a, b and c show the gene and amino acid sequences of the human protein obtained (SEQ ID NOs: 75 to 100).
  • Fig. 14 we succeeded in creating an artificial protein population in which four motifs appeared frequently, and in particular, the BH4—BH3 motif appeared frequently. In this way, by changing the amount ratio of single-stranded DNA to be mixed during the DNA polymerization reaction, the ratio of the motif contained in the artificial protein population can be controlled.
  • Figure 15 compares the frequency of BH1-BH3 motifs in the artificial protein population (C) and the artificial protein population (D).
  • Example 6 28 clone genes whose protein expression was confirmed were introduced into human breast cancer cell line MCF-7, and the effect on the growth of MCF-7 was examined.
  • a 96-well cell culture plate was seeded with lxlO 4 MCF-7 per well and cultured at 37 degrees for 24 hours. Thereafter, plasmid 0.2 / zg encoding the gene of 28 clones was introduced into MCF-7 using Lipofectamine (20 OO lnvitrogen). 48 hours after gene introduction, the proliferation activity was quantified using tetrazorium salt WST-1 which is an indicator of mitochondrial metabolic activity (Roche). Do not code a control protein! Compared to the growth activity when an empty vector was introduced, one clone out of 28 clones has a D29 force that significantly inhibits the growth of MCF-7. (Approximately 40% growth inhibitory effect over control).
  • TUNEL staining an index for detecting apoptotic cells, was performed (Roche).
  • TUNEL staining fragmented DNA in the early stage of apoptosis can be detected with fluorescently labeled nucleotides.
  • D29 was able to induce apoptosis in MCF-7 to the same extent as the natural cell death-inducing protein Bax (Fig. 17).
  • A10 one of the clones in which cell growth inhibition was not observed by WST-1, was found to be unable to induce apoptosis in MCF-7.
  • the artificial protein population produced by random polymerization of the apoptosis-control motif sequence by this method was found to contain functional artificial proteins capable of inducing apoptosis in breast cancer cells.
  • the clone whose expression was confirmed in Example 6 contained a cell death inhibitory BH4 motif as well as a cell death promoting BH3 motif. Therefore, the possibility of obtaining a cell death-suppressing functional protein having a function opposite to that of the artificial cell death promoting protein of Example 7 from these clones was examined.
  • the genes of 28 clones whose protein expression was confirmed and the natural cell death-promoting protein BIM were introduced into the human cervical cancer cell line HeLa, and the effects of the clones on the growth inhibitory activity of BIM were examined.
  • the cell culture plates 96 Ueru were seeded with 0. 5xl0 4 of HeLa per Ueru were incubated at 37 ° for 24 hours.
  • the artificial gene population production method of the present invention has made it possible to produce an artificial gene population in which a large number of motif sequences are randomly polymerized, which was not possible with the conventional artificial gene population production method. Moreover, it has become possible to produce an artificial protein population encoded by the artificial gene population using the artificial gene population. According to the method for producing an artificial gene population of the present invention, it is possible to randomly polymerize a plurality of types of DNAs, provided that the motif sequence is encoded by at least a part of the gene group, provided that the similarity is high. In addition, by randomly polymerizing single-stranded DNA that avoids the appearance of stop codons, DNA sequences with long translation reading frames (ORF) can be randomly polymerized.
  • ORF long translation reading frames
  • an arbitrary motif sequence is changed based on a sequence such as a functional motif sequence, a structural motif sequence, an artificial peptide sequence obtained by evolutionary molecular engineering, or a sequence designed based on protein engineering knowledge.
  • a sequence such as a functional motif sequence, a structural motif sequence, an artificial peptide sequence obtained by evolutionary molecular engineering, or a sequence designed based on protein engineering knowledge.

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Abstract

Pour créer un gène fonctionnel artificiel ou une protéine fonctionnelle artificielle, il est prévu de fournir une population de gènes artificiels obtenus via la polymérisation aléatoire de séquences de motifs de plusieurs types, une population de protéines artificielles codée par la population de gènes artificiels décrite précédemment et une méthode pour la préparer. Une population de gènes artificiels obtenue via la polymérisation aléatoire des séquences de motif de plusieurs types est préparée en construisant au moins trois types de séquences d'ADN, ayant chacun un ADN à brin unique contenant un codage de séquence d'une séquence de motif arbitraire, en y transférant une séquence de base complémentaire de manière à permettre la formation d'une paire de bases complémentaire dans une partie des séquences terminales des séquences d'ADN à brin unique l'une avec l'autre, puis en traitant un mélange de réaction liquide, qui est préparé en mélangeant les ADN à brin unique ainsi construits de plusieurs types à un ratio arbitraire, avec la polymérase d'ADN de sorte que la polymérisation aléatoire se poursuive parmi l'ADN codant les séquences de motif. En traduisant la population de gènes artificiels et en la convertissant en protéines artificielles, on peut préparer une population de protéines artificielles codée par les séquences de motifs de plusieurs types.
PCT/JP2005/013913 2004-07-30 2005-07-29 Procédé de préparation de population de gènes artificiels et de population de protéines artificielles par polymérisation aléatoire des séquences de motif de plusieurs types WO2006011589A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09322775A (ja) * 1996-06-10 1997-12-16 Kagaku Gijutsu Shinko Jigyodan 高分子マイクロ遺伝子重合体の作成方法
JP2001352990A (ja) * 2000-06-16 2001-12-25 Japan Science & Technology Corp 多機能塩基配列及びそれを含む人工遺伝子

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09322775A (ja) * 1996-06-10 1997-12-16 Kagaku Gijutsu Shinko Jigyodan 高分子マイクロ遺伝子重合体の作成方法
JP2001352990A (ja) * 2000-06-16 2001-12-25 Japan Science & Technology Corp 多機能塩基配列及びそれを含む人工遺伝子

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAITO H. ET AL: "Synthesis of Functional Proteins by Mixing Peptide Motifs.", CHEMISTRY AND BIOLOGY., vol. 11, no. 6, 2004, pages 765 - 773, XP002992959 *

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