JP4568723B2 - Artificial gene and artificial protein populations by random polymerization of multiple motif sequences - Google Patents

Description

  The present invention relates to an artificial gene group in which multiple types of motif sequences are randomly polymerized, an artificial protein group encoded by the artificial gene group, and a method for producing the same, for the purpose of creating functional artificial genes or functional artificial proteins.

  In recent years, research on elucidating the functions of human genome genes and proteins has been progressing mainly in the field of drug discovery, etc. As post-genomic research, analysis of new gene functions and protein functions have progressed on a large scale. As a result, the functions of many genes and proteins are being clarified. With the elucidation of the functions of genes and proteins, expectations and motivation to design and create artificial genes and proteins with reasonably new functions have increased with the accumulation of knowledge. This research into “designing and creating artificial genes and proteins with reasonably new functions” was born as protein engineering in the 1980s (Science, 219, 666-671, 1983). However, although several artificial proteins have been created since then, they have not yet reached the level where new proteins can be freely designed.

  In the 1990s, evolutionary molecular engineering (in vitro molecular evolution) has become a major trend in the field of artificial molecule creation. In this evolutionary molecular engineering, instead of rationally designing artificial molecules, there is a strategy to obtain a molecule with the desired function / activity by “selection” from a random sequence group prepared in advance. Has been adopted (Science, 67,938-947,1997). In other words, evolutionary molecular engineering is: (1) Select a few molecules with the desired activity from a combinatorial molecular population (DNA or protein) with various sequence diversity; (2) Gene amplification reaction of the gene The same reaction is repeated after amplification by (PCR), and (3) an artificial molecule having the desired reaction activity is evolved. At present, this technique is attempting to create functional artificial proteins using gene-protein fusion technologies such as ribosome display and mRNA display. However, the conventional techniques have the following problems. (I) An artificial protein that has sequence diversity and has not yet been established as an effective construction method of a DNA population suitable for a target experiment is (ii) an artificial protein that can be selected by ribosome (mRNA) display; However, it is not suitable for the selection of molecules that are limited to enzyme activity and function in vivo.

  Successful creation of an artificial genetic diversity population (library) that serves as the screening population when creating artificial proteins with unusual functions that do not exist in nature through evolutionary molecular engineering Hold the key. For this reason, it is indispensable to develop artificial genes and artificial protein population creation methods for the purpose of effectively creating functional molecules. Methods for preparing artificial gene populations include error prone PCR (PCR Methods Appl, 2: 28-33, 1992), DNA shuffling (Nature, 370: 389-391, 1994), or natural gene sequences. Chemical synthesis methods in which some or all are randomized are currently widely used.

  The error prone PCR method is a method of preparing a mutant population by randomly introducing base substitutions into a parent gene by PCR amplification in the presence of manganese ions using the parent gene as a template. However, when the base substitution mutation introduction efficiency 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. In addition, in this error prone PCR method, 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. For this reason, the error prone PCR method is usually performed under such a condition that a mutation introduction rate is low, that is, a substitution rate of 2 to 3 bases per gene and a mutation rate of 1 residue as an amino acid are introduced. For this reason, it is considered difficult to create an artificial molecule that greatly changes its function and substrate affinity.

  In the DNA shuffling method, a mutant population can be prepared by homologous recombination in vitro using a gene DNA encoding a protein and one or more similar DNAs having sequence homology. However, since the DNA shuffling method is based on homologous recombination, it is limited to recombination between relatively similar sequences. That is, it is difficult to shuffle between DNAs having low sequence similarity. Furthermore, in order to obtain the target mutant population, a series of complicated operations such as DNA cleavage and ligation are required.

  The method of using a gene population in which part or all of the natural gene sequence is randomized by chemical synthesis has been effective in the production of functional nucleic acids such as artificial RNA enzymes (ribozymes). It has not yet 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 a functional protein from a random sequence population. , And so on.

  In recent years, attention has been focused on the role of short sequences (motif sequences) that play an important role in the function and structure of nucleic acids and proteins. As a technique for creating an artificial gene population using a motif sequence, a technique for producing a polymer microgene polymer invented by the present inventor (Japanese Patent No. 34159995; Proc. Natl. Acad. Sci. USA, 94: 3805- 3810, 1997). By using this technique, it is possible to prepare an artificial gene from which all the translation reading frame stop codons are excluded. This method is a method of preparing a large translation reading frame by polymerizing a short DNA sequence (microgene) from which a stop codon has been eliminated in advance in tandem. Furthermore, a microgene serving as a repetitive unit is a motif sequence corresponding to a desired function or structure in three reading frames using the multifunctional base sequence design method invented by the present inventor (Japanese Patent Laid-Open No. 2001-352990). Therefore, an artificial gene population with high potential can be prepared.

  However, problems to be overcome include: (1) The number of motifs that can be introduced into an artificial gene population is limited to three corresponding to the number of reading frames; (2) To create an artificial gene population with sequence diversity Depends on random frame shifts that occur during the microgene polymerization reaction, and (3) it is difficult to adjust the appearance frequency of motif sequences according to the target experiment. In addition, since the above-described microgene polymer production method (Japanese Patent No. 3419995) aims to create a repetitive polymer of microgenes, a gene population in which a plurality of types of motif sequences are randomly polymerized is produced. Not reached.

  From the above background, in order to effectively create artificial gene diversity populations (libraries) that are the key to success when creating artificial proteins with unusual functions that do not exist in nature through evolutionary molecular engineering A random recombination technique for DNA encoding at least a part of a plurality of motif sequences, and a recombination technique and an open reading frame (ORF) that does not require sequence similarity throughout the DNA used Development of a combinatorial artificial gene population and artificial protein population creation technology that can freely control the appearance frequency of a motif sequence in accordance with a target experiment is required.

Japanese Patent No. 3415999. JP 2001-352990 A. Science, 219, 666-671, 1983. Science, 67,938-947,1997. PCR Methods Appl, 2: 28-33, 1992. Nature, 370: 389-391, 1994. Trends Biochem. Sci. 26: 100-106, 2001. Proc. Natl. Acad. Sci. USA, 94: 3805-3810, 1997.

  An object of the present invention is to effectively create a functional artificial gene or a functional artificial protein, an artificial gene group in which multiple types of motif sequences are randomly polymerized, an artificial protein group encoded by the artificial gene group, and the Providing a production method, and furthermore, at least part of it encodes a motif sequence, can randomly polymerize DNA that is not subject to high overall sequence similarity, and avoids the appearance 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 can randomly polymerize DNA sequences having a long translation reading frame (ORF) by randomly polymerizing the single-stranded DNA.

  As a result of intensive studies to solve the above problems, the inventor constructed at least three kinds of single-stranded DNAs containing sequences encoding arbitrary motif sequences, and determined the terminal sequence of the single-stranded DNA sequence. Complementary base pair combinations by introducing base sequences that can form complementary base pairs with each other, mixing the various single-stranded DNAs at an arbitrary ratio, and allowing DNA polymerase to act It is possible to proceed a random polymerization reaction between DNAs encoding many types of motif sequences depending on the frequency of base pair formation depending on the concentration of each of the various single-stranded DNAs, etc. A DNA sequence having a long translational reading frame (ORF) and random recombination between DNAs encoding a motif sequence in at least a part thereof, which is not subject to high sequence similarity. It found that dam polymerization is possible, thereby completing the present invention.

  That is, the present invention constructs at least three or more DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and part of the terminal sequences of the single-stranded DNA sequence are complementary to each other. A complementary base sequence is introduced so that base pairs can be formed, and 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, thereby encoding a motif sequence. The process consists of proceeding a random polymerization reaction between DNAs to produce an artificial gene group in which a large number of motif sequences are randomly polymerized. As 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.

  In the present invention, 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 preferably 6 bases or more. . At both ends of the complementary base pair, one or more bases that are not paired with the partner base are present in the range of 1 to 3 bases, preferably in the range of 1 to 3, thereby increasing the efficiency of the polymerization reaction by the DNA polymerase. In the present invention, a single-stranded DNA containing a sequence encoding an arbitrary motif sequence is designed based on a functional motif sequence, a structural motif sequence, an artificial peptide sequence obtained by evolutionary molecular engineering, or protein engineering knowledge. A microgene constructed on the basis of the determined sequence. When constructing the microgene, the stop codon is excluded in advance in each translation reading frame within the single-stranded DNA sequence to be synthesized. Can do. Further, in the present invention, by adding a restriction enzyme recognition sequence to a single-stranded DNA encoding many kinds of motif sequences, the frequency of appearance of motif sequences used in random polymerization reactions can be directly monitored by restriction enzyme reactions. In addition, an artificial gene population can be prepared.

  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 is further translated by translating the artificial gene population into an artificial protein. It is possible to produce an artificial protein population in which is inserted randomly. In producing the artificial protein population, in the present invention, an artificial gene population is prepared by randomly polymerizing single-stranded DNAs encoding multiple types of motif sequences without inserting base mutations or deletions. By translating, an artificial protein population in which a large number of motif sequences are randomly inserted can be produced without depending on frame shift. Further, in the present invention, a single-stranded DNA encoding a plurality of motif sequences is randomly polymerized while inserting base mutations and deletions to produce an artificial gene population, and the artificial gene population is translated to produce frame shifts. Depending on the above, an artificial protein population in which a large number of motif sequences are randomly inserted can be prepared. Thus prepared artificial gene populations in which multiple types of motif sequences of the present invention are randomly polymerized, or artificial protein populations into which various motif sequences are randomly inserted, functional genes or functions for those populations. Functional artificial genes or functional artificial proteins can be created by screening sex proteins.

That is, the present invention specifically relates to [1] (1) constructing at least three kinds of DNA sequences of a single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and (2) the single-stranded DNA. Complementary base sequence so that part of the 3 'end sequence of the DNA sequence forms a complementary base pair with each other , and at least one base that does not pair with the partner base exists at both ends of the complementary base pair. (3) Random polymerization reaction between DNAs encoding motif sequences by allowing a DNA polymerase to act on a reaction mixture in which various single-stranded DNAs constructed by the method are mixed at a controlled concentration A method for producing an artificial gene group in which a large number of motif sequences are randomly polymerized, wherein the occurrence frequency of motif sequences used in random polymerization reactions is controlled, and [2] arbitrary mochi By constructing at least 4 types of DNA sequences of single-stranded DNA containing a sequence encoding a sequence, and allowing a DNA polymerase to act on a reaction mixture in which the single-stranded DNA is mixed at a controlled concentration, A method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized, or [ 3 ] complementary bases, wherein random polymerization reaction is performed between DNAs encoding motif sequences The number of pairs is 6 bases or more, and a method for producing an artificial gene group in which a large number of motif sequences according to [1] or [2] are randomly polymerized, or [ 4 ] complementary base pairs The multiple species motif sequence according to any one of [1] to [3] above, wherein the bases that do not pair with the partner base are present at both ends in any number of bases 1 to 3. Heavy The method for manufacturing a artificial gene population, and characterized in that to control the frequency of occurrence of [5] also by adjusting the combination of complementary base pairs of single-stranded DNA of a wide, motif sequences utilized by random polymerization reaction A method for producing an artificial gene group in which the multiple motif sequences according to any one of [1] to [ 4 ] are randomly polymerized, and [ 6 ] the concentration of various single-stranded DNAs added to the random polymerization reaction system. The method comprises a method for producing an artificial gene population in which a large number of motif sequences according to any one of [1] to [ 5 ] are randomly polymerized, which varies within a range of 0.2 μM to 20 μM.

In the present invention, [ 7 ] various constructed single-stranded DNAs are mixed with the reaction solution, and the complementary base sequence of the introduced single-stranded DNA is used as a template to act from the 3 ′ end to the 5 ′ end. A large number of the above-mentioned [1] to [ 6 ], wherein a random polymerization reaction is allowed to proceed between DNAs encoding motif sequences by the action of a thermostable DNA polymerase having an exonuclease activity. [ 8 ] A single-stranded DNA containing a sequence encoding an arbitrary motif sequence has been obtained from functional motif sequences, structural motif sequences, and evolutionary molecular engineering. [1] characterized in that it is a microgene constructed based on an artificial peptide sequence or a sequence designed based on protein engineering knowledge. ] To [ 7 ] a method for preparing an artificial gene population in which a large number of motif sequences are randomly polymerized, or [ 9 ] a single-stranded DNA sequence to be synthesized in the course of constructing a microgene. A method for producing an artificial gene population in which a large number of motif sequences according to [ 8 ] are randomly polymerized and [ 10 ] a sequence encoding an arbitrary motif sequence, wherein a stop codon is excluded in frame By adding a restriction enzyme recognition sequence to a single-stranded DNA that encodes multiple types of motif sequences, single-stranded DNA can directly monitor the frequency of occurrence of motif sequences used in random polymerization reactions. Construction of an artificial gene population in which a large number of motif sequences according to any one of the above [1] to [ 9 ] are randomly polymerized, which is constructed METHODS or Ranaru.

Furthermore, the present invention provides a host cell with a vector incorporating a gene produced by a method for producing an artificial gene population in which a large number of motif sequences according to any one of [ 11 ] and [1] to [ 10 ] are randomly polymerized. [ 12 ] Inserting nucleotide mutations and deletions into single-stranded DNAs encoding multiple types of motif sequences, or a method for preparing artificial protein populations that are randomly inserted with various motif sequences characterized by introduction and expression A plurality of motif sequences according to the above [ 11 ], wherein the artificial gene population is prepared by random polymerization and the artificial gene population is translated to obtain a diverse molecule population independent of frame shift. A method for preparing randomly inserted artificial protein populations, [ 13 ] single-stranded DNA encoding multiple motif sequences, inserting nucleotide mutations and deletions, A variety of motif sequences according to the above [ 11 ], wherein an artificial gene population is produced by random polymerization and a diversity molecular population is obtained depending on a frame shift by translating the artificial gene population. inserted a manufacturing method and an artificial protein population [14] the [1] to [10] produced by the manufacturing method of any one of a number species motif sequence was randomly polymerized artificial gene cluster, or the A functional artificial gene or functional artificial protein characterized by screening a functional gene or a functional protein for an artificial protein population into which various motif sequences according to any one of [11] to [13] are randomly inserted It consists of the creation method.

It is a figure which shows the outline of artificial protein population preparation using the motif arrangement | sequence of this invention. In the Example of this invention, it is a figure which shows the electrophoretic diagram of the design (upper) of single stranded DNA containing a restriction enzyme recognition sequence, and random polymerization reaction. The 3 ′ end of KY-1354 was designed to form a complementary base pair with the 3 ′ end of KY-1355-KY-1357. As a result of electrophoresis, the polymerization reaction did not proceed with KY-1354 alone (lane 1), but the polymerization reaction proceeded by mixing DNA in various combinations (lanes 2-6). M represents a marker DNA. In the Example of this invention, it is a figure which shows the result of confirmation of multiple types of DNA random polymerization by a restriction enzyme reaction. It is a figure which shows the arrangement | sequence of the DNA polymerization product which has a restriction enzyme recognition motif in the Example of this invention. In the Example of this invention, it is a figure which shows the design (A) and design (B) of single stranded DNA which has BH1-BH4 motif which exists in the apoptosis control type | mold protein. In design A, the 3 ′ end of KY-1372 was designed to form a complementary base pair with the 3 ′ ends of KY-1374, KY-1375, and KY-1377. In design B, the 3 ′ ends of KY-1372 and KY-1379 were designed to form complementary base pairs with the 3 ′ ends of KY-1374 and KY-1375. It is a figure which shows the electrophoretic diagram of the single strand DNA polymerization reaction containing the BH1-BH4 motif in the Example of this invention. . It was confirmed that the four types of single-stranded DNA synthesized in Design A and Design B both proceeded with the polymerization reaction. In the Example of this invention, it is a figure which shows the result of the confirmation of the single strand DNA random polymerization containing the BH1-BH4 motif by a restriction enzyme reaction. It was confirmed from the electrophoretic diagram after the restriction enzyme reaction that each motif was polymerized randomly. a, b and c are diagrams showing sequences of an artificial gene population obtained by design A and an artificial protein population which is a translation product thereof, according to an embodiment of the present invention. It is a figure which shows the result of the confirmation of the expression in the mammalian cell of the artificial protein in the Example of this 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. In the Example of this invention, it is a figure which shows the arrangement | sequence of the artificial protein group which is the artificial gene group obtained by the design B, and its translation product. In the Example of this invention, it is a figure which shows the result of the electrophoretic diagram of the design (C, D) of a single strand DNA which has BH1-BH4 motif which exists in an apoptosis control type protein, and a single strand DNA polymerization reaction. In design (C), four types of single-stranded DNA (KY-1372, KY-1389, KY-1391) were mixed in equal amounts and polymerized, whereas in design (D), KY-1372: KY- 1389: KY-1390: KY-1391 = 2: 2: 0.4: 0.3 The ratio of KY-1372 and KY-1389 encoding the BH4 and BH3 motifs was increased and added to the polymerization reaction system. It was. In the Example of this invention, it is a figure which shows the confirmation result of the single strand DNA random polymerization containing the BH1-BH4 motif by a restriction enzyme reaction. In designs C and D, partial cleavage by three restriction enzymes was confirmed. FIGS. 4A and 4B are diagrams showing sequences of an artificial gene population obtained by design C and an artificial protein population that is a translation product of the artificial gene population in Examples of the present invention. a, b, and c are diagrams showing sequences of an artificial gene population obtained by design D and an artificial protein population that is a translation product thereof, according to an example of the present invention. In the Example of this invention, it is a figure which shows the result of having compared the appearance frequency of the BH1-BH3 motif in an artificial protein population (C) and an artificial protein population (D). In the protein population (D) prepared by increasing the concentration of single-stranded DNA (KY-1389) encoding the BH3 motif, the appearance frequency of the BH3 motif increased. It is a figure which shows the various localization patterns in the human cancer cell of the artificial protein in the Example of this invention. In the Example of this invention, it is the figure which quantified the number of the apoptotic cells induced | guided | derived to the breast cancer cell line MCF-7 by introduction | transduction of the gene which codes the acquired artificial protein D29. The effect was equivalent to the natural apoptosis-inducing protein Bax, and about 30% of the cells were apoptosis-positive cells. It was found that such apoptosis-positive cells were not confirmed in the cells into which the gene for the control artificial protein A10 was introduced. In the Example of this invention, it is a figure which shows that apoptosis is induced | guided | derived effectively in the cell which expresses D29. A double staining by a confocal microscope (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. In the Example of this invention, it is the figure which quantified the inhibitory activity of the growth inhibition induced | guided | derived to the cervical cancer cell line HeLa by introduction | transduction of the gene which codes the obtained artificial protein A10 or D16. Although the number of HeLa cells is decreased by natural apoptosis-inducing protein BIM, or anticancer drugs etoposide (VP-16) or staurosporine (STS) (quantified by WST-1, see control pcDNA), the introduction of A10 or D16 gene Proliferative activity was partially restored. This growth inhibitory inhibitory effect could be observed in the same manner even in cells into which a plasmid encoding the gene of Bcl-xL, which is a natural apoptosis inhibitory protein, was introduced. In the Example of this invention, it was the figure which showed that the obtained artificial protein A10 or D16 can suppress the apoptosis induced by STS. 24 hours after the gene transfer, the medium was replaced with a medium containing 125 nM STS, and the cells were further cultured for 23 hours. As a result of staining the apoptotic cells by the TUNEL method (red), apoptosis was effectively induced in the cells into which the control pcDNA or D29 was introduced, whereas in the cells into which the apoptosis-inhibiting Bcl-xL gene was introduced, TUNEL The number of positive cells decreased. Apoptosis was partially suppressed in the cells into which the A10 or D16 gene was introduced. In the Example of this invention, it is the figure which showed that the cell which expressed the artificial protein D16 acquired becomes apoptosis negative. As in FIG. 20, after fixing the cells 23 hours after STS administration, the artificial protein was stained green with Myc primary antibody and FITC secondary antibody, and apoptotic cells were double stained with TMR-red label (red). . It was found that apoptosis was suppressed in cells expressing D16 or Bcl-xL. It was confirmed that apoptosis was positive in cells expressing D29.

  The present invention relates to an artificial gene population in which a large number of motif sequences are randomly polymerized for the purpose of effectively creating a functional artificial gene or functional artificial protein, an artificial protein population encoded by the artificial gene population, and a method for producing the same To provide. The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to the present invention includes (1) constructing at least three or more DNA sequences of a single-stranded DNA containing a sequence encoding an arbitrary motif sequence. (2) introducing complementary base sequences so that part of the terminal sequences of the single-stranded DNA sequences can form complementary base pairs with each other, and (3) various single-stranded DNAs constructed by the method DNA polymerase is allowed to act on the reaction mixture mixed at an arbitrary ratio, and a random polymerization reaction between the DNAs encoding the motif sequences is allowed to proceed. It consists of making.

  The method for producing the artificial gene population of the present invention will be specifically described along with the operations with examples. As shown in FIG. 1, first, a plurality of types (multiple types) of single-stranded DNAs containing a target motif sequence are synthesized. In the present invention, synthesis is performed so that a part of the 3′-side sequences of plural kinds of single-stranded DNAs can form complementary base pairs. The number of complementary base pairs is preferably 6 bases or more. However, synthesis is performed so that one or more bases (preferably 1-3 bases) that do not pair with the partner base exist at both ends of the complementary base pair. By this operation, the efficiency of the polymerization reaction can be increased. Moreover, there is no restriction | limiting in the kind, arrangement | sequence, length, and combination of multiple types of single stranded DNA to add, The multiple types of single stranded DNA which codes arbitrary sequences can be utilized for this reaction. Furthermore, there is no limitation on the combination of single-stranded DNAs that form complementary base pairs.

  For example, when the reaction is performed using four types of single-stranded DNA, the 3 ′ sequences of the remaining three types of single-stranded DNA form complementary sequences with respect to the 3 ′ sequence of one type of single-stranded DNA. Alternatively, it may be designed so that the 3 ′ sequences of the remaining two types of single-stranded DNA can form a complementary sequence equally to two types of single-stranded DNA having a homologous 3 ′ end sequence. Good. The concentration of single-stranded DNA added to the reaction system can vary from 0.2 μM to 20 μM, and the concentration of single-stranded DNA desired to be used for the polymerization reaction is increased and added to the reaction system. That is, by adjusting the combination of complementary base pairs and the single-stranded DNA concentration, the appearance frequency of the motif sequence used in the random polymerization reaction can be controlled. In designing a plurality of types of single-stranded DNAs, it is desirable that the length and Tm of each sequence be close to each other.

  These complementary regions of single-stranded DNA function as a template for PCR reaction using a thermostable polymerase. When a PCR reaction is carried out by acting a thermostable DNA polymerase containing an exonuclease activity that acts in the direction from the 3 ′ end to the 5 ′ end in the presence of such multiple single-stranded DNAs (3 ′ → 5 ′), A double-stranded DNA polymer population in which a plurality of types of single-stranded DNA containing a motif sequence are randomly extended and linked is synthesized. The PCR conditions are as follows: DNA polymerase (for example, Vent polymerase) is used, for example, at 94 ° C. for 10 seconds and at 55-75 ° C. for 60 seconds until DNA polymerization can be confirmed (30 to 65 cycles). ) Finally, carry out at 69 ° C. for 7 minutes. Before performing the above reaction, it is preferable to further carry out the reaction at 94 ° C. for 10 minutes and at 69 ° C. for 10 minutes. Furthermore, the elongation reaction temperature is preferably set to a value close to Tm-5 ° C. of the single-stranded DNA.

  In this way, complementary regions of multiple types of single-stranded DNA form base pairs in a random combination, and as a result of repeating extension and polymerization reactions, the DNA encoding the motif sequence is randomly shuffled. A double-stranded artificial DNA population is created. The method of the present invention is different from the method for producing a polymer microgene polymer invented by the present inventor (Japanese Patent No. 3415995; Proc. Natl. Acad. Sci. USA, 94: 3805-3810, 1997). A sequence diversity population can be generated without depending on base substitution, insertion, or deletion that occurs between DNA strands during the polymerization reaction.

  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, as described above, complementary bases of various single-stranded DNAs can be used. It is possible to control the appearance frequency of the motif sequence used in the random polymerization reaction by adjusting the combination of pairs and / or the concentration of each type of single-stranded DNA. In addition, in the method for producing an artificial gene population of the present invention, a large number of motif sequences are arbitrarily selected and randomly polymerized to produce an artificial gene population and an artificial protein population. When constructing artificial gene populations, constructing 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 produce an artificial gene population by predicting a certain degree of function with reasonableness.

  An artificial protein population produced by the method for producing an artificial gene population of the present invention, into which various motif sequences are randomly inserted, is introduced into a host cell and expressed by introducing a vector incorporating the gene into various motifs. Artificial protein populations with randomly inserted sequences can be generated. Vectors, host cells, and gene expression methods known in this field are used as vectors, host cells, and gene expression methods used in the production of the artificial protein population. An artificial gene population in which a large number of motif sequences prepared by the method of the present invention are randomly polymerized or an artificial protein population into which various motif sequences are randomly inserted is a functional gene for the artificial gene population or artificial protein population. Alternatively, functional artificial genes or functional artificial proteins can be created by screening functional proteins.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
Prior to describing the examples, 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. It consists of five schemes: study, (4) transformation and sequencing of random polymers into E. coli, and (5) expression of artificial proteins and screening of functional artificial proteins in E. coli or mammalian cells. When it is desired to obtain an artificial gene population (when the expression of an artificial protein is not required), the reaction is completed in steps (1) to (4). In this example, an artificial gene and an artificial protein population are generally prepared by random polymerization of four types of motif sequences. However, the number of motifs that can be used in the reaction is not limited to four types (the number of the larger number is also theoretical). Is possible). Specific experimental examples are given below.

[Preparation of artificial gene population by random polymerization of four kinds of single-stranded DNA containing restriction enzyme recognition motif]
(Design of single-stranded DNA containing restriction enzyme recognition sequence)
As single-stranded DNA containing a functional sequence, four types of single-stranded DNA containing a restriction enzyme recognition sequence were synthesized as follows. KY-1354 (SEQ ID NO: 5'-GGG GAATTC GGC GGGA-3 '), KY-1355 (SEQ ID NO: 5'-GGG AAGCTT ACCCGCCA-3'), KY-1356 (SEQ ID NO: 5'- GGG TCTAGA ACCCGCCA-3 '), KY-1357 (SEQ ID NO: 5'-GGG AGATCT ACCCGCCA-3'). Here, the fourth to ninth sequences (underlined) of KY-1354, KY-1355, KY-1356, and KY-1357 are the restriction enzyme recognition sequences GAATTC (EcoRI), AAGCTT (HindIII), TCTAGA ( XbaI) and AGATCT (BglII). In addition, the 10th to 15th 6 bases (5'-GGCGGG-3 ') of KY-1354 are the 11th to 16th 6 bases (5'-GGCGGG-3') of KY-1355, KY-1356 and KY-1357 (5 ' It was designed to form complementary base pairs with -CCCGCC-3 '). In addition, adenine (A) was introduced at the 3 ′ end of KY-1355, KY-1356, and KY-1357 and at the 10th position so that a complementary base pair of 6 bases or more could not be formed with KY-1354. In addition, the four types of single-stranded DNA are such that the lengths (16-17 bases), Tm (˜58 ° C.), and GC content (65-69%) of these four types of single-stranded DNAs are close to each other. Designed.

(Random polymerization reaction of single-stranded DNA containing restriction enzyme recognition sequence)
First, 50 μL of the single-stranded DNA random polymerization reaction solution was prepared. This reaction solution contains 10 x ThermoPol Reaction Buffer (NEW ENGLAND BioLabs, 1x ThermoPol Reaction Buffer: 20 mM 2-amino-2-hydroxymethyl-1,3, -propanediol hydrochloride (hereinafter tris-hydrochloric acid) pH 8.8, 2 Vent DNA polymerases (2 units / μL, NEW ENGLAND BioLabs) with 5 μL, 350 μM dNTP, 3 ′ → 5 ′ exonuclease activity, 10 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X-100) 6 μL and 4 types of single-stranded DNA (KY-1354-KY-1357) were mixed in the following 6 types. (1) KY-1354: 20 pmol only (1 DNA), (2) KY-1354: 20 pmol + KY-1355: 20 pmol (2 DNA), (3) KY-1354: 20 pmol + KY-1356: 20 pmol (DNA2 Species), (4) KY-1354: 20 pmol + KY-1357: 20 pmol (2 types of DNA), (5) KY-1354: 20 pmol + KY-1355: 10 pmol + KY-1356: 10 pmol (3 types of DNA), (6) KY-1354: 20 pmol + KY-1355: 6.7 pmol + KY-1356: 6.7 pmol + KY-1357: 6.7 pmol (4 types of DNA).

  These reaction solutions were put into a 200 μL thin wall PCR tube (Toyobo, Osaka), and a polymerization reaction was performed between the above-mentioned single-stranded DNAs by a temperature cycle with a thermal cycler PCR-2400 (Perkin Elmer, Norwalk). As a pretreatment for the polymerization reaction, a reaction was performed at 94 ° C. for 10 minutes and at 69 ° C. for 10 minutes. The polymerization reaction temperature was set to 55 ° C. in consideration of being below the Tm value (58 ° C.) of KY-134-1357. 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. 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 a 1.0% TAE agarose gel (Agarose ME Iwai Chemical Tokyo) and a Mupid electrophoresis apparatus (Cosmo Bio, Tokyo) to confirm the polymerization reaction (FIG. 2). ). By this reaction, it was confirmed that DNA of several kilobase pairs or more was polymerized by this method.

(Confirmation of random DNA polymerization by restriction enzyme reaction)
In order to confirm random polymerization of single-stranded DNA containing a restriction enzyme recognition sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. 50 μL of five types of reaction solutions containing different restriction enzymes were prepared as follows. 5 μL of the above DNA polymerization reaction solution, 10 × restriction enzyme buffer B (HindIII, EcoRI), M (BglII), or H (XbaI) 5 μL (Roche Diagnostics, Basel), restriction enzyme (Roche Diagnostics) Basel) (1) no enzyme, (2) HindIII (10 units / μL) 2 μL, (3) BglII (10 units / μL) 2 μL, (4) XbaI ( 40 units / μL) was added at 2 μL, and (5) EcoRI (10 units / μL) was added at 2 μL. The cleavage reaction was allowed to proceed at 37 ° C. for 90 minutes. In order to confirm the DNA cleavage reaction, 5 μL was extracted from the 50 μL reaction solution and subjected to electrophoresis (100 V, 15 minutes) using a 1.0% TAE agarose gel. (Figure 3). As shown in FIG. 3, the DNA polymer in 1-2 is cleaved with a restriction enzyme sequence contained in any single-stranded DNA used for the polymerization reaction, and a DNA polymer composed of a plurality of types of single-stranded DNA. Confirmed that it was cleaved by multiple restriction enzymes. From this, it was found that random polymerization and extension reaction occur between single-stranded DNAs containing restriction enzyme recognition motifs.

(Transformation to E. coli and sequencing of DNA polymerization products)
The random polymerization reaction solution between the above DNA sequences was cloned and sequenced using a commercially available Zero Blunt TOPO PCR Cloning Kit (Invitrogen, CA). The ligation reaction is performed on a reaction scale of 6 μL, and this reaction solution contains 1 μL of DNA polymerization product, 1 μL of Salt Solution, 1 μL of TOPO vector, and 3 μL of ultrapure water. After the reaction solution was reacted at room temperature for 30 minutes, 2 μL of the reaction solution was mixed with Top-10 cell (Invitrogen) and transformed. 18 clones containing the insert were selected, the plasmid was purified by QIAGEN mini kit (Qiagen), and the sequence was determined by the capillary sequencer CEQ2000XL DNA analyzer (Beckman) by the dye termination method using DTCS cycle sequence reaction kit (Beckman). . One clone (EHB # 7: SEQ ID NO: 5) obtained from a polymer obtained by mixing three kinds of single-stranded DNAs of KY-1354, KY-1355, and KY-1357, KY-1354, KY-1355, FIG. 4 shows three clones (EHBX1 to EHBX3: SEQ ID NOs: 6, 7, and 8) obtained from a polymer in which four kinds of single-stranded DNAs of KY-1356 and KY-1357 were mixed. As shown in FIG. 4, an artificial gene population in which DNAs containing 3 or 4 types of restriction enzyme recognition sequences were randomly polymerized at an arbitrary ratio could be obtained.

[Production of artificial protein population (A) by random polymerization of BH1-BH4 motif sequence present in apoptosis-regulated protein]
(Selection of apoptosis control motif BH1-BH4)
The Bcl-2 family protein family is known as a natural protein that controls programmed spontaneous cell death (apoptosis). Bcl-xl belonging to the family is known to be important for suppressing apoptosis, and Noxa is important for promoting apoptosis. Here, we try to use the partial sequence of BH1, BH2, and BH4 motifs in human Bcl-xl protein and the partial sequence of BH3 motif in human Noxa protein as motif sequences for creating artificial protein populations. It was. Portions encoding the following peptide sequences were selected from the BH1-BH4 motif, respectively. BH1: ELFRDGVN, BH2: ENGGWDTF, BH3: LRRFGDKLN, BH4: RELVVDFL.

(Design of single-stranded DNA encoding BH1-BH4 motif sequence: A)
In order to create an artificial protein group A composed of BH1-BH4 motifs, a multi-functional nucleotide sequence design method invented by Shiba et al. ). Here, four types of single-stranded DNA are designed in another reading frame of the gene encoding the BH1-BH4 motif so as to encode a peptide having a high α-helix property to help stabilize the artificial protein. (FIG. 5). Here, KY-1372 (SEQ ID NO: 9), KY-1377 (SEQ ID NO: 10), KY-1375 (SEQ ID NO: 11), and KY-1374 (SEQ ID NO: 12) are the restriction enzyme recognition sequence GTCGAC (SalI), It was designed to include AAGCTT (HindIII), AGATCT (BglII), and GAATTC (EcoRI) (FIG. 5, design A, underlined).

  Also, the 3 ′ end 7 bases (5′-GGCGGGG-3 ′) of KY-1372 are complementary to the 3 ′ end 7 bases (5′-CCCCGCC-3 ′) of KY-1377, KY-1375, and KY-1374. It was designed to form a target base pair. In addition, adenine (A) and cytosine (C) were inserted into the 9 ′ base from the 3 ′ end and the 3 ′ end of KY-1372 so that an interaction of 7 base pairs or more was not formed. Furthermore, when designing these four types of single-stranded DNA, the length (40-45), GC base content (55-65%), intramolecular hairpin formation ability, and Tm are close to each other. Designed.

(Random polymerization reaction of single-stranded DNA containing BH1-BH4 motif)
First, 50 μL of the single-stranded DNA random polymerization reaction solution was prepared. The composition of this reaction solution is 5 μL of 10 × ThermoPol Reaction Buffer (same as in Example 1), 350 μM dNTP, 2.6 μL of Vent DNA polymerases (2 units / μL, NEW ENGLAND BioLabs), and 4 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. As a pretreatment for the polymerization reaction, a reaction was performed 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.

  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 a 1.0% TAE agarose gel (Agarose ME Iwai Chemical Tokyo) and a Mupid electrophoresis apparatus (Cosmo Bio, Tokyo) to confirm the polymerization reaction (FIG. 6). ). Further, as a result of optimization of the reaction so that single-stranded DNAs encoding BH1-BH4 motifs were randomly polymerized at similar frequencies, KY-1374: KY-1375: KY-1377: KY-1372 = The single-stranded DNA amount ratio was set to 2: 1: 100: 100. By this reaction, it was confirmed that DNA of several kilobase pairs or more was polymerized by this method.

(Confirmation of random DNA polymerization containing BH1-BH4 motif by restriction enzyme reaction)
In order to confirm the random polymerization of the single-stranded DNA containing the BH1-BH4 motif sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. 50 μL of five types of reaction solutions containing different restriction enzymes were prepared as follows. 5 μL of DNA polymerization reaction solution, 10 × restriction enzyme buffer H (SalI, or EcoRI), or 5 μL of M (HindIII, or BglII), (Roche Diagnostics, Basel), restriction enzyme (Roche Diagnostics, Basel) (2) SalI (40 units / μL) 2 μL, (3) HindIII (10 units / μL) 2 μL, (4) EcoRI (10 units / μL) Was added under the condition of 2 μL, and (5) BglII (10 units / μL) was added at 2 μL. The cleavage reaction was allowed to proceed at 37 ° C. for 90 minutes. In order to confirm the DNA cleavage reaction, 5 μL was extracted from the 50 μL reaction solution and subjected to electrophoresis (100 V, 15 minutes) using a 1.0% TAE agarose gel (FIG. 7). As shown in FIG. 7, it was found that a DNA polymer composed of single-stranded DNA containing a BH1-BH4 motif sequence was partially cleaved by all the restriction enzymes used. From this, a random polymerization reaction was confirmed between the four types of single-stranded DNA.

(Transformation to E. coli, screening, and sequencing of DNA polymerization products)
The DNA polymerization reaction product containing the BH1-BH4 motif was cloned and sequenced using a commercially available pcDNA3.1Directional TOPO Expression Kit (Invitrogen, CA). Here, an artificial gene was ligated to a mammalian expression vector (pcDNA3.1D / V5-His-TOPO, Invitrogen) for screening of a functional artificial protein in a mammalian cell system. The ligation reaction is performed on a 6 μL scale, and this reaction solution contains 1 μL of DNA polymerization reaction product, 1 μL of Salt Solution, 1 μL of TOPO vector, and 3 μL of ultrapure water. After the reaction solution was reacted at room temperature for 30 minutes, 2 μL of the reaction solution was mixed with Top-10 cell (Invitrogen), allowed to stand on ice for 30 minutes, and transformed. From the screening PCR, 10 types of clones containing the insert were selected, the plasmid was purified by QIAGEN mini kit (Qiagen), and the capillary sequencer CEQ2000XL DNA analyzer (Beckman) by the dye termination method using DTCS cycle sequence reaction kit (Beckman). ) To determine the sequence (SEQ ID NOs: 13-42).

  The gene sequence and amino acid sequence of the obtained artificial protein are shown in FIG. 8 (SEQ ID NOs: 13-42). From FIG. 8, it was found that an artificial protein population in which artificial DNAs encoding BH1-BH4 motifs were randomly inserted could be created. In addition, in “Design A of single-stranded DNA containing BH1-BH4 motif sequence”, a motif is introduced depending on the frame shift that occurs during DNA polymerization reaction in order to create a library with higher sequence diversity. The gene was designed in such a way (if the frame shift does not occur, the reading frame will shift). As shown in FIG. 8, many reading frame arrangements different from the reading frame encoding the BH1-BH4 motif appeared. As described above, in this method, a library can be prepared using a plurality of motif sequences, but it is possible to increase sequence diversity by using a sequence of another translation reading frame.

(Confirmation of expression of artificial protein in mammalian cells)
In order to confirm the expression of the sequenced artificial protein in mammalian cells, a plasmid encoding the gene of MxA6 (see FIGS. 8, a, b, and c) is used as MCF-7, which is one of representative human breast cancer cell lines. The gene was introduced into. As a control, a 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 the histidine-tagged artificial protein was confirmed by immunostaining with Alexa-His-antibody (Qiagen) (FIG. 9). As shown in FIG. 9, the expression of the artificial protein was observed in the cells into which the MxA6 gene was introduced, whereas the expression was not observed in the control, and the localization thereof was found to match that of the mitochondria. Using such mammalian cell systems or E. coli expression systems, expression of artificial protein populations and screening of functional artificial proteins can be performed.

[Production of artificial protein population (B) by random polymerization of BH1-BH4 motif sequence present in apoptosis-regulated protein]
In the production of the artificial protein population (A) of Example 2, KY-1372 encoding a BH4 motif is a single-stranded DNA (KY-1377, KY-1375, KY-1374) encoding a BH3, BH2 or BH1 motif. Designed to form complementary base pairs (1: 3 correspondence). In the production of the artificial protein population (B), the single-stranded DNA encoding the BH3 or BH4 motif is designed to form a complementary base pair with the single-stranded DNA encoding the BH2 or BH1 motif (2: 2). (Correspondence) (Figure 5). Thus, the ratio of single-stranded DNAs forming complementary base pairs can be freely adjusted. Other methods are basically the same as the production of the artificial protein population (A).

(Design of single-stranded DNA containing BH1-BH4 motif sequence: B)
In order to create an artificial protein population B composed of BH1-BH4 motifs, four types of single-stranded DNAs KY-1372, KY-1379 (BH3 motif, SEQ ID NO: 43), KY-1375, and KY-1374 were used as Shiba et al. It was designed by the multifunctional base sequence design method invented in (JP 2001-352990). Here, KY-1372, KY-1379, KY-1375, and KY-1374 were designed to include restriction enzyme recognition sequences GTCGAC (SalI), CTCGAG (XhoI), AGATCT (BglII), and GAATTC (EcoRI), respectively. (Figure 5 Design B, underlined). Also, KY-1372 or KY-1379 3 ′ end 7 bases (5′-GGCGGGG-3 ′) is complementary to KY-1375 and KY-1374 3 ′ end 7 bases (5′-CCCCGCC-3 ′) It was designed to form a target base pair. In addition, adenine (A) and cytosine (C) were inserted into the 9th base from the 3 ′ end and the 3 ′ end of KY-1372 and KY-1379, respectively, so that an interaction of 7 base pairs or more was not formed. .

(Random polymerization reaction of single-stranded DNA containing BH1-BH4 motif)
First, 50 μL of the single-stranded DNA random polymerization reaction solution was prepared. Except that XhoI was used for examining the polymerization reaction of KY-1379 encoding XhoI, the preparation was performed in the same manner as in the section on preparation of artificial protein population (A). The polymerization reaction was confirmed by 1% agarose gel.

 (Figure 6). Moreover, as a result of optimizing the reaction so that the BH1-BH4 motifs were randomly polymerized at similar frequencies, KY-1374: KY-1375: KY-1379: KY-1372 = 4: 1: 10: 1 Thus, the BH1-BH4 amount ratio was set.

(Confirmation of random DNA polymerization containing BH1-BH4 motif by restriction enzyme reaction)
In order to confirm the random polymerization of the single-stranded DNA containing the BH1-BH4 motif sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. The reaction is the same as in the section on the production of the artificial protein population (A) except that XhoI was added to the restriction enzyme. For each of the five reaction solutions, (1) no enzyme, (2) SalI (40 units / μL) 2 μL, (3) XhoI (10 units / μL) 2 μL (4) EcoRI (10 units / μL) 2 μL, (5) BglII (10 units / μL) was added under the condition of 2 μL. The cleavage reaction was allowed to proceed at 37 ° C. for 90 minutes. In order to confirm the DNA cleavage reaction, 5 μL was extracted from the 50 μL reaction solution and subjected to electrophoresis (100 V, 15 minutes) using a 1.0% TAE agarose gel. (Figure 7). As shown in FIG. 7, it was found that a DNA polymer composed of single-stranded DNA containing a BH1-BH4 motif sequence was partially cleaved by all the restriction enzymes used. From this, a random polymerization reaction of four kinds of single-stranded DNAs could be confirmed.

(Transformation to E. coli, screening, and sequencing of DNA polymerization products)
Transformation and sequencing were performed in the same manner as in the section on the production of the artificial protein population (A). The gene sequence and amino acid sequence of the artificial protein are shown in FIG. 10 (SEQ ID NOs: 44-53).

[Production of artificial protein population (C) by random polymerization of BH1-BH4 motif sequence present in apoptosis-regulated protein]
In the production of the artificial protein populations (A, B) in Example 2 and Example 3, the BH1-BH4 motif is an artificial protein depending on the frame shift caused by random base deletion or insertion occurring 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). Here, the design of the single-stranded DNA was modified so that the BH1-BH4 motif was randomly polymerized without depending on the frame shift. Other methods are basically the same as the production of the artificial protein population (A). In Example 4, it is expected that more BH1-BH4 motifs appear in the artificial protein population without depending on the frame shift.

(Design of single-stranded DNA containing BH1-BH4 motif sequence: C)
In order to create an artificial protein group C composed of BH1-BH4 motifs, 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 in which CC is added to the 5 ′ end of KY-1377, KY-1374, and KY-1375, respectively. As a result, 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 contain restriction enzyme recognition sequences GTCGAC (SalI), AAGCTT (HindIII), GAATTC (EcoRI), and AGATCT (BglII), respectively (FIG. 11). Design C, underlined). In addition, the 3 ′ end 7 bases (5′-GGCGGGG-3 ′) of KY-1372 has a complementary base pair with the 3 ′ end 7 bases (5′-CCCCGCC-3 ′) of KY-1389-KY-1391. Designed to form. In addition, adenine (A) and cytosine (C) were inserted into the 9th base from the 3 ′ end and the 3 ′ end of KY-1372 so that an interaction of 7 base pairs or more was not formed.

(Random polymerization reaction of single-stranded DNA containing BH1-BH4 motif)
50 μL of the single-stranded DNA random polymerization reaction solution was prepared in the same manner as in the preparation of the artificial protein population (A). As for the single-stranded DNA to be added, an equal amount of single-stranded DNA was added to the polymerization reaction system such that KY-1372: KY-1389: KY-1390: KY-1391 = 1: 1: 1: 1. The polymerization reaction was carried out at 72 ° C. and 45 cycles, and the polymerization was confirmed by 1% agarose gel (FIG. 11).

(Confirmation of random DNA polymerization containing BH1-BH4 motif by restriction enzyme reaction)
In order to confirm the random polymerization of the single-stranded DNA containing the BH1-BH4 motif sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. The reaction conditions are the same as in the section for producing the artificial protein population (A). For each of the five reaction solutions, (1) no enzyme, (2) HindIII (10 units / μL) 2 μL, (3) EcoRI (10 units / μL) 2 μL, (4) BglII (10 units / μL) 2 μL It was added on condition that. As shown in FIG. 12, it was found that a DNA polymer composed of single-stranded DNA containing a BH1-BH4 motif sequence was partially cleaved by all the restriction enzymes used. From this, a random polymerization reaction of four kinds of single-stranded DNAs could be confirmed.

(Transformation to E. coli, screening, and sequencing of DNA polymerization products)
Transformation and sequencing were performed in the same manner as in the section on the production of the artificial protein population (A). The gene and amino acid sequence of the artificial protein obtained by Design C are shown in FIGS. 13a and 13b (SEQ ID NOs: 57-74). As shown in FIG. 13, the present inventors succeeded in obtaining an artificial protein population in which four BH1-BH4 motif sequences appear with high frequency.

[Production of artificial protein population (D) by random polymerization of BH1-BH4 motif sequence present in apoptosis-regulated protein]
As a result of the production of the artificial protein population (C) of Example 4, it was found that the obtained protein population had a relatively low frequency of appearance of the BH3 motif. Therefore, in Example 5, the amount ratio of the single-stranded DNA added to the DNA polymerization reaction is as follows: KY-1372: KY-1389: KY-1390: KY-1391 = 2: 2: 0.4: 0.3 The proportion of KY-1372 and KY-1389 encoding BH4 and BH3 motifs was 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, FIG. 12 shows the digestion with restriction enzymes, and FIGS. 14a, 14b, and 14c show the gene and amino acid sequence of the obtained artificial protein (SEQ ID NOs: 75-100), respectively. As shown in FIG. 14, four motifs appeared with high frequency, and in particular, an artificial protein population with a high appearance frequency of BH4-BH3 motif was successfully created. Thus, the ratio of the motif contained in the artificial protein population can be controlled by changing the amount ratio of the single-stranded DNA mixed in the DNA polymerization reaction. FIG. 15 shows a comparison of the appearance frequency of the BH1-BH3 motif in the artificial protein population (C) and the artificial protein population (D).

[Expression and localization diversity of artificial proteins in human cancer cells]
Forty-one clones were randomly selected from the artificial protein populations A, B, C, and D prepared in Examples 2, 3, 4, and 5, and the gene was introduced into the human breast cancer cell line MCF-7. MCF-7 was fixed in methanol 24 hours after gene introduction, and the expression of an artificial protein with a Myc epitope tag (EQKLISEEDL; SEQ ID NO: 101) was confirmed by immunostaining with Myc antibody. As a result, 28 out of 41 clones (68%), the effective expression of the protein could be observed in human cells. In addition, as a result of examining its intracellular localization, it was found that there were those present in the cytoplasm (11 clones), those present in the nucleus (5 clones), those present in mitochondria (5 clones), and others (7 clones). (Fig. 16). As described above, it was found that the artificial protein obtained by this method is effectively expressed in mammalian cells and has the ability to localize to various organelles in the cells.

[Creation of functional artificial proteins that induce cell death in cancer cells]
In 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 1 × 10 ⁴ MCF-7 per well and cultured at 37 ° C. for 24 hours. Thereafter, 0.2 μg of a plasmid encoding the gene of 28 clones was introduced into MCF-7 using Lipofectamine 2000 (Invitrogen). 48 hours after gene introduction, the proliferation activity was quantified using the tetrazolium salt WST-1 which is an indicator of mitochondrial metabolic activity (Roche). As a result of comparison with the growth activity when an empty vector not encoding a control protein was introduced, it was found that one clone out of 28 clones, D29, significantly inhibited the growth of MCF-7 (as a control). About 40% growth inhibitory effect).

  In order to examine whether this growth inhibitory activity is due to programmed spontaneous cell death (apoptosis), TUNEL staining, which is an index for detecting apoptotic cells, was performed (Roche). In TUNEL staining, fragmented DNA in the early stage of apoptosis can be detected with fluorescently labeled nucleotides. As a result, it was found that D29 can induce apoptosis in MCF-7 to the same extent as the natural cell death-inducing protein Bax (FIG. 17). On the other hand, it was found that one of the clones A10 in which cell growth inhibition was not observed by WST-1 could not induce apoptosis in MCF-7. Furthermore, as a result of examining the state of cells expressing the artificial protein in detail using a confocal microscope, a clear correlation between cells expressing D29 and TUNEL positive cells was observed (FIG. 18). Thus, it was found that the artificial protein population prepared by random polymerization of the apoptosis-control motif sequence by this method contains functional artificial proteins that can induce apoptosis in breast cancer cells.

[Creation of cell death-suppressing functional artificial protein using the same protein population]
The clone whose expression was confirmed in Example 6 contained a cell death-suppressing BH4 motif as well as a cell death promoting BH3 motif. Therefore, the possibility of acquiring a cell death-suppressing functional protein having a function opposite to that of the cell death promoting artificial 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 effect of the clones on the growth inhibitory activity of BIM was examined. A 96-well cell culture plate was seeded with 0.5 × 10 ⁴ HeLa per well and cultured at 37 ° C. for 24 hours. Thereafter, 100 ng of a plasmid encoding 28 clone genes and 50 ng of a plasmid encoding BIM were introduced into HeLa using Lipofectamine 2000 (Invitrogen). Proliferation activity was quantified with tetrazolium salt WST-1 48 hours after gene transfer (Roche). As a result of comparison with the growth activity when a vector encoding GFP as a control was introduced, it was found that two clones A10 and D16 out of 28 clones significantly suppressed the growth inhibition by BIM (FIG. 19). ). Such a growth inhibition inhibitory effect could be observed even in cells into which a plasmid encoding the gene of Bcl-xL, which is a natural apoptosis inhibitory protein, was introduced. Furthermore, it was also found that growth inhibition by two types of anticancer agents, etoposide (VP-16) and staurosporine (STS), was partially suppressed in HaLa cells into which A10 and D16 were introduced (FIG. 19).

In order to examine whether this cell death inhibitory activity is due to inhibition of apoptosis, TUNEL staining was performed (Roche). About 1 × 10 ⁵ HeLa cells were transfected with 0.4 μg of the plasmid encoding the artificial protein, 24 hours later, the medium was replaced with a medium containing 125 nM STS, and further incubated for 23 hours. As a result, it was found that apoptosis induced by STS was significantly suppressed in HeLa cells into which A10 or D16 had been introduced (FIG. 20). In contrast, almost all cells into which the plasmids encoding empty vector pcDNA and D29 were introduced were TUNEL positive. Furthermore, as a result of examining the state of cells expressing the artificial protein in detail using a confocal microscope, a clear correlation between cells expressing D16 and TUNEL-negative cells was observed (FIG. 21). This apoptosis inhibitory effect was also observed in cells expressing Bcl-xL, a natural apoptosis inhibitor protein. Thus, it was found that the artificial protein population produced by this method can create not only an apoptosis-inducing type but also a suppressive functional artificial protein.

  The method for producing an artificial gene population of the present invention makes 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 method for producing an artificial gene population. 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 becomes possible to randomly polymerize a plurality of types of DNAs that do not require high sequence similarity over the entire coding sequence of a motif sequence, and a stop codon. By randomly polymerizing single-stranded DNA that avoids the appearance of DNA, random polymerization of DNA sequences having a long translation reading frame (ORF) became possible. Furthermore, arbitrary motif sequences are 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. By randomly selecting many types of motif sequences and randomly polymerizing them to create artificial gene populations and artificial protein populations, we add rational predictability to functionality and randomly select multiple types of motif sequences. It was possible to produce artificial gene populations and artificial protein populations that were polymerized in the same manner.

  From this, the possibility of creating a powerful artificial gene diversity group (library) that is the key to success in the creation of artificial proteins with extraordinary functions that do not exist in nature through evolutionary molecular engineering Increased. Furthermore, in the present invention, the function of multiple motif sequences is selected, the combination of complementary base pairs of various single-stranded DNAs and / or the concentration of each of various single-stranded DNAs are adjusted, and randomly selected. It is possible to control the appearance frequency of motif sequences used in polymerization reactions, and to create artificial gene populations by predicting certain functions and controlling random polymerization reactions. became.

Claims (14)

(1) constructing at least three kinds of DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and (2) part of 3 ′ terminal sequences of the single-stranded DNA sequences are mutually A complementary base sequence was introduced so that one or more bases forming a complementary base pair and not paired with the other base existed at both ends of the complementary base pair , and (3) constructed by the method Appearance of motif sequences that are used in random polymerization reactions by random polymerization reaction between DNAs encoding motif sequences by allowing DNA polymerase to act on a reaction mixture in which various single-stranded DNAs are mixed at controlled concentrations A method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized, wherein the frequency is controlled to proceed. Constructing at least four kinds of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and allowing DNA polymerase to act on a reaction mixture in which the single-stranded DNA is mixed at a controlled concentration The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to claim 1, wherein random polymerization reaction between DNAs encoding the motif sequences is allowed to proceed. The number of complementary base pairs is 6 bases or more, The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to claim 1 or 2 . The multi-motif sequence according to any one of claims 1 to 3, wherein a base that does not pair with the partner base is present at both ends of the complementary base pair in any number of bases of 1 to 3. A method for producing a randomly polymerized artificial gene population. Adjust the combination of complementary base pairs of single-stranded DNA of a wide, numerous according to any one of claims 1-4, characterized in that to control the frequency of occurrence of the motif sequences utilized by random polymerization reaction A method for producing an artificial gene population in which seed motif sequences are randomly polymerized. The multiple motif sequences according to any one of claims 1 to 5 , wherein the concentration of various single-stranded DNAs added to the random polymerization reaction system varies within a range of 0.2 to 20 µM. Method for producing artificial gene populations. Heat-resistant DNA containing exonuclease activity acting from the 3 ′ end to the 5 ′ end using the constructed single-stranded DNA mixed with the reaction solution and using the complementary base sequence of the introduced single-stranded DNA as a template The artificial gene population in which the multiple motif sequences according to any one of claims 1 to 6 are randomly polymerized by allowing a polymerase to act to cause random polymerization reaction between DNAs encoding the motif sequences. Manufacturing method. A single-stranded DNA containing a sequence encoding an arbitrary motif sequence is converted into a functional motif sequence, a structural motif sequence, an artificial peptide sequence obtained from evolutionary molecular engineering, or a sequence designed based on protein engineering knowledge. The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to any one of claims 1 to 7 , wherein the gene is a microgene constructed based on the gene. In the construction of micro-gene, in a single-stranded DNA sequence to synthesize, advance in the translation reading frame, a number species motif sequence of claim 8, wherein the keep eliminate stop codons are randomly polymerized artificial How to create a gene population. Frequency of appearance of motif sequences used in random polymerization reactions by adding restriction enzyme recognition sequences to single-stranded DNAs containing sequences encoding arbitrary motif sequences to single-stranded DNAs encoding multiple types of motif sequences 10. The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to any one of claims 1 to 9 , which is constructed so that can be directly monitored by restriction enzyme reaction. A vector incorporating a gene produced by the method for producing an artificial gene population in which the multiple motif sequences of any one of claims 1 to 10 are randomly polymerized is introduced into a host cell and expressed. Of artificial protein population in which the motif sequences are randomly inserted. A single-stranded DNA encoding multiple motif sequences is randomly polymerized without insertion of base mutations or deletions, and an artificial gene population is created. 12. The method for producing an artificial protein population in which various motif sequences are randomly inserted according to claim 11 , wherein a molecular population is obtained. Diversity molecules depending on the frameshift by creating an artificial gene population by randomly polymerizing single-stranded DNA encoding multiple types of motif sequences while inserting base mutations and deletions, and translating the artificial gene population 12. The method for producing an artificial protein population in which various motif sequences are randomly inserted according to claim 11, wherein the population is obtained. An artificial gene group in which a large number of motif sequences are randomly polymerized, produced by the production method according to any one of claims 1 to 10 , or various motifs produced by the method according to any one of claims 11 to 13. A method for creating a functional artificial gene or a functional artificial protein, comprising screening a functional gene or a functional protein for an artificial protein population into which sequences are randomly inserted .