WO1998005764A1 - Groupement d'acides nucleiques et son procede d'obtention - Google Patents

Groupement d'acides nucleiques et son procede d'obtention Download PDF

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WO1998005764A1
WO1998005764A1 PCT/DK1997/000316 DK9700316W WO9805764A1 WO 1998005764 A1 WO1998005764 A1 WO 1998005764A1 DK 9700316 W DK9700316 W DK 9700316W WO 9805764 A1 WO9805764 A1 WO 9805764A1
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nucleic acid
blocks
gene
acid pool
terminal
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PCT/DK1997/000316
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English (en)
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Yoshiaki Miyota
Shiro Fukuyama
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Novo Nordisk A/S
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Priority to AU35394/97A priority Critical patent/AU3539497A/en
Publication of WO1998005764A1 publication Critical patent/WO1998005764A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the present invention relates to a nucleic acid pool to be produced by ligating oligonucleotides at random, and a method for producing it, and also to a genetic product to be produced by expressing the nucleic acid existing in the pool as a gene..
  • W095/22625 disclosed is a method for forming novel genes by dividing a plurality of genes at random and homologously recombining them to reconstruct novel genes.
  • this is one method for forming chimera genes.
  • the genes to be formed by this method are similar to the original genes, and the former shall have the essential base sequences of the latter.
  • it is difficult to desire the impartation of some additional functions to organisms which they could not gain during the steps of their evolution.
  • One method for this may be to prepare a nucleic acid pool that covers all base combinations.
  • the total number of the base sequences that may code for a relatively small protein with 100 amino acids (300 bp) is an enormous number of 4 300 (about 10 180 ) , and it is in fact impossible to prepare the nucleic acid pool that may cover all of them.
  • their sub-structures which are referred to as modules were specifically noted, and an attempt was made to change the sequencing of the base sequence blocks corresponding to the individual modules to thereby produce mutants having different module sequences (Viva Origino, Vol. 23 , No. 1 (1995) 86-87) . In this attempt, however, the base sequences were re-sequenced merely individually for the individual mutants. No one has heretofore attempted the formation of a nucleic acid pool covering all re-sequenced molecules and the collection of genes capable of expressing products having intended properties from the pool.
  • the subject matter of the present invention is to provide a method for efficiently obtaining base sequences that exist in spaces greatly different from those of naturally-existing base sequences, and also to provide genetic products to be obtained by expressing, as genes, the nucleic acid sequences that are obtained in that manner and that do not exist naturally.
  • the sequence space of a gene includes the full-length sequence thereof to be theoretically constituted by a combination of four bases, A, G, C and T.
  • a base sequence that codes for a protein composed of a number "n" of amino acids shall be constructed by selecting and sequencing any desired one of the four bases for a total of 3n-times, therefore including 4 3n combinations.
  • a protein composed of 100 amino acids shall include different base sequences of about lO* ⁇ 8 types as so mentioned hereinabove.
  • amino acid substitution is nothing but the essential means that organisms have carried out during the steps of their evolution or, that is, such is the imitation of organisms and is to search only around the sequences that organisms already examined. In addition, there is a probability that the sequences thus obtained will be those that were already weeded out in the past.
  • the present inventors have considered that, in order to be greatly apart from the sequencing spaces that organisms already examined, if we carry out such matters that could not have been carried out by organisms, the purpose will be attained.
  • the present invention provides a specific nucleic acid pool that is mentioned below, a method for producing it to be mentioned below, and also a genetic product to be obtained by expressing, as a gene, the nucleic acid existing in the nucleic acid pool, as is mentioned below.
  • a nucleic acid pool comprising two or more different nucleic acid, which is constructed by dividing all or a part of one or more genes into 3 or more blocks followed by ligating all or a part of these blocks into sequences that are different from the sequence or sequences of the original, non-divided gene or genes.
  • nucleic acid pool according to the previous 1) , which contains 10 or more different nucleic acids.
  • nucleic acid pool according to the previous 1) or 2) , which contains all the nucleic acids with different sequences as constructed by re-sequencing a number, n, of said blocks (where n is the number of the different blocks as formed by the division) .
  • nucleic acid pool according to any one of the previous 1) to 3) , wherein the gene is a gene coding for a protein, and the amino acid sequence as encoded by each block is the same as the amino acid sequence as encoded by the corresponding part on the original gene.
  • nucleic acid pool according to any one of the previous 1) to 4) wherein the gene is a gene coding for an enzymatic function or a control gene for it.
  • the nucleic acid pool according to the previous 5) wherein the gene is a gene coding for any one of proteases, lipases, cellulases, amylases, catalases, xylanases, oxidases, dehydrogenases, oxygenases and reductases.
  • the nucleic acid pool according to any one of the previous 1) to 6) wherein the gene is one derived from prokaryotes.
  • nucleic acid pool according to the previous 7) wherein the gene is one derived from bacillus bacteria.
  • the nucleic acid pool according to the previous 8) wherein the gene is a protease API21 gene.
  • each block is an oligonucleotide.
  • nucleic acid pool according to the previous 10) , wherein the nucleic acid is a single-stranded polynucleotide.
  • nucleic acid pool according to the previous 10) , wherein the nucleic acid is a double-stranded polynucleotide.
  • a method for producing a nucleic acid pool comprising two or more different nucleic acids which comprises dividing all or a part of one or more genes into three or more oligonucleotide blocks or synthesizing oligonucleotides corresponding to said blocks, followed by ligating all or a part of these blocks into sequences that are different from those on genes.
  • the method for producing a nucleic acid pool according to the previous 13) which comprises the following steps a) to c) : a) a step of preparing 3 or more blocks of single-stranded oligonucleotides having base sequences that correspond to all or a part of one or more genes through division of one or more genes or through synthesis of oligonucleotide chains having said base sequences; b) a step of adding a ribonucleotide to its 3' -terminal of each with a deoxyribonucleotide at the 3 ' -terminal of the oligonucleotide chain blocks as obtained in the previous step a), while adding a phosphoryl group to its 5 ' -terminal of each thereof with a hydroxyl group at the 5 '-terminal; and c) a step of ligating in any desired sequence the oligonucleotide chain blocks as obtained in the previous step b) , by reacting the 3
  • a method for producing a double-stranded nucleic acid pool which comprises converting the single-stranded nucleic acids as obtained in any one of the previous 14) to 20 into double-stranded ones through polymerase reaction.
  • nucleic acid pool means a high-density mixture of two or more different nucleic acids. Nucleic acids are single-stranded or double-stranded poly- ucleotides.
  • the nucleic acid pool of the present inention can cover a specific number or more, for example, 10 or more different nucleic acid molecules having different structures. It is desirable that, when the mixture, nucleic acid pool is directly used in biochemical operation or reaction, it is in such a form that all the plural nucleic acid components constituting it can be reacted. However, the form of the mixture, nucleic acid pool is not specifically defined, and the nucleic acid pool may be either in solution or dry mixture.
  • the nucleic acid pool of the present invention is characterized in that it is constructed by dividing all or a part of one or more genes into 3 or more blocks followed by ligating all or a part of these blocks into sequences that are different from the sequence or sequences of the original, non- divided gene or genes, and is therefore characterized in that it comprises a plurality of different nucleic acids having base sequences that are different from the original, non-divided base sequence or sequences.
  • the step of "ligating all or a part of the divided blocks into sequences that are different from the original, non-divided sequence or sequences" as referred to herein includes (i) re-sequencing of the blocks in a sequence that is different from the original, non-divided se- uence, (ii) ligation of a plurality of the same blocks continuously or discontinuously, (iii) re-ligation of the blocks except at least one block, and (iv) combination of these (i) to (iii) .
  • the operation for dividing a gene into plural blocks and re-sequencing these in any desired order that is employed in the present invention is hereinafter referred to as "shuffling".
  • one DNA has a sequence composed of a number, n, of blocks, as represented by a formula (1): A - al - a2 - . . . . - a n - B (9) wherein the starting end A and/or the terminal end B may be omitted, this may be shuffled according to the invention to give a mixture of nucleic acids to be represented by a formula (2) : A - al' - a2' - . . . . - a x - B (2) wherein al ' , a2 ' , . . . , a x are blocks that are independently selected from the group of al, a2 , . . .
  • the total number of the blocks al', a2 • , . . . , a x may not be the same as the total number of the blocks ⁇ l ; ⁇ & f • * • / ci ⁇ •
  • one or more genes In order to make the shuffling effective, one or more genes must be divided into 3 or more blocks. If divided into 2 blocks, only one re-sequenced form can be obtained and many different nucleic acids cannot be obtained. If so, the effectiveness of the nucleic acid pool of the invention is poor. Preferably, one or more genes are divided into 5 or more blocks.
  • the blocks (such as al, a2, . . . . , an in the above- mentioned formula (1)), which are the units to be shuffled, are oligonucleotides or polynucleotides composed of 2 or more nucleotides (hereinafter referred to as "oligonucleotides") . If the length of each block is too short, the operation with the blocks is complicated. In general, therefore, each block is preferably composed of 21 or more nucleotide units, more preferably 45 or more nucleotide units. The uppermost limit of the block length is not specifically defined, provided that the block length is shorter than the length of one gene.
  • the block length is preferably within the range of from 5 to 30 % of the length of a gene.
  • the division of a gene into blocks may be effected at any sites of the gene. Though not excluding the division of a gene into the constitutive exons or segment blocks that correspond to the domains or modules of the protein which the gene codes for, there is a probability that the shuffling at such sites would have been examined in the natural world in the past. In order to obtain base sequences that have not heretofore been examined in the natural world, it is desirable that the division of a gene is effected inside the constitutive exons or at the sites corresponding to the inside of the domains or modules of the protein which the gene codes for.
  • the gene to be shuffled is a gene that codes for a protein
  • the gene blocks, oligonucleotides each have the same reading frame before and after the division of the gene. Namely, it is desirable that the gene blocks to be shuffled are so designed that they are translated to always give the corresponding amino acid sequences, irrespective of their relative positions in the shuffled sequence.
  • the re-sequencing of the divided blocks to be conducted through the shuffling thereof in the present invention is to ligate a desired number of the blocks, al, a2 , . . . , an, while allowing the ligation of two or more same blocks in series and allowing the deletion of some blocks, as so mentioned hereinabove.
  • the nucleic acid pool of the invention to be obtained by the ligation covers at least all nucleic acids each composed of nearly the same number of blocks as the number of the divided blocks. For example, when a gene is divided into 5 blocks, al, a2 , a3 , a4 and a5, it is desirable that the nucleic acid pool obtained covers substantially all different combinations each comprised of these 5 blocks.
  • the nucleic acid pool obtained covers all simple re-sequences, such as al-a3-a2-a4-a5 (where the order of a2 and a3 was altered) and al-a4-a2-a3-a5 (where s2, a3 and a4 were re-sequenced), more preferably complex re-sequences comprising a plurality of same blocks, such as al-a3-a2-al-a5, in addition to such simple re- sequences.
  • the number of the former simple re-sequences is n!
  • the nucleic acid pool as obtained by shuffling a gene according to the present invention thus can cover such an extremely large number of nucleic acid molecules having different base sequences, each of which is different from the base sequence of the original gene.
  • the kind of the gene to be shuffled is not specifically defined.
  • Employable herein is any and every gene that is composed of polynucleotide chains and contains a coding region necessary for expressing a protein or RNA.
  • the nucleotide unit may contain any molecule of deoxyribonucleotides or ribonucleotides.
  • genes coding for proteins especially enzymes, or control genes for enzymatic functions.
  • enzymes include proteases, lipases, cellulases, amylases, catalases, xylanases, oxidases, dehydrogenases, oxygenases and reductases .
  • the kind of the gene to which the present invention is directed is not specifically defined but shall be such that, when it is introduced into a suitable host, the host can produce the genetic product through expression of the gene.
  • genes as cloned from living organisms include artificially synthesized genes, and even genes as cloned from living organisms and artificially mutated.
  • prokaryotes with definite enzyme producibility .
  • prokaryotes mentioned are bacillus bacteria.
  • One example of the genes derived from such bacteria is a protease API21 gene derived from Bacillus NKS-21 (FERM BP-93-1) (Japanese Patent Application Laid-Open No. 5-91876, Sequence Number 1) .
  • the present invention also provides a method for producing a nucleic acid pool comprising two or more different nucleic acids each having a base sequence that is different from the base sequence of the original, non-divided gene, which comprises dividing all or a part of one or more genes into three or more oligonucleotide blocks, followed by ligating all or a part of these blocks into sequences that are different from the sequence or sequences of the original, non-divided gene or genes.
  • the division of a gene into blocks can be conducted by any desired method that satisfies the above-mentioned conditions necessary to the nucleic acid pool.
  • the division of a gene can be conducted by the use of restriction enzymes.
  • any desired restriction enzymes can be used, including, for example, EcoRI, Hindlll, BamHI, Pstl, Kpnl, Xbal, Smal, Sad, Clal, Alul, Haelll and Rsal.
  • each block of the gene can be obtained through synthesis in accordance with the above-mentioned conditions. To re-sequence these blocks, they are blended and ligated, for example, using a ligase.
  • One preferred method of producing a single-stranded nucleic acid pool of the present invention is to ligate plural blocks each with a ribonucleotide at the 3 '-terminal, using an RNA ligase.
  • This method comprises the following steps a) to c): a) a step of preparing 3 or more blocks of single-stranded oligonucleotides having base sequences that correspond to all or a part of one or more genes through division of one or more genes or through synthesis of oligonucleotide chains having said base sequences; b) a step of adding a ribonucleotide to its 3 '-terminal of each with no ribonucleotide at the 3 '-terminal of the oligonucleotide chain blocks as obtained in the previous step a), while adding a phosphoryl group to its 5 '-terminal of each thereof with no phosphoryl group at the 5 '-terminal; and c) a step of
  • Fig. 1 schematically illustrated are the above- mentioned steps for shuffling one gene.
  • one gene is divided into four blocks (al, a2, a3 , a4) .
  • the base and the nucleic acids are represented only by the corresponding base sequences.
  • A, G, C and T are nucleotide units comprising the corresponding bases.
  • rG means GMP; and
  • P) and (OH) mean the phosphoryl group and the hydroxyl group, respectively, existing at the terminals of each nucleotide chain.
  • the division of the gene can be effected, using restriction enzymes.
  • the divided blocks are denatured under heat or with an alkaline or the like into single-stranded oligonucleotide.
  • single-stranded oligonucleotides are synthesized using ordinary devices and according to ordinary methods.
  • Step b) Where the blocks as obtained in the step a) each have a 3 '-terminal deoxyribonucleotide, a ribonucleotide is added to the 3 '-terminal (step b) .
  • This addition can be effected by reacting a terminal deoxynucleotidyl transferase on each said block in the presence of a nucleoside triphosphate (ATP, GTP, CTP, UTP) .
  • AMP, GMP, CMP, UMP includes the base corresponding to the nucleoside triphosphate used (A, G, C, U) .
  • each block may have a desired 3'- terminal ribonucleotide.
  • GMP this is represented by rG underlined in Fig. 1
  • the nucleoside triphosphate is used in an amount of from 2 to 10 times or so, by mol, relative to mol of each block.
  • the reaction temperature may be from 30 to 40sc or so; and the reaction time may be from 30 minutes to 2 hours or so.
  • a phosphoryl group is added to the 5'- terminal of each block.
  • This addition can be effected, using a polynucleotide kinase in the presence of ATP.
  • ATP is used in an amount of from 2 to 10 times or so, by mol, relative to mol of each block.
  • the reaction temperature may be from 30 to 402C or so; and the reaction time may be from 10 minutes to 1 hour or so.
  • the pH is most suitably from 7 to 9 or so.
  • the ligation of oligonucleotide chain blocks in the step c) can be effected by reacting an RNA ligase on the mixture of blocks thus obtained in the previous step, in the presence of
  • Useful divalent ions are magnesium ions and manganese ions, of which preferred are magnesium ions.
  • employable is an RNA ligase.
  • the RNA ligase is an enzyme that catalyzes the ligation of a 5 ' -phosphoryl terminated polynucleotide and a 3 ' -hydroxyl terminated polynucleotide.
  • the substrate for such an RNA ligase is naturally an RNA, but the enzyme can effectively catalyze the ligation of a 5 ' -phosphoryl terminated polydeoxyribonucleotide and a polydeoxyribonucleotide having a ribonucleotide only at its 3' -terminal.
  • a T4 RNA ligase is a T4 RNA ligase.
  • the reaction is conducted generally in a buffer, at a pH of from 7 to 9 and at a temperature of from 10 to 40 ⁇ C over a period of from 30 to 180 minutes.
  • the oligonucleotides may be reacted in a solution comprising 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.1 mM ATP, 10 mg/liter BSA, 1 mM hexaammine cobalt chloride (HCC) and 25 % polyethylene glycol 6000, at 25 ⁇ C for 60 minutes or longer.
  • the amino acid sequences to be encoded by the blocks vary, depending on the shuffled sites of the blocks. In some cases, however, it is often desirable that the shuffling does not result in the change in the amino acid sequence to be encoded by each block, as so mentioned hereinabove. For this purpose, a modified method as schematically illustrated in Fig. 2 will be effective.
  • blocks each having a base sequence of from the (3p+l)th to the (3q+2)th, as counted from the starting point of the reading frame on a gene are prepared in the first step (step a'), and a ribonucleotide that corresponds to the (3q+3)th base, as counted in the same manner as above, or a ribonucleotide corresponding to a base that does not change the amino acid to be encoded at said site is added to each said block at its 3 '-terminal in the step b')
  • a ribonucleotide is not added at its 3 '-terminal for the reasons mentioned below.
  • GMP this is represented by rG underlines in Fig. 2
  • AMP this is represented by rA underlined in Fig. 2
  • TTT terminal deoxynucleotidyl transferase
  • employable is a method of preparing ribonucleotide-terminated blocks only. As a result of this step, the amino acid sequences to be encoded by these blocks shall be the same as those on the original gene.
  • the blocks are phosphorylated with a polynucleotide kinase (PNK) in the same manner as in the above- mentioned step b) (refer to the latter half of the step b) ) .
  • PNK polynucleotide kinase
  • some blocks may not be processed with a ribonucleotide at the 3 '-terminal. If the blocks not processed so are treated with an RNA ligase, any other block could no more be ligated to these blocks at the 3'- terminal. As a result, all the nucleic acids in the nucleic acid pool obtained shall have substantially any of these blocks at the 3 '-terminal. In the same manner, if some particular blocks are not phosphorylated at the 5 '-terminal, all the nucleic acids in the nucleic acid pool obtained shall have substantially any of such specific blocks at the 5 '-terminal.
  • nucleic acid pool comprising nucleic acids which have predetermined particular blocks positioned at the terminals while having random re-sequences in the intermediate part.
  • This method is especially advantageous in producing protein mutants having particular amino acid sequences at the terminals or for the purpose of expressing particular control functions.
  • the molecules as obtained in the process mentioned in the previous (1) are single-stranded ones, which can be converted into double-stranded ones through genetic treatment thereof to be mentioned below.
  • the block mixture is made to contain a block having a 5 ' -phosphoryl group but not having a 3 ⁇ -ribonucleotide group (this block is referred to as oligonucleotide A; in Fig. 2, a4 corresponds to this block). Accordingly, all the nucleic acids that constitute the nucleic acid pool to be produced shall be substantially terminated by oligonucleotide A at the 3 '-terminal, as so mentioned in the last in the previous (2) .
  • the nucleic acid blocks are subjected to ordinary DNA-extending reaction, using, as a primer, a decamer (10-mer) or higher oligonucleotide, preferably a heptamer (17-mer) or higher oligonucleotide, that is complementary to oligonucleotide A.
  • a decamer (10-mer) or higher oligonucleotide preferably a heptamer (17-mer) or higher oligonucleotide, that is complementary to oligonucleotide A.
  • a decamer 10-mer
  • oligonucleotide preferably a heptamer (17-mer) or higher oligonucleotide, that is complementary to oligonucleotide A.
  • employable is any and every enzyme that catalyzes the DNA-extending reaction, such as Taq polymerase, Klenow fragment, DNA polymerase I or the like.
  • PCR polymerase chain reaction
  • oligonucleotide B an additional oligonucleotide having a 3 ' -ribonucleotide group but not having a 5 ' -phosphoryl group (hereinafter referred to as oligonucleotide B) is added to the block mixture, in addition to the above-mentioned oligonucleotide A, during the process of preparing the pool. Accordingly, all the molecules that constitute the pool shall have oligonucleotide A at the 3 '-terminal and oligonucleotide B at the 5 '-terminal.
  • the nucleic acid blocks are subjected to PCR, using, as primers, a 10-mer or higher oligonucleotide, preferably a 17-mer or higher oligonucleotide, that is complementary to oligonucleotide A, and a 10-mer or higher oligonucleotide, preferably a 17-mer or higher oligonucleotide, that is complementary to oligonucleotide B, whereby the nucleic acid blocks are converted into double-stranded ones while being amplified at the same time. Therefore, this process is advantageous for the following operation.
  • the oligonucleotide A and/or B may be the same as those existing on the original gene, or, if desired, may also be others which the original gene does not have.
  • the resulting double-stranded nucleic acid is blunted, and then ligated to any desired vector, preferably an expression vector, such as pKK223-3, using a DNA ligase.
  • any desired vector preferably an expression vector, such as pKK223-3, using a DNA ligase.
  • the polynucleotide A and B positioned at the both terminals of the nucleic acid may be made to have suitable restriction enzyme recognizing sites.
  • the nucleic acid may be ligated to a suitable vector, using the defined restriction enzymes.
  • the vector library thus produced in the manner mentioned above is introduced into a suitable host, in which the genetic information is expressed.
  • the intended genetic product with favorable properties and also the gene coding for it can be obtained.
  • Any and every ordinary host can be used herein.
  • Preferred examples of the host include cells of E . coli , bacillus bacteria, yeasts, and lactic acid bacteria. If desired, in-vitro transcription systems and translation systems are also employable herein. In those cases, the genetic information can be expressed even when the nucleic acid is not ligated to a vector.
  • the "genetic information” as referred to herein indicates the information on a gene which is carried by a DNA or RNA and which is translated into a protein or is transcribed into RNA in a suitable living body by the DNA or RNA for itself or after having been ligated to any other DNA or RNA.
  • the genetic information that is expected to be expressed according to the method of the present invention is not specifically defined, but includes, for example, those on various genetic products, such as enzymes, antibodies, hormones receptor proteins and ribozymes, and those on various control functions of, for example, operators, promoters and attenuators .
  • Example 1 Production of Single-Stranded Nucleic Acid Pool
  • a nucleic acid pool was produced in accordance with the process mentioned below, based on the wild-type alkali protease (Japanese Patent Application-Laid Open No. 5-91876) as cloned from a protease API21 (Bacillus NKS-21; FERM BP-93-1) having a sequence of Sequence Number 1.
  • Step a) Preparation of Oligonucleotide Blocks
  • Oligo A, oligo 1 to 3 , and oligo B are parts of the protease API21 gene. Their positions are as mentioned above. These oligonucleotides were synthesized in a DM trityl-on condition (that is, while the 5 ' -hydroxyl group was protected with dimethoxytrityl group) , and purified through an OPC column. The reagents used herein were obtained from Perkin Elmer Co.
  • Step b Processing of Oligonucleotide Blocks (2-1) Addition of Ribonucleotide:
  • HCC hexaam ine cobalt chloride
  • BSA bovine serum albumin
  • Oligo 1 and oligo 2 were processed in the same manner as above.
  • Oligo 3 was processed in the same manner as above, but using ATP in place of UTP.
  • oligo Ar oligo lr, oligo 2r and oligo 3r.
  • oligo lr 500 pmols of oligo lr, 1 nmol of ATP and 10 units of polynucleotide kinase were dissolved in the standard solution having the same composition as above to make 10 ⁇ l in total. The resulting solution was left at 37 - ⁇ C for 1 hour. Oligo 2r, oligo 3r and oligo B were processed in the same manner as above. These polynucleotides thus formed are referred to as oligo lpr, oligo 2pr, oligo 3pr and oligo Bp.
  • Oligo B' (its sequence is represented by Sequence Number 7) which is complementary to oligo B was synthesized in the same manner as in Example 1.
  • the DNAs' constituting the single-stranded nucleic acid pool as obtained in Example 1 were all or, that is, without being separated into the individual DNAs, mixed with 10 pmols of oligo B', and added to tris-HCl buffer containing MgCl2 and DTT (dithiothreitol) to make 20 ⁇ l in total.
  • the resulting mixture was finally comprised of 10 mM tris-HCl buffer (pH 7.5), 7 mM MgCl2, and 0.1 mM of DTT. This was heated at 752C for 5 minutes, and then cooled to 30ec. Next, l unit of Klenow fragment was added thereto, and kept at 37 - ⁇ C for 2 hours, whereby the single-stranded nucleic acids were converted into double-stranded ones.
  • a plasmid pSDT812 Japanese Patent Application Laid-Open No. 1-1415966 , which has been prepared by inserting a gene of a wild-type alkali protease as cloned from Bacillus NKS-21 into a plasmid pHSG396 at its Clal-cleaving site, was digested with a restriction enzyme Clal, and then blunted with a commercially- available blunting reagent (Blunting Kit, manufactured by Takara Shuzo Co.).
  • pHY812 formed in the above was digested with restriction enzymes Hindlll and Sphl, and then processed with BAP (a) .
  • the DNAs constituting the double- stranded nucleic acid pool as formed in Example 3 were digested all at a time with restriction enzymes Hindlll and Sphl (b) .
  • (a) and (b) were ligated in the same manner as above, using the ligation kit.
  • With the resulting DNA cells of E. coli JM105 were transformed. The resulting transformants were screened on an L-plate containing ampicillin.
  • Example 4 From the colonies that had been selected in Example 3, a plasmid DNA was prepared, which was then digested with Hindlll and Sphl to check as to whether or not it gave a fragment of about 160 bp after the digestion. The base sequences of 95 clones that had given the fragment having the intended length were analyzed, which verified the shuffling of the gene blocks corresponding to oligo 1, oligo 2 and oligo 3. Table 1 shows different types of shuffling, and the number of clones with each type.
  • Type of Shuffling Number Type of Shuffling Number of
  • the plasmid DNAs as produced in Example 4 were mixed. Using the resulting DNA mixture, cells of Bacillus subtilis U0T0999 were transformed. Tetracycline-resistant transformants were selected. 300 transformants were replicated on a skim milk-containing medium plate, on which were found clear zones around the colonies of 15 transformants. Accordingly, it is understood that the enzyme which the shuffled gene codes for can be selected depending on its activity. A plasmid DNA was prepared from these 15 transformants that had formed the clear zones, and then sequenced. From the base sequence thus identified, it is understood that the blocks of the plasmid DNA prepared herein were sequenced in the same order as in the wild-type plasmid DNA.
  • RNAs From 10 transformants (one forms clear zones, while nine do not) as obtained in Example 5, and also from the host, Bacillus subtilis UOT0999 which does not have the plasmid, full-length RNAs were prepared. These were processed with a ribonuclease-free deoxyribonuclease, in order to remove the influence of the plasmid on the hybridization to be effected later on. Next, using oligo B' as the probe, these were subjected to Northern hybridization. As a result, all lanes corresponding to the RNA of the transformants gave detectable bands, but no band was detected on the lanes corresponding to the RNA of the host.
  • nucleic acid pool capable of covering various base sequences which are substantially apart from the naturally-existing base sequence spaces. Therefore, it is possible to obtain excellent genetic products, such as proteins and enzymes, which could not be obtained in conventional methods and which were not examined by organisms in the past.
  • the method of the present invention for producing a nucleic acid pool it is possible to obtain a mixture of nucleic acids while optionally shuffling the constitutive blocks at random in the intermediate parts but fixing the terminal sequences to be predetermined, desired ones, and it is also possible to shuffle the constitutive blocks without changing the amino acid sequence which each block codes for. Therefore, as compared with a method of producing a completely-randomized nucleic acid pool, there is a high possibility that useful genetic products can be produced according to the method of the present invention.
  • Fig. 1 is a schematic view showing one embodiment of the method for producing a nucleic acid pool of the present invention.
  • Fig. 2 is a schematic view showing another embodiment of the method for producing a nucleic acid pool of the present invention.
  • Fig. 3 is a restriction enzyme cleavage map of plasmid pHY812, in which the alkali protease gene derived from Bacillus NKS-21 has been ligated to plasmid pHY300PLK.

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Abstract

L'invention porte sur l'obtention d'un certain nombre de séquences de base existant dans des espaces sensiblement séparés des espaces de séquences de base existant naturellement; sur un groupement de deux ou plus de deux acides nucléiques différents constitué par division de tout ou partie d'un ou plusieurs gènes en trois blocs ou plus de trois blocs, puis par liaison de tout ou partie de ces blocs en séquences différentes de la ou des séquences de l'original, sur un ou des gènes non divisés, et le procédé d'obtention du groupement; et sur un produit génétique obtenu par expression des informations génétiques comprises dans le groupement d'acides nucléiques.
PCT/DK1997/000316 1996-08-07 1997-07-23 Groupement d'acides nucleiques et son procede d'obtention WO1998005764A1 (fr)

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AU35394/97A AU3539497A (en) 1996-08-07 1997-07-23 Nucleic acid pool and method for producing the same

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JP8208423A JPH1066577A (ja) 1996-08-07 1996-08-07 核酸プールおよびその製造方法
JP8/208423 1996-08-07

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011818A1 (fr) * 1997-08-28 1999-03-11 Isao Karube Procede de detection d'acides nucleiques ou de polypeptides hautement fonctionnels
US6984488B1 (en) * 1996-11-07 2006-01-10 Ramot At Tel-Aviv University Ltd. Determination and control of bimolecular interactions
EP2270234A1 (fr) 1997-12-08 2011-01-05 California Institute of Technology Procédé pour créer des séquences de polynucléotides et polypeptides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018645A1 (fr) * 1991-04-16 1992-10-29 Diagen Institut Für Molekularbiologische Diagnostik Gmbh Procede de preparations de nouveaux biopolymeres
WO1995017413A1 (fr) * 1993-12-21 1995-06-29 Evotec Biosystems Gmbh Procede permettant une conception et une synthese evolutives de polymeres fonctionnels sur la base d'elements et de codes de remodelage
WO1995022625A1 (fr) * 1994-02-17 1995-08-24 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018645A1 (fr) * 1991-04-16 1992-10-29 Diagen Institut Für Molekularbiologische Diagnostik Gmbh Procede de preparations de nouveaux biopolymeres
WO1995017413A1 (fr) * 1993-12-21 1995-06-29 Evotec Biosystems Gmbh Procede permettant une conception et une synthese evolutives de polymeres fonctionnels sur la base d'elements et de codes de remodelage
WO1995022625A1 (fr) * 1994-02-17 1995-08-24 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6984488B1 (en) * 1996-11-07 2006-01-10 Ramot At Tel-Aviv University Ltd. Determination and control of bimolecular interactions
WO1999011818A1 (fr) * 1997-08-28 1999-03-11 Isao Karube Procede de detection d'acides nucleiques ou de polypeptides hautement fonctionnels
EP2270234A1 (fr) 1997-12-08 2011-01-05 California Institute of Technology Procédé pour créer des séquences de polynucléotides et polypeptides

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AU3539497A (en) 1998-02-25

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