WO2015016124A1 - Β-glucuronidase modifiée - Google Patents

Β-glucuronidase modifiée Download PDF

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WO2015016124A1
WO2015016124A1 PCT/JP2014/069539 JP2014069539W WO2015016124A1 WO 2015016124 A1 WO2015016124 A1 WO 2015016124A1 JP 2014069539 W JP2014069539 W JP 2014069539W WO 2015016124 A1 WO2015016124 A1 WO 2015016124A1
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glucuronidase
improved
amino acid
acid sequence
gene
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Japanese (ja)
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哲也 四方
武志 角南
雄大 西川
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独立行政法人科学技術振興機構
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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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01031—Beta-glucuronidase (3.2.1.31)

Definitions

  • the present invention relates to an improved ⁇ -glucuronidase whose activity expression is accelerated as compared with the wild type, and an improved ⁇ -glucuronidase whose enzyme molecular activity (k cat ) is improved as compared with the wild type.
  • k cat enzyme molecular activity
  • the ⁇ -glucuronidase gene has been conventionally used as a particularly useful reporter gene in plant cells that do not have it.
  • ⁇ -glucuronidase gene When a ⁇ -glucuronidase gene is used as a reporter gene, ⁇ -glucuronidase is expressed from the gene. ⁇ -glucuronidase does not show activity in the monomer state, but only becomes tetramer to show activity, and its detection becomes possible.
  • the ⁇ -glucuronidase gene is used as a reporter gene, it is known that the speed of this tetramer formation process is the rate-limiting step of detection.
  • Previous studies have revealed that the expression of ⁇ -glucuronidase activity has a problem that it takes a considerable amount of time depending on the cell size (for example, T. Matsuura et al. , “Kinetic Analysis of ⁇ -Galactosidase and ⁇ -Glucuronidase Tetramerization Coupled with Protein Translation”, Journal of Biological Chem.
  • ⁇ -glucuronidase gene when ⁇ -glucuronidase gene is introduced into a cell as a reporter gene, the concentration varies dramatically if the cell size is different. If the cells are large and the local concentration of the gene is low, there is a problem that it takes time before detection. This is considered to be due to the fact that when the cell size is large, even if the same amount of ⁇ -glucuronidase monomer is expressed, its local concentration is low, making it difficult to associate and form a tetramer. It is done.
  • An object of the present invention is to provide a new and improved mutated ⁇ -glucuronidase having a high enzyme activity compared to the wild type and easy to detect.
  • the improved ⁇ -glucuronidase of the present invention is characterized in that a mutation is introduced into the amino acid sequence of the ⁇ -glucuronidase monomer and has methionine or serine at the 561st position from the N-terminal side of the amino acid sequence. .
  • the improved ⁇ -glucuronidase of the present invention preferably has methionine at the 561st position from the N-terminal side of the amino acid sequence and leucine at the 520th position from the N-terminal side of the amino acid sequence.
  • the improved ⁇ -glucuronidase of the present invention preferably has serine at the 561st position from the N-terminal side of the amino acid sequence and has alanine at the 444th position from the N-terminal side of the amino acid sequence.
  • the improved ⁇ -glucuronidase of the present invention preferably has the amino acid sequence of SEQ ID NO: 10, 12, 14, 16, 18 or 20.
  • the present invention also provides an improved ⁇ -glucuronidase in which a mutation is further introduced into the above-described improved ⁇ -glucuronidase, which has at least one of the following (a) to (d): -Also provides for glucuronidase.
  • the improved ⁇ -glucuronidase of the present invention preferably has all of the above (a) to (d).
  • the improved ⁇ -glucuronidase of the present invention preferably has the amino acid sequence of SEQ ID NO: 27 or 29.
  • the improved ⁇ -glucuronidase of the present invention those having methionine at the 561st position from the N-terminal side of the amino acid sequence have a much faster tetramer formation rate than wild-type ⁇ -glucuronidase. Thus, detection can be performed more quickly.
  • those having serine at the 561st position from the N-terminal side of the amino acid sequence have a fast fluorescence intensity amplification, and the substrate hydrolysis rate compared to the wild type. Is high (has a large k cat ).
  • the improved ⁇ -glucuronidase of the present invention provides a new and improved ⁇ -glucuronidase that has evolved from the wild type, has higher activity, and is easy to detect. Is done.
  • FIG. 2 is a diagram schematically showing a method for improving an enzyme in terms of evolutionary engineering, which is preferably used for producing the improved ⁇ -glucuronidase (GUS) of the present invention.
  • the time course of the GUS catalytic reaction (substrate hydrolysis) accompanying the translation reaction from the GUS gene (100 pM DNA) in a test tube is shown in comparison with the wild-type GUS and the improved GUS (C1102, C1110) of the present invention. It is a graph. It is a graph which compares and compares the wild-type GUS and the improved GUS (C1102, C1110) of this invention about the reaction rate in 60 minutes after reaction start.
  • FIG. 6 (a) is a graph showing the results of nucleotide sequence analysis of the obtained improved ⁇ -glucuronidase
  • FIG. 6 (a) is a graph showing the results of nucleotide sequence analysis of the obtained improved ⁇ -glucuronidase
  • FIG. 6 (a) shows the average value of non-synonymous substitutions of improved ⁇ -glucuronidase (per clone) (the vertical axis is the average variation) Number [/ clone], the horizontal axis is the number of selection steps (number of rounds), and FIG. 6 (b) shows the number of enriched non-synonymous substitutions (50% or more of all clones have in common) (Vertical axis is the number of rich mutations, and the horizontal axis is the number of selection steps (round number)).
  • FIG. 6 (c) is the concentration ratio of non-synonymous substitutions (of clones with mutations).
  • FIG. 5 is a graph showing the results of in-tube ⁇ -glucuronidase synthesis reaction (DNA start) for wild type, C12, and C24.
  • FIG. 6 is a graph showing the results of in-tube ⁇ -glucuronidase synthesis reaction (RNA start) for wild type, C12, and C24.
  • FIG. 1 is a diagram schematically showing a method for improving an enzyme in terms of evolutionary engineering, which is preferably used for producing the improved ⁇ -glucuronidase (GUS) of the present invention.
  • This method is a method proposed by the present applicant in, for example, Japanese Patent Application Laid-Open No. 2012-210170, and uses a liposome encapsulating a gene library and a cell-free protein synthesis system (cell-free transcription and translation system), and a cell sorter. Details will be described later in the experimental examples.
  • a gene library in which random mutations are introduced into a gene (DNA or RNA) encoding a target enzyme using a known genetic engineering technique is prepared.
  • a gene DNA or RNA
  • a cell-free protein synthesis system are encapsulated in a liposome that is an artificial lipid bilayer vesicle. If the gene is DNA, it is transcribed into RNA, and if the gene is RNA, the gene is expressed in the liposome as it is. At that time, for example, the substrate and the expressed enzyme are reacted to evaluate the activity of the enzyme.
  • a fluorescent cell sorter is used to quantitatively evaluate the activity of the enzyme, and a liposome containing a more active enzyme is selected.
  • a gene (DNA or RNA) encoding an enzyme contained in the selected liposome is taken out, further introduced with a mutation by a known genetic engineering technique, and amplified to prepare a gene library. This is taken as one round, and this gene library is also encapsulated in a liposome together with a cell-free protein synthesis system and expressed, and rounds are repeated. This makes it possible to produce a so-called improved enzyme having a high activity for each round.
  • the improved ⁇ -glucuronidase of the present invention is produced by using the above-described method using ⁇ -glucuronidase as a target enzyme and a gene encoding the enzyme.
  • ⁇ -glucuronidase as a target enzyme and a gene encoding the enzyme.
  • SEQ ID NO: 1 the nucleotide sequence of plasmid DNA (pET_GUS) encoding wild-type ⁇ -glucuronidase is shown in SEQ ID NO: 1
  • amino acid sequence of wild-type ⁇ -glucuronidase expressed from the DNA is shown in SEQ ID NO: 2.
  • a DNA library in which mutations are randomly introduced into such a wild-type ⁇ -glucuronidase gene by a known appropriate point mutation introduction method is prepared.
  • a DNA fragment (gus_wt) containing a T7 promoter sequence and a region encoding ⁇ -glucuronidase was added to primers (1), (2) (base sequence A PCR using DNA polymerase (for example, 30 cycles), and using this gus_wt as a template and the same primers (1) and (2), for example, DNA for random mutation introduction
  • primers (1), (2) base sequence A PCR using DNA polymerase (for example, 30 cycles)
  • DNA for random mutation introduction A DNA library into which mutations are randomly introduced can be prepared by an error-prone PCR method using a polymerase (for example, Mutzyme II DNA polymerase, Agilent Technologies).
  • a reaction solution for protein synthesis including the DNA into which the mutation has been introduced and a cell-free protein synthesis system is prepared and encapsulated in liposomes.
  • a cell-free protein synthesis system a commercially available one can be used without particular limitation, and a preferred example is PUREsystem (Gen Frontier).
  • a liposome the unilamellar liposome produced by the well-known method by allowing a W / O emulsion to pass through an oil-water interface can be used conveniently.
  • the mutated ⁇ -glucuronidase is expressed in the liposome.
  • active ⁇ -glucuronidase active ⁇ -glucuronidase
  • active ⁇ -glucuronidase active ⁇ -glucuronidase
  • PFB-FDGlcU 5- (pentafluorobenzoylamino) fluorescein di- ⁇ -D-glucuronide
  • liposomes containing DNA encoding active ⁇ -glucuronidase are collected with a cell sorter using green fluorescence as an index.
  • the encapsulated DNA is extracted from the collected liposomes.
  • QIAquick PCR Purification Kit Qiagen
  • the extracted DNA is amplified by PCR using the primers (1), (2) and DNA polymerase described above, and only the DNA of the desired length is recovered and purified by agarose gel electrophoresis.
  • the purified DNA is also introduced with a mutation, encapsulated in a liposome together with a cell-free protein synthesis system, active ⁇ -glucuronidase is detected, and DNA encoding the active ⁇ -glucuronidase is recovered from the liposome. Evolve and evolve rounds.
  • the improved ⁇ -glucuronidase of the present invention is an improved ⁇ -glucuronidase in which a mutation is introduced into the amino acid sequence of a ⁇ -glucuronidase monomer, which is a methionine or methionine at the 561st position from the N-terminal side of the amino acid sequence. It has serine. Wild-type ⁇ -glucuronidase has leucine (Leu / L) at position 561 from the N-terminal side, but in the improved ⁇ -glucuronidase of the present invention, this leucine is methionine (Met / M) or serine (Ser / L). S). Interestingly, the effect of the improved ⁇ -glucuronidase varies depending on whether 561 has methionine or serine.
  • FIG. 2 shows the ⁇ -glucuronidase catalyzed reaction (substrate hydrolysis) accompanying the translation reaction from the ⁇ -glucuronidase gene in the test tube of the improved ⁇ -glucuronidase of the present invention actually produced in Experimental Example 1 described later.
  • FIG. 4 is a diagram showing the time course in comparison with wild-type ⁇ -glucuronidase.
  • FIG. 3 is a graph comparing the wild-type ⁇ -glucuronidase and the improved ⁇ -glucuronidase of the present invention with respect to the reaction rate 60 minutes after the start of the reaction. As is clear from FIGS.
  • C1102 is an improved ⁇ -glucuronidase having methionine at the 561st position from the N-terminal side of the amino acid sequence (the nucleotide sequence is shown in SEQ ID NO: 9 and the amino acid sequence is shown in SEQ ID NO: 10, respectively).
  • C1110 base sequence shown in SEQ ID NO: 11 and amino acid sequence shown in SEQ ID NO: 12
  • C1110 expresses catalytic activity much faster than wild-type ⁇ -glucuronidase (the catalytic activity can be observed more than 20 minutes faster).
  • FIG. 5 shows the ⁇ -glucuronidase catalyzed reaction (substrate hydrolysis) accompanying the translation reaction from the ⁇ -glucuronidase gene in the test tube of the improved ⁇ -glucuronidase of the present invention actually produced in Experimental Example 1 described later.
  • a modified ⁇ -glucuronidase having serine at position 561 from the N-terminal side of the amino acid sequence (e301 (base sequence is SEQ ID NO: 13, amino acid sequence)
  • E304 base sequence is shown in SEQ ID NO: 15 and amino acid sequence is shown in SEQ ID NO: 16
  • e314 base sequence is shown in SEQ ID NO: 17 and amino acid sequence is shown in SEQ ID NO: 18
  • e321 The nucleotide sequence is SEQ ID NO: 19 and the amino acid sequence is SEQ ID NO: 20, respectively. This is also shown). From the results shown in FIG.
  • the present invention provides a new and improved ⁇ -glucuronidase that has evolved from the wild type in terms of enzyme activity, has high activity, and is easy to detect.
  • the improved ⁇ -glucuronidase of the present invention when it has methionine at the 561st position from the N-terminal side of the amino acid sequence, it preferably has leucine at the 520th position from the N-terminal side of the amino acid sequence. Specifically, it is particularly preferable to have the amino acid sequence shown in SEQ ID NO: 10 or 12 (that is, C1102, C1110 described above).
  • the improved ⁇ -glucuronidase of the present invention when it has serine at the 561st position from the N-terminal side of the amino acid sequence, it preferably has an alanine at the 444th position from the N-terminal side of the amino acid sequence. Specifically, it is particularly preferable to have the amino acid sequence shown in SEQ ID NO: 14 or 20 (that is, e301, e321 described above). In addition, as another preferred specific example in the case of having serine at the 561st position from the N-terminal side of the amino acid sequence, there is also a case of having the amino acid sequence shown in SEQ ID NO: 16 or 18 (that is, e304 and e314 described above). .
  • the present invention further provides ⁇ -glucuronidase obtained by repeating the above-described rounds and further evolving ⁇ -glucuronidase having methionine at the 561st position from the N-terminal side of the amino acid sequence.
  • the further evolved ⁇ -glucuronidase of the present invention has methionine at the 561st position from the N-terminal side of the amino acid sequence and leucine at the 520th position. And at least one of the following (a) to (d).
  • Such an improved ⁇ -glucuronidase exhibits much higher enzyme activity, such as the ability to detect activity even at a DNA concentration of about 50 pM, which makes it difficult to detect enzyme activity in the wild type.
  • Such an improved ⁇ -glucuronidase of the present invention preferably has all of the above (a) to (d) as C12 and C24 obtained in Experimental Example 2 described later. It is particularly preferred to have the amino acid sequence shown in 27 or SEQ ID NO: 29.
  • ⁇ Experimental example 1> Screening of active ⁇ -glucuronidase (GUS) gene (1-1) Preparation of gene library A random mutant library (mt_gus) of GUS gene for use in gene screening experiments was prepared by error-prone PCR. First, from a plasmid DNA (pET_gus) encoding wild type ⁇ -glucuronidase (base sequence shown in SEQ ID NO: 1), a DNA fragment (gus_wt) containing a T7 promoter sequence and a region encoding ⁇ -glucuronidase was PCR (30 cycles).
  • pET_gus plasmid DNA
  • gus_wt a DNA fragment containing a T7 promoter sequence and a region encoding ⁇ -glucuronidase was PCR (30 cycles).
  • the DNA polymerase was KOD FX (Toyobo), primer (1) (GAGTGCCGCCCGCAAGCTTTTA: SEQ ID NO: 3) and primer (2) (CCCGCGGAATATATACGAACTCACTATAGGTCGACTGTAGATATATGATTGATTAATATTTGTATATATTTTGTTATA Using gus_wt as a template, a primer (1) and a primer (2) were used to prepare a ⁇ -glucuronidase gene into which mutation was introduced by DNA polymerase for random mutagenesis (Mutazime II DNA polymerase, Agilent Technologies).
  • reaction solution for protein synthesis a reaction solution containing a cell-free protein synthesis system (PURE system: prepared by the inventors) and mt_gus was prepared. Reagents necessary for the preparation, the solution being prepared, and the reaction solution after the preparation were stored on ice. The composition of the reaction solution is shown in Table 1.
  • the main reagents include RNase inhibitor (Promega), T7 RNA polymerase (Takara Bio), transferrin Alexa 647 (Life Technologies Japan (former Invitrogen)), PFB-FDGlcU (5- (pentafluorbenzobenzolamino) fluorescein di- ⁇ -D-glucuronide) ) (Life Technologies Japan (former Invitrogen)) was used.
  • the DNA concentration in the reaction solution was 8 pM (in the case of screening for recovering liposomes from the 80 fL region) or 100 pM (in the case of screening for recovering liposomes from the 1 fL region).
  • a liquid paraffin solution (5 mg / mL, weight ratio 9: 1) of phospholipid (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, yorkipolar lipid) and cholesterol (Nacalai Tesque) was prepared. did.
  • the reaction solution for protein synthesis was added to the lipid solution at a rate of 5 vol%, and the mixture was vigorously stirred with a vortex mixer to prepare a W / O emulsion.
  • the W / O emulsion was layered on a buffer for liposome external solution, and this was centrifuged at 18000 ⁇ g (cooled to 4 ° C. during centrifugation) to obtain a liposome dispersion.
  • DNase I RNase-free, Takara Bio
  • 2.5 ⁇ L (12.5 units) per 100 ⁇ L of this dispersion was added to decompose the DNA existing outside the liposome.
  • the 80 fL region was set to a region where the fluorescence intensity of the reaction product> 1000 and the liposome size was 50 fL to 250 fL.
  • the 1 fL region was set to a region where the fluorescence intensity of the reaction product was> 1000 and the liposome size was 0.5 fL to 2 fL. Since the DNA concentration at the time of encapsulation was 8 pM (targeting 80 fL size) and 100 pM (targeting 1 fL size) so that the number of encapsulated genes per liposome was about one molecule, recovery of liposomes from the 80 fL region and the 1 fL region The liposomes were collected independently.
  • This plasmid DNA was introduced into competent cells (XL 10-Gold Ultracompetent Cells, Stratagene) and cultured on LB Agar plates containing ampicillin, and then colonies were recovered. Plasmid DNA was extracted from the colonies recovered using the QIAprep Spin Miniprep Kit. Introduction of the target gene in the recovered plasmid DNA was confirmed by agarose electrophoresis after treatment with restriction enzymes (NotI, BglII).
  • the amino acid sequence of the improved ⁇ -glucuronidase in which this C1102 was expressed was compared to the wild type ⁇ -glucuronidase, the 33rd arginine (Arg / R) from the N-terminal to the cysteine (Cys / C), and the 254th Valine (Val / V) is phenylalanine (Phe / F), 520th methionine (Met / M) is leucine (Leu / L), 561th leucine (Leu / L) is methionine (Met / M) (The amino acid sequence is shown in SEQ ID NO: 10).
  • the 30th T from the 5 ′ side is C
  • the 654th T is C
  • the 1050th T is C
  • the 1558th A is T
  • the 1681st The T of A was changed to A (the base sequence is shown in SEQ ID NO: 11).
  • the amino acid sequence of the improved ⁇ -glucuronidase in which this C1110 was expressed was such that the 520th methionine (Met / M) from the N-terminal side was leucine (Leu / L) and the 561st compared to the wild-type ⁇ -glucuronidase.
  • the leucine (Leu / L) in the above was changed to methionine (Met / M) (the amino acid sequence is shown in SEQ ID NO: 12).
  • the 113 th C from the 5 ′ side is T
  • the 663 th A is T
  • the 1133 th A is G
  • the 1328 th G is A
  • the 1331 th The 1458th A was changed to G
  • the 1551st C was changed to T
  • the 1682th T was changed to C
  • the 1686th C was changed to T (the base sequence is shown in SEQ ID NO: 13).
  • the amino acid sequence of the improved ⁇ -glucuronidase in which this e301 was expressed was compared with the wild-type ⁇ -glucuronidase, the 38th alanine (Ala / A) from the N-terminal side to the valine (Val / V).
  • Glutamine (Gln / Q) is histidine (His / H)
  • 378th glutamic acid (Glu / E) is glycine (Gly / G)
  • 443rd cysteine (Cys / C) is tyrosine (Tyr / Y).
  • the 444th valine (Val / V) was changed to alanine (Ala / A) and the 561st leucine (Leu / L) was changed to serine (Ser / S) (the amino acid sequence is shown in SEQ ID NO: 14). .
  • the 156th C from the 5 'side is T
  • the 579th C is T
  • the 705th A is G
  • the 1109th A is G
  • the 1352th C was changed to T
  • 1359th C was changed to A
  • 1425th C was changed to T
  • 1530th A was changed to G
  • 1682th T was changed to C (the base sequence is shown in SEQ ID NO: 15).
  • the amino acid sequence of the improved ⁇ -glucuronidase in which this e304 was expressed was such that the 370th lysine (Lys / K) from the N-terminal side to arginine (Arg / R) compared to the wild-type ⁇ -glucuronidase, the 451st The alanine (Ala / A) was changed to valine (Val / V) and the 561st leucine (Leu / L) was changed to serine (Ser / S) (the amino acid sequence is shown in SEQ ID NO: 16).
  • the 174th A from the 5 ′ side is T
  • the 619th G is A
  • the 676th A is T
  • the 756th G is C
  • the 1317th The T of A was changed to A
  • the 1525 th A was changed to G
  • the 1682 th T was changed to C
  • the 1800 th A was changed to G (the base sequence is shown in SEQ ID NO: 17).
  • the amino acid sequence of the improved ⁇ -glucuronidase in which this e314 was expressed was compared to the wild type ⁇ -glucuronidase, the 207th valine (Val / V) from the N-terminal side to the methionine (Met / M).
  • Threonine (Thr / T) is serine (Ser / S)
  • 509 threonine (Thr / T) is alanine (Ala / A)
  • 561 leucine (Leu / L) is serine (Ser / S) (The amino acid sequence is shown in SEQ ID NO: 18).
  • the 126 th C from the 5 ′ side is T
  • the 660 th T is A
  • the 1087 th A is G
  • the 1165 th T is C
  • the 1254 th The G of A was changed to A
  • the 1331st T was changed to C
  • the 1682th T was changed to C
  • the 1734th G was changed to A (the base sequence is shown in SEQ ID NO: 19).
  • the amino acid sequence of the improved ⁇ -glucuronidase in which this e321 was expressed was such that the 220th aspartic acid (Asp / D) from the N-terminal side to glutamic acid (Glu / E) was 363 as compared to the wild-type ⁇ -glucuronidase.
  • Isoleucine (Ile / I) is valine (Val / V)
  • 444th valine (Val / V) is alanine (Ala / A)
  • 561 leucine (Leu / L) is serine (Ser / S).
  • the amino acid sequence is shown in SEQ ID NO: 20).
  • a DNA fragment containing a T7 promoter sequence and a region encoding ⁇ -glucuronidase was used as a template with plasmid DNA encoding the cloned improved ⁇ -glucuronidase as a primer (4) (CCCGCGGAAATAATACGACTCAC: SEQ ID NO: 22) and primer (5)
  • a modified ⁇ -glucuronidase with His-tag was prepared by PCR reaction (30 cycles, DNA polymerase was KOD FX (Toyobo)) using (GAGTGCGGCCCAAGTCCATGGGTGGGTGTGTTGTTTGCCTCCCTGCTGCGTTTT: SEQ ID NO: 23).
  • This gene fragment was electrophoresed using E-Gel Clone Well (0.8% agarose gel, Life Technologies Japan (former Invitrogen)), and only the DNA fragment of the desired length was recovered and purified, and then the gene was obtained by PCR. The fragment was amplified (20 cycles, DNA polymerase was KOD FX (Toyobo)).
  • An improved ⁇ -glucuronidase gene fragment with His-tag was inserted into a DpnI-treated vector (a vector was prepared using pET_gusA as a template) by the In-fusion method (In-Fusion HD Cloning Kit, Clontech), and His-tag Plasmid DNA into which the attached gene was introduced was prepared.
  • This plasmid DNA was introduced into competent cells (XL 10-Gold Ultracompetent Cells, Stratagene) and cultured on LB Agar plates containing ampicillin, and then colonies were collected. Plasmid DNA was extracted from colonies collected using QIAprep Spin Miniprep Kit.
  • nucleotide sequence was decoded by the Dye Terminator method to confirm the introduction of the improved ⁇ -glucuronidase gene with His-tag.
  • the concentration gradient was as follows.
  • the concentrated sample was filtered through a 0.45 ⁇ m filter, and then applied to a HiLoad 16/600 Superdex 200 pg column equilibrated with a gel filtration buffer, followed by gel filtration.
  • the buffer composition used for gel filtration is shown below.
  • the initial reaction rate for each substrate concentration was determined from the change in fluorescence intensity over time.
  • the k cat and K m of wild-type ⁇ -glucuronidase and improved ⁇ -glucuronidase were evaluated.
  • FIG. 2 shows the time course of ⁇ -glucuronidase catalyzed reaction (substrate hydrolysis) associated with the translation reaction from the ⁇ -glucuronidase gene (100 pM DNA) in a test tube, and the improved ⁇ -glucuronidase of the present invention. It is a graph which compares and shows glucuronidase (C1102, C1110), a vertical axis
  • shaft is reaction product amount (fluorescence intensity) (a unit is arbitrary), and a horizontal axis is reaction time (minute).
  • FIG. 3 is a graph showing a comparison of the wild-type ⁇ -glucuronidase and the improved ⁇ -glucuronidase (C1102, C1110) of the present invention with respect to the reaction rate 60 minutes after the start of the reaction. Decomposition rate (after normalization).
  • Tables 4 and 5 show comparison of characteristic parameters (k cat , K m ) of wild type ⁇ -glucuronidase and improved ⁇ -glucuronidase (C1102, C1110), and Table 4 shows TG-bGlcU ( Table 5 shows the results of evaluation using PFB-FDGlcU as a substrate when evaluation was performed using Tokyo Green- ⁇ GlcU (Na), Sekisui Medical) as a substrate.
  • Table 6 also shows k t1 k t2 (rate at which ⁇ -glucuronidase is synthesized from DNA), k 1 2 k 2 (four) for wild-type ⁇ -glucuronidase and the improved ⁇ -glucuronidase (C1102, C1110) of the present invention. ( Mer formation rate) and k cat (enzyme activity).
  • FIG. 4 schematically shows the meanings of k t1 , k t2 , k 1 , k 2 , and k cat .
  • C1102 and C1110 which are improved ⁇ -glucuronidases having methionine at the 561st position from the N-terminal side of the amino acid sequence, are compared with wild-type ⁇ -glucuronidase. It can be seen that the catalytic activity is remarkably rapidly developed (the catalytic activity can be observed more rapidly than 20 minutes). This is because in the improved ⁇ -glucuronidase, the rate of tetramer formation is markedly higher than that of wild-type ⁇ -glucuronidase (as shown in Table 6, at least for C1110, k 1 2 k 2 (tetramer). This is considered to be because the formation speed) is about 10 times faster.
  • FIG. 5 shows the time course of ⁇ -glucuronidase catalyzed reaction (substrate hydrolysis) associated with the translation reaction from the ⁇ -glucuronidase gene (100 pM DNA) in vitro as well as C1102 and C1110 as improved ⁇ -glucuronidase. Also shown are improved ⁇ -glucuronidases (e301, e304, e314, e321) having serine at the 561st position from the N-terminal side of the amino acid sequence. From the results shown in FIG.
  • ⁇ Experimental example 2> An improved ⁇ -glucuronidase was created using liposomes as a micro reaction field. First, 100 ⁇ L of chloroform was added to 10 mg of lipid (POPC: Cholesterol 9: 1 (mass ratio)) and mixed well. Next, 2 mL of liquid paraffin was added and mixed well. The mixture was warmed at 80 ° C. for 30 minutes to volatilize chloroform and removed from the solvent as much as possible. Inner liquid, outer liquid, and diluted liquid were prepared on ice.
  • the encapsulating liquid is a reaction liquid encapsulated inside the liposome, and its composition is as follows.
  • DNA of various concentrations ( ⁇ -glucuronidase gene is encoded downstream of the T7 promoter: the nucleotide sequence of the gene encoding wild-type ⁇ -glucuronidase used in this experiment is SEQ ID NO: 24, and the amino acid sequence is SEQ ID NO: 25 Show.) ⁇ 330 mM sucrose ⁇ 1 ⁇ M Transfer from human serum, Alexa Fluor 647 conjugate (Life Technologies Japan) ⁇ 50 ⁇ M 5- (Pentafluorobenzoylamino) fluorescein di-beta-D-glucuronide (abbreviated as “PFB-FDGlcU”) (Life Technologies Japan) ⁇ 1 mM Phenylethyl beta-D-thiogalactopyranoside (PETG) ⁇ 5mU / ⁇ L T7 RNA polymerase (Takara Bio) ⁇ Cell-free protein synthesis system (PUREsystem)
  • the PFB-FDGlcU is a reagent for detecting ⁇ -
  • PFB-fluorescein (abbreviated as “PFB-F”), which is a green fluorescence product, is used. Arise.
  • PUREsystem The detailed composition of the cell-free protein synthesis system (PUREsystem) is described in Kita et al. , Replication of genetic information with self-encoded replicate in liposomes, Chembiochem, 9 (15), 2403-2410 (2008).
  • the external solution is a solution that exists outside the liposome during the synthesis of ⁇ -glucuronidase in the liposome, and is composed of 333 mM glucose, 1 mM PETG, and a low molecular component of a cell-free protein synthesis system.
  • glucose was added to the external liquid instead of sucrose in order to give a difference in specific gravity between the internal and external liposomes.
  • PETG and the low molecular weight component of the cell-free protein synthesis system leak from the liposome, they were also added to the external solution.
  • the diluent is a diluent used to adjust the concentration before measuring and fractionating the liposomes with a fluorescent cell sorter. 50 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate, 13 mM Mg (OAc) 2 Consists of
  • the DNA for the next round was prepared in a tube by RT-PCR using the selected RNA as a template.
  • a random mutation was artificially introduced into the ⁇ -glucuronidase gene by GeneMorph II Random Mutagenesis kit (Agilent Technologies).
  • evolution experiments were conducted up to round 44, and cloning and nucleotide sequence analysis were performed.
  • FIG. 6 is a graph showing the results of nucleotide sequence analysis of the obtained improved ⁇ -glucuronidase.
  • FIG. 6 (a) shows the average value (per clone) of non-synonymous substitutions of the improved ⁇ -glucuronidase (vertical). The axis is the average number of mutations [/ clone], and the horizontal axis is the number of selection steps (number of rounds).
  • FIG. 6 (b) shows the number of enriched non-synonymous substitutions (more than 50% of all clones 6 is a graph showing the number of rich mutations (the vertical axis is the number of rich mutations, the horizontal axis is the number of selection steps (number of rounds)), and FIG. (Ratio of clones having) (vertical axis is rich ratio (% total), horizontal axis is the number of selection steps (round number)).
  • C12 Two particularly active ones were selected and named “C12” and “C24”, respectively.
  • the analyzed base sequence of C12 is shown in SEQ ID NO: 26, and the amino acid sequence is shown in SEQ ID NO: 27, respectively.
  • the 59th aspartic acid (Asp / D) from the N-terminal side is glycine (Gly / G), and the 64th alanine (Ala / A) is valine (Val).
  • the analyzed base sequence of C24 is shown in SEQ ID NO: 28, and the amino acid sequence is shown in SEQ ID NO: 29, respectively.
  • the 59th aspartic acid (Asp / D) from the N-terminal side is asparagine (Asn / N), and the 64th alanine (Ala / A) is valine (Val).
  • ⁇ -glucuronidase synthesis and substrate degradation reaction were carried out using DNA and RNA as starting materials, respectively.
  • DNA of various concentrations ( ⁇ -glucuronidase gene (wild type, C12 or C24) is encoded downstream of the T7 promoter) ⁇ 330 mM sucrose ⁇ 1 ⁇ M Transfer from human serum, Alexa Fluor 647 conjugate (Life Technologies Japan) ⁇ 50 ⁇ M 5- (Pentafluorobenzoylamino) fluorescein di-beta-D-glucuronide (PFB-FDGlcU) (Life Technologies Japan) ⁇ 1 mM Phenylethyl beta-D-thiogalactopyranoside (PETG) ⁇ 5mU / ⁇ L T7 RNA polymerase (Takara Bio) ⁇ Cell-free protein synthesis (PUREsystem) Using the above reaction solution, the following flow reaction (T7 RNA polymerase transcribes mRNA from DNA.
  • T7 RNA polymerase transcribes mRNA from DNA.
  • FIG. 7 is a graph showing the results of in-tube ⁇ -glucuronidase synthesis reaction (DNA start) for wild type, C12, and C24.
  • FIG. 7 (a) is for 50 pM DNA
  • FIG. 7 (b) is 1000 pM DNA. Shows the case. 7 (a) and (b), the vertical axis represents the green fluorescence intensity, the horizontal axis represents the reaction time (minutes), and from the results shown in FIGS. 7 (a) and (b), C12 and C24 are It can be seen that it has a high activity in a much shorter time.
  • RNA synthesis using various concentrations of RNA DNA for ⁇ -glucuronidase synthesis (wild type, C12 or C24) using T7 RNA polymerase and standard buffer (Takara Bio), using RNeasy Mini Kit (Qiagen) Purified product.
  • RNA DNA for ⁇ -glucuronidase synthesis (wild type, C12 or C24) using T7 RNA polymerase and standard buffer (Takara Bio), using RNeasy Mini Kit (Qiagen) Purified product.
  • RNA DNA for ⁇ -glucuronidase synthesis (wild type, C12 or C24) using T7 RNA polymerase and standard buffer (Takara Bio), using RNeasy Mini Kit (Qiagen) Purified product.
  • ⁇ ⁇ -glucuronidase forms a homotetramer with activity.
  • ⁇ PFB-FDGlcU by ⁇ -glucuronidase. Is decomposed to produce a green fluorescent product (PFBF).
  • PFBF green fluorescent product
  • the green fluorescence intensity (Ex. 492 nm, Em. 516 nm) derived from the reaction product PFB-F is measured by Mx3005P qPCR System (Agilent Technology). did.
  • FIG. 8 is a graph showing the results of in-tube ⁇ -glucuronidase synthesis reaction (RNA start) for wild type, C12, and C24.
  • FIG. 8 (a) is for 50 pM RNA
  • FIG. 8 (b) is 500 pM RNA.
  • FIG. 8 (c) shows the case of 5000 pM RNA.
  • the vertical axis represents the green fluorescence intensity
  • the horizontal axis represents the reaction time (minutes). From the results shown in FIGS. 8 (a), (b) and (c) It can be seen that C12 and C24 have a higher activity in a much shorter time than the wild type.

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Abstract

L'invention concerne une β-glucuronidase modifiée dans laquelle une mutation a été introduite dans la séquence d'acides aminés d'un monomère de β-glucuronidase, et dans laquelle la β-glucuronidase modifiée est mutée et nouvelle et comporte une méthionine ou une sérine à la 561ème position à partir de l'extrémité N-terminale de la séquence d'acides aminés, et étant donné qu'elle est une β-glucuronidase modifiée, présente une activité enzymatique élevée par comparaison avec la forme sauvage, et est facilement détectée.
PCT/JP2014/069539 2013-07-29 2014-07-24 Β-glucuronidase modifiée WO2015016124A1 (fr)

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US9719075B2 (en) 2014-09-29 2017-08-01 Integrated Micro-Chromatography Systems Mutant Staphylococcus beta-glucuronidase enzymes with enhanced enzymatic activity
US9909111B2 (en) 2016-03-21 2018-03-06 Integrated Micro-Chromatography Systems Mutant lactobacillus beta-glucuronidase enzymes with enhanced enzymatic activity
US9920306B2 (en) 2014-09-29 2018-03-20 Integrated Micro-Chromatography Systems, Llc Mutant β-glucuronidase enzymes with enhanced enzymatic activity
US11268079B2 (en) 2018-08-01 2022-03-08 Integrated Micro-Chromatography Systems, Inc. Compositions of beta-glucuronidase enzyme blends with enhanced enzymatic activity and methods of preparation thereof
US11421210B2 (en) 2018-10-08 2022-08-23 Integrated Micro-Chromatography Systems, Inc. Chimeric and other variant beta-glucuronidase enzymes with enhanced properties

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9719075B2 (en) 2014-09-29 2017-08-01 Integrated Micro-Chromatography Systems Mutant Staphylococcus beta-glucuronidase enzymes with enhanced enzymatic activity
US9920306B2 (en) 2014-09-29 2018-03-20 Integrated Micro-Chromatography Systems, Llc Mutant β-glucuronidase enzymes with enhanced enzymatic activity
US9909111B2 (en) 2016-03-21 2018-03-06 Integrated Micro-Chromatography Systems Mutant lactobacillus beta-glucuronidase enzymes with enhanced enzymatic activity
US11268079B2 (en) 2018-08-01 2022-03-08 Integrated Micro-Chromatography Systems, Inc. Compositions of beta-glucuronidase enzyme blends with enhanced enzymatic activity and methods of preparation thereof
US11807879B2 (en) 2018-08-01 2023-11-07 Integrated Micro-Chromatography Systems, Inc. Compositions of beta-glucuronidase enzyme blends with enhanced enzymatic activity and methods of preparation thereof
US11421210B2 (en) 2018-10-08 2022-08-23 Integrated Micro-Chromatography Systems, Inc. Chimeric and other variant beta-glucuronidase enzymes with enhanced properties

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