JP4437652B2 - Recombinant bilirubin oxidase and method for producing the same - Google Patents

Description

  The present invention relates to bilirubin oxidase, particularly a recombinant bilirubin oxidase, which is an important enzyme as a reagent for clinical laboratory analysis, and a method for producing the same, and further to quantification of bilirubin as a clinical analysis of recombinant bilirubin oxidase. It relates to a measurement method.

  Bilirubin oxidase has been used in clinical laboratory analysis for the purpose of quantifying bilirubin. Conventionally, bilirubin oxidase was extracted from the incomplete filamentous fungus Myrothecium verrucaria MT-1 and partially purified. Enzyme preparation [hereinafter referred to as wild-type enzyme (wtBO). ] Has been used.

  However, this wild type enzyme has a problem that the enzyme activity is unstable. For this reason, as one proposal for stabilizing bilirubin oxidase, a method of adding a combination of an amino acid and an organic acid salt (see, for example, Patent Document 1) or a method of solving by adding polycarboxylic acids ( For example, see Patent Document 2).

On the other hand, determination of the base sequence of DNA encoding the gene of bilirubin oxidase, construction of an expression system using yeast incorporating the DNA encoding the gene, and collection of the expressed enzyme have also been attempted (for example, patent documents). 3 and non-patent document 1).
JP-A-6-284886 JP-A-8-66196 Japanese Patent Laid-Open No. 5-199882 Koikeda, S., Ando, K., Kaji, H., Inoue, T., Murao, S., Takeuti, K., and Samejima, T., Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast. (1993) J. Biol. Chem. 268, 18801-09.

  However, in the method of adding an additive for stabilization as in the former case, the additive may adversely affect the measurement, and sufficient stabilization is not necessarily realized. On the other hand, in the case of the latter, based on a publicly available method, the expression level of bilirubin oxidase is low, and it has not been put into practical use for clinical laboratory analysis by isolating and purifying the expressed enzyme. That is, until now, recombinant bilirubin oxidase important for clinical examination has not been supplied to the market.

  As described above, since the bilirubin oxidase conventionally used in clinical examinations has the disadvantage of being unstable, there is a method for supplying bilirubin oxidase having more stable activity and suitable for the conditions of clinical examination. It was necessary. Therefore, the present invention has been proposed in view of such circumstances, and an object thereof is to provide a recombinant bilirubin oxidase having a more stable activity than the conventionally used bilirubin oxidase, It aims at providing the quantitative measurement method of bilirubin using this. It is another object of the present invention to provide a method for producing bilirubin oxidase, which can easily isolate and purify such recombinant bilirubin oxidase and can be mass-produced.

The bilirubin oxidase of the present invention is a recombinant bilirubin oxidase, and the bilirubin oxidase gene derived from the incomplete filamentous fungus Myrothecium verrucaria MT-1 described in SEQ ID NO: 1 is designated as pichia (pichia). ) It is obtained by introducing and expressing in Pichia pastoris GS115 strain which is a yeast of the genus, and the amino terminal sequence is YVEFVAQISPQ.

For example, by transforming by introducing a bilirubin oxidase gene (SEQ ID NO: 1) derived from the imperfect filamentous fungus Myrothercium verrucaria MT-1 using Pichia yeast as a host cell. Compared with the wild-type enzyme (wtBO), which is a conventional enzyme preparation, or as a recombinant enzyme (rBO _Aspergillus ) with a conventional incomplete filamentous fungus as a host, it is stable and difficult to inactivate. Recombinant bilirubin oxidase is realized.

On the other hand, the method for producing bilirubin oxidase of the present invention is a method for producing recombinant bilirubin oxidase, which is an incomplete filamentous fungus Myrotesium verkaria MT-1 described in SEQ ID NO: 1. The derived bilirubin oxidase gene is transformed by introducing Pichia pastoris GS115, which is a yeast of the genus Pichia, as a host animal.

  By using yeast as a host cell and introducing the gene for transformation, the above-mentioned stable recombinant bilirubin oxidase can be produced in large quantities and efficiently. Selection of a yeast strain as a host is performed for the purpose of increasing production efficiency.

  Furthermore, the method for quantitatively measuring bilirubin according to the present invention is characterized in that bilirubin contained in an aqueous solution, particularly blood, serum, plasma, bile fluid or urine, is quantified using the aforementioned stable recombinant bilirubin oxidase. And

  By using the stable recombinant bilirubin oxidase described above, bilirubin present in the aqueous solution can be easily quantified. The enzyme can also be used as a clinical test reagent for measuring bilirubin contained in blood, bile fluid and the like.

  According to the present invention, a recombinant bilirubin oxidase having a more stable enzyme activity than before can be obtained by using yeast as a host cell for gene recombination. Moreover, according to the method for producing bilirubin oxidase of the present invention, a large amount of the stable bilirubin oxidase can be provided by a very simple and economical method. Therefore, for example, in clinical laboratories, important clinical laboratory measurements using bilirubin oxidase as a reagent can be performed at low cost.

  Hereinafter, bilirubin oxidase to which the present invention is applied, a method for producing the enzyme, and a method for quantitatively measuring bilirubin will be described.

In order to produce the recombinant bilirubin oxidase of the present invention, first, total RNA was extracted from the imperfect filamentous fungus Myrothecium verrucaria MT-1 and designed from the bilirubin oxidase cDNA sequence described in Non-Patent Document 1. Using the synthetic oligonucleotide primer, bilirubin oxidase cDNA is synthesized by reverse transcription reaction. After amplification of the cDNA by PCR, the amplified gene fragment is ligated to a plasmid to construct a primary plasmid incorporating the enzyme gene. Candidate plasmids used herein, the ease of operation, pBluescriptII SK ^- (Stratagene, Inc.) and the like. Here, it is desirable to analyze the base sequence of the primary plasmid and confirm that the inserted bilirubin oxidase gene is identical to the previously reported sequence.

  In order to introduce a large amount of bilirubin oxidase by introducing the constructed plasmid containing the bilirubin oxidase gene into a heterologous host cell, a vector suitable for the target host cell is reconstructed. After introducing the constructed vector into a host cell, for example, by electroporation, selection of the gene-introduced cell is performed. Here, as a host animal, yeast is desirable from the viewpoint of mass expression, and yeast of the genus Pichia is particularly desirable. Further, as an expression vector, pPIC9K (manufactured by Invitrogen) is desirable, and it is further desirable to provide a selection marker in the vector in order to facilitate selection of gene-transferred cells. Examples of selection markers include drug resistance genes.

The following text describes the case where yeast is used as the host. The transgenic cell (yeast) can be cultured, and the recombinant bilirubin oxidase present in the culture supernatant can be recovered. Here, the culture medium used for the culture is preferably a BMM minimal medium containing 0.2 mM CuCl ₂ , and further, it is desirable that methanol having a final concentration of 0.5% be present as an expression induction substrate.

  Recombinant bilirubin oxidase can be purified from the collected culture supernatant. Examples of the purification method include adsorption on a hydrophobic column, separation by salt gradient reverse gradient chromatography, and gel filtration chromatography, and a combination of these results in good separation and purification results.

  The molecular weight of the recombinant bilirubin oxidase after separation can be measured by SDS-PAGE method or gel filtration method. Further, by analyzing the amino terminal sequence of the enzyme, it can be confirmed that the amino acid terminal sequence is as designed. Furthermore, the optimum pH and pH stability of the enzyme can be verified by measuring the reaction rate while varying the pH value in the buffer solution. In addition, the optimum temperature and thermal stability of the enzyme can be verified by measuring changes in enzyme activity while varying the temperature in the buffer.

  Recombinant bilirubin oxidase produced by the above operations has higher stability than wild-type enzyme, and it is used as a clinical test reagent in aqueous solution, especially in the blood, serum, plasma, bile fluid or urine. This is extremely useful when performing quantification.

  Next, specific examples of the present invention will be described based on experimental results. Needless to say, the present invention is not limited to these examples.

1. CDNA cloning of bilirubin oxidase cDNA imperfect filamentous fungus Myrothecium verrucaria MT-1 strain was cultured according to the method of Non-Patent Document 1, then collected and wet cells were ground under liquid nitrogen. Using Dynabeads mRNA DIRECT Kit (manufactured by Dynal) from about 100 mg of frozen bacterial powder, 20 μl of total mRNA extraction solution was prepared according to the attached manual. Using the total amount, a reverse transcription reaction was performed with a First-Strand cDNA Synthesis Kit (Amersham Biosciences) according to the manual. Synthetic oligonucleotide primers BO-EcoN (SEQ ID NO: 2) and BO-PstC (sequence) designed from the base sequence of bilirubin oxidase cDNA described in Non-Patent Document 1 using 5 μl of the reverse transcription reaction solution (total volume 33 μl) as a template PCR (polymerase chain reaction) was performed using No. 3).

  Primer BO-EcoN is obtained by adding an EcoRI cleavage sequence (underlined) to the 5 ′ side of the codon encoding the amino terminal Val of mature bilirubin oxidase. Primer BO-PstC is obtained by adding a PstI cleavage sequence (underlined) to the 3 ′ side of the termination codon of mature bilirubin oxidase. PCR reaction solution composition is 5 μl of reverse transcription reaction solution, 5 μl of primer BO-EcoN, 5 μl of primer BO-PstC, 4 μl of dNTP mixture, 5 μl of 10X EX Taq buffer, Takara EX Taq DNA polymerase (manufactured by Takara Biochemicals) 0.25 μl, distilled water 30.75 μl, total volume 50 μl. The PCR reaction was carried out for 26 cycles with denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 1 minute, extension reaction at 72 ° C. for 5 minutes as one cycle. As a result, an amplified gene DNA fragment encoding 1,620 bp mature bilirubin oxidase was obtained.

Next, the PCR amplified gene fragment restriction enzymes EcoRI, was digested with PstI, digested with the same enzymes, purified plasmid pBluescriptII SK ^- linked to (Stratagene Co.) to prepare a plasmid PBSBO. The base sequence of pBSBO was analyzed by Hitachi SQ5500E DNA Sequencer using Thermo Sequenase Primer Cycle Sequencing Kit (manufactured by Amersham Biosciences), and it was confirmed that the bilirubin oxidase gene was identical to the previously reported sequence.

2. Construction of heterologous expression system using bilirubin oxidase with Pichia pastoris After digesting pBSBO with restriction enzymes EcoRI and Not I, the product DNA was separated by agarose gel electrophoresis, and then Prep-A-Gene DNA Purification Kit (Bio-Rad And a bilirubin oxidase gene fragment (1,650 bp, SEQ ID NO: 1) was extracted and purified. This extracted DNA fragment was digested with the same restriction enzyme and ligated to a purified plasmid pPIC9K (manufactured by Invitrogen) to prepare a plasmid for yeast transformation (pPICBO).

Fig. 1 shows a schematic diagram of the fabricated pPICBO. In the figure, reference numeral 1 is an ampicillin resistance gene (amp ^r ), reference numeral 2 is a replication origin (ColE1ori), reference numeral 3 is an alcohol oxidase gene fragment (3′AOX1), and reference numeral 4 is , Kanamycin resistance gene (kan ^r ), 5 is a histidine production gene (his4), 6 is a transcription termination sequence (terminator), and 7 is a bilirubin oxidase gene (bo) 8 is an α factor secretion signal coding sequence (α-signal), and 9 is an alcohol oxidase gene fragment (5′AOX1). According to the manual of Multi-Copy Pichia Expression Kit (manufactured by Invitrogen), pPICBO was opened with restriction enzyme Bpu1102I, and yeast Pichia pastoris GS115 (manufactured by Research Corporation Technologies, Inc.) was transformed by electroporation. This transformed yeast is a His ⁺ (histidine non-requiring) strain.

3. Culture of transformant and screening of transformant An appropriate amount of the transformed yeast (His ⁺ ) was applied to an MD agar plate medium (Table 1), and cultured at 30 ° C. overnight to be sufficiently grown. After sterilizing water was added to this plate and transformed yeast was suspended, 10 ⁵ cells per one plate were applied to a YPD agar plate medium (Table 1) containing an appropriate amount of antibiotic G418 and cultured at 30 ° C.

  A transformed yeast grown on a YPD agar plate containing 25 mg / ml G418 was transformed again with pPICBO opened with the restriction enzyme Bpu1102I, and colonies showing resistance to 1.5 mg / ml G418 by the same procedure. (G418 highly resistant strain) was screened.

4). Expression of Recombinant Bilirubin Oxidase (rBO _Pichia ) The G418 highly resistant strain was inoculated into 4 ml of YPD liquid medium, and shaken in a test tube at 30 ° C. for 24 hours to prepare a seed culture. 4 ml of the seed culture yeast suspension was inoculated into 100 ml of YPD liquid medium in a 500 ml Sakaguchi flask and pre-cultured by shaking culture at 30 ° C. for 24 hours. The precultured yeast was collected by centrifugation, resuspended in an appropriate amount of BMM minimal liquid medium, and then inoculated in a total amount into 1 L of BMM minimal medium (5 L Erlenmeyer flask) containing 0.2 mM CuCl ₂ . The culture was shaken at 25 ° C. for 5 days while adding 0.5% final concentration of methanol as an expression induction substrate every 24 hours. By this methanol induction, recombinant bilirubin oxidase (rBO _Pichia ) was induced and released into the culture medium. Bacteria were removed from the total 16 L culture solution by centrifugation, and the culture supernatant was recovered to obtain a crude enzyme solution.

5. RBO _Pichia Purification of the addition of a final concentration of 30% ammonium sulfate in the culture supernatant of, TOYOPEARL-Butyl 650M column pre-equilibrated with 50mM potassium phosphate buffer containing 30% ammonium sulfate (pH6.0) (φ4 6 × 16 cm, manufactured by TOSOH). After washing the column with the same buffer, rBO _Pichia was eluted by a linear concentration gradient method (total volume 1 L) in which the ammonium sulfate concentration of the buffer was changed from 30% to 0%. The active fractions were collected and concentrated by ultrafiltration. Next, the concentrated active fraction was layered on a HiLoad Superdex 200 pg 26/60 column (φ2.6 × 60 cm, manufactured by Amersham Biosciences) equilibrated with 100 mM potassium phosphate buffer (pH 6.0). Gel filtration chromatography was performed using the same buffer at a flow rate of 2 ml / min. The active fractions were collected and tested for purity by SDS electrophoresis. As a result, a single band having a molecular weight of 66 kDa was confirmed, and this fraction was used as a purified enzyme preparation. The yield of the purified enzyme was 30 mg per liter of the culture solution, and a large amount of highly active enzyme could be obtained very easily compared with the conventional method.

6). Using the amino terminal sequence of rBO _Pichia and 130 pmol of a molecular weight purified enzyme preparation, the amino terminal sequence was determined using an Applied Biosystems Model 476A protein sequencer. As a result, the sequence of YVEFVAQISPQ was obtained. This sequence consists of four residues of YVEF corresponding to the restriction enzyme EcoRI recognition sequence from the Ste13 signal cleavage site in the α factor secretion signal coding sequence encoded by pPIC9K. It matched the sequence added to the amino terminal sequence VAQISPQ- of the mature protein. Therefore, it was revealed that the purified enzyme had the designed amino terminal sequence. When the molecular weight of the purified enzyme was determined using a Superose 12 HR 10/30 column (φ1.0 × 30 cm, manufactured by Amersham Biosciences), it was 65 kDa, which is a good 66 kDa, which is the molecular weight measurement result obtained by SDS gel electrophoresis. Matched. Therefore, the purified enzyme preparation rBO _Pichia was a monomeric soluble protein.

7). Spectroscopic properties of rBO _Pichia
(A) Absorption spectrum: The absorption spectrum of rBO _Pichia in 100 mM potassium phosphate buffer (pH 6.0) is shown in FIG. spectrum of RBO _Pichia are described previously (... Gotoh Y., et al, J. Biochem 106, 621-626, 1989) similar to the spectrum of wtBO and conventional recombinant enzyme RBO _Aspergillus of, Cys (S ^- ) Showed an absorption maximum at 600 nm corresponding to charge transfer absorption from Cu ²⁺ , and its molecular extinction coefficient (ε = 4,300) showed the same value as wtBO. A shoulder peak near 330 nm derived from type 3 copper was also observed.

(B) EPR spectrum: The EPR spectrum of rBO _Pichia in 100 mM potassium phosphate buffer (pH 6.0) is shown in FIG. The rBO _Pichia EPR spectrum is in good agreement with the rBO _Aspergillus spectrum, the parameters for type 1 copper are g _// = 2.22, A _// = 8.33x10 ^-3 cm ^-1 , the parameters for type 2 copper are g _{/ /} = 2.31, A _// = 10.2x10 ^-3 cm ^-1 and no change.

(C) Atomic absorption spectrum: As a result of atomic absorption spectrum analysis, rBO _Pichia was found to contain 3.5 copper ions per molecule. Therefore, like wtBO and rBO _Aspergillus , it was considered that each enzyme molecule contained a total of four copper ions: one type 1 copper, one type 2 copper, and two type 3 coppers. From the above spectroscopic measurement results, it became clear that rBO _Pichia has the same copper active center structure as wtBO and rBO _Aspergillus .

8). Enzyme Chemical Properties of BO _Pichia Standard (a) Enzyme activity: Enzyme activity was determined by using a substrate solution of 50 mM Tris-H ₂ SO ₄ buffer (pH 7.8) / 30 μM bilirubin and absorption at 440 nm at 37 ° C. It was measured by monitoring the decrease over time. The amount of enzyme that oxidizes 1 μmol of bilirubin in 1 minute was defined as 1 unit. For protein quantification, BCA Protein Assay Reagent (manufactured by PIERCE) was used, and the concentration was determined by a calibration curve prepared using bovine serum albumin as a standard. The activity of the purified rBO _Pichia was 26 U / mg, which was equal to or better than the reported wtBO and rBO _Aspergillus . Further, when the substrate concentration dependency of the reaction rate was examined, a sigmoid curve as shown in FIG. 4 was shown. Further, from the Hill plot, it became clear that rBO _Pichia showed a Hill constant of 1.56 and a high positive cooperativity compared to wtBO with a Hill constant of 1.14.

(A) Optimum pH and pH stability: MES-Bicine-CAPS mixed buffer adjusted to 0.2 M ionic strength was used to plot the reaction rate at pH 6 to pH 11. As shown in FIG. 5, rBO _Pichia A bell-shaped curve having a peak at around pH 7.5 was obtained for both of wtBO and wtBO, and the optimum pH of both enzymes was consistent. Moreover, as a result of comparing the pH stability of rBO _Pichia in the treatment at 25 ° C. for 10 minutes with wtBO using the same buffer (pH 4 to pH 12), both enzymes were inactivated at pH 6 to pH 11 as shown in FIG. Was not found, and was found to be stable in this pH range.

(C) Thermal stability: As a result of investigating the thermal stability of rBO _Pichia in 50 mM Tris-H ₂ SO ₄ buffer (pH 7.8) No decrease in rBO _Pichia activity was observed. In addition, as a result of examining the time course of enzyme activity during heat treatment at 60 ° C. in 50 mM Tris-H ₂ SO ₄ buffer (pH 7.8), the activity half-life (t _1/2 ) of rBO _Pichia was as long as 90 minutes. The value is 6 times 15 times wtBO. From these results, it is clear that rBO _Pichia has higher thermal stability than wtBO.

  As described above, according to the method for producing a recombinant bilirubin oxidase of the present invention, a heat-stable recombinant enzyme can be easily obtained compared to a wild-type enzyme or a recombinant enzyme produced by a conventional technique. And it became clear that it can be manufactured in large quantities. Further, the stability exhibited by the recombinant bilirubin oxidase of the present invention was analyzed by molecular weight measurement, amino acid sequence analysis, and the like, and as a result, was considered to be caused by differences in sugar chain modification.

  In addition, since the recombinant bilirubin oxidase of the present invention is more stable than the conventional wild-type enzyme, it is contained in an aqueous solution, particularly blood, serum, plasma, bile fluid, or urine as a reagent for clinical testing. This is extremely useful when quantifying bilirubin.

It is the gene schematic of a bilirubin oxidase expression plasmid. It is an absorption spectrum of recombinant bilirubin oxidase. It is an EPR spectrum of recombinant bilirubin oxidase. It is a graph which shows the substrate saturation curve with respect to bilirubin of a recombinant bilirubin oxidase. It is a graph which shows the influence of pH with respect to the activity of recombinant bilirubin oxidase. It is a graph which shows the pH stability of recombinant type bilirubin oxidase.

Explanation of symbols

1 Ampicillin resistance gene (amp ^r )
2 Replication start point (ColE1ori)
3 Alcohol oxidase gene fragment (3'AOX1)
4 Kanamycin resistance gene (kan ^r )
5 Histidine production gene (his4)
6 Transcription termination sequence (terminator)
7 Bilirubin oxidase gene (bo)
8 α-factor secretion signal coding sequence (α-signal)
9 Alcohol oxidase gene fragment (5'AOX1)

Claims (4)

Recombinant bilirubin oxidase, and the bilirubin oxidase gene derived from the incomplete filamentous fungus Myrothecium verrucaria MT-1 described in SEQ ID NO: 1 is Pichia pastoris, a yeast of the genus Pichia A bilirubin oxidase obtained by introducing and expressing in the GS115 strain, wherein the amino terminal sequence is YVEFVAQISPQ . A method for producing a recombinant bilirubin oxidase, wherein a bilirubin oxidase gene derived from the incomplete filamentous fungus Myrothecium verrucaria MT-1 described in SEQ ID NO: 1 is assigned to the genus Pichia. A method for producing bilirubin oxidase, comprising transforming by introducing a yeast Pichia pastoris GS115 strain as a host animal . A method for quantitatively measuring bilirubin, characterized in that bilirubin in an aqueous solution is quantified using the bilirubin oxidase according to claim 1 . The method for quantifying bilirubin according to claim 3 , wherein the aqueous solution is blood, serum, plasma, bile, or urine.

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