CN106397578A - Production method of hemoprotein - Google Patents
Production method of hemoprotein Download PDFInfo
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- CN106397578A CN106397578A CN201610616389.1A CN201610616389A CN106397578A CN 106397578 A CN106397578 A CN 106397578A CN 201610616389 A CN201610616389 A CN 201610616389A CN 106397578 A CN106397578 A CN 106397578A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/80—Cytochromes
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Abstract
The present invention provides a production method of hemoprotein. The method comprises administering at least one selected from the group consisting of a heme precursor and a heme analog to a lepidopteran insect individual to which a polynucleotide encoding an amino acid sequence encoding a heme protein is introduced, And a step of obtaining the hemoglobin protein from the lepidoptera individual.
Description
【Technical field】
The present invention relates to the production method of hemoprotein.
【Background technology】
Carry out using the volume production of the useful human protein of microbial cell etc. in the past.Using volume productions such as Bacillus coli cells
The technology of the Cytochrome P450 as the hemoprotein related to drug metabolism etc. is also one of them.United States Patent (USP) Shen
2005-0130116 disclosure please be disclose, in escherichia expression system, by interpolation as the similar body of ferroheme
The method of the Cytochrome P450 of bag ferroheme in hemin manufacture.
【Content of the invention】
【Invention technical task to be solved】
As U.S. Patent Application Publication No. 2005-0130116 using cultured cells in the medium as necessary
Technology in, have the necessity applying substantial amounts of hemin.The present inventor is not as using culture medium as necessary expression system
System, pays close attention to silkworm expression system.The present inventor with silkworm expression system manufacture hemoprotein recombinant when, for by virus
The yield of the protein of method induced expression of infection etc., follows the trail of the endogenic ferroheme synthesis having less than silkworm, knot
Fruit finds, has the problem of the protein mainly producing shortcoming ferroheme.Culture expression in Escherichia coli or insect cell etc.
In system, take by adding, in culture medium, the method that co-factor supplements ferroheme, but do not carry out in silkworm expression system like that
Discussion.
【Solve the technical scheme of problem】
The present inventor repeat discussion with keen determination it was found that by recombinant protein expression induction after Lepidoptera elder brother
Worm is individual to apply ferroheme precursor or ferroheme analog, and the modulation of the hemoprotein of active form of interior bag ferroheme becomes
May, thus can solve above-mentioned problem, thus completing the present invention.
That is, provided the production method of hemoprotein by the present invention, it includes:To having imported coding hemoprotein
The polynucleotides of amino acid sequence lepidopterous insects individual apply selected from ferroheme precursor and ferroheme analog at least 1
Kind, described lepidopterous insects individuality makes the operation that hemoprotein produces;And individual from described lepidopterous insects, obtain
The operation of described hemoprotein.
In addition, providing the composition containing Cytochrome P450, cytochrome P450 reductase and cytochromes by the present invention
Production method, it includes polynucleotides and Codocyte pigment to the amino acid sequence having imported Codocyte cytochrome p 450
1st lepidopterous insects of the polynucleotides of the amino acid sequence of P450 reductase are individual and imported the many of Codocyte pigment
The respective administration of the 2nd lepidopterous insects individuality of nucleotides is selected from least a kind of ferroheme precursor and ferroheme analog, makes
The operation of hemoprotein is produced in described lepidopterous insects individuality;Individual from described 1st and the 2nd lepidopterous insects, each
Obtain the 1st microsomal fraction containing Cytochrome P450 and cytochrome P450 reductase and the 2nd microsome containing cytochromes
The operation of fraction;Make described 2nd microsomal fraction solubilising, obtain the operation of solubilized fraction;By described 1st microsomal fraction and institute
State solubilized fraction mixing, obtain the operation of composition.
【Invention effect】
The active form of bag ferroheme in producing in the protein production systems using lepidopterous insects is provided by the present invention
Hemoprotein method.
【Brief description】
【Fig. 1】It is the Vector map of carrier pM01.
【Fig. 2】It is the figure showing the extinction spectrum purifying CYP3A4 protein solution.
【Fig. 3】It is the photo of the hemin distribution in the silkworm chrysalis after showing hemin administration.
【Fig. 4】It is the microsome level showing from the pupa modulation applying hemin after virus infection for the 4th day or the 5th day
The coordinate diagram of the CYP3A4 metabolic activity dividing.
【Fig. 5】It is shown in after virus has infected, applying within the 3rd day or the 4th day the microsome level of the pupa modulation of hemin
The coordinate diagram of the CYP3A4 metabolic activity dividing.
【Fig. 6】It is the CYP3A4 metabolism showing the microsomal fraction from the pupa modulation applying amino-laevulic acid and iron ion
The coordinate diagram of activity.
【Fig. 7】It is the CYP3A4 generation of the microsomal fraction showing the pupa modulation from the amino-laevulic acid applying various concentration
Thank to the coordinate diagram of activity.
【Fig. 8】It is the microsomal fraction showing from the pupa modulation applying hemin and/or amino-laevulic acid
The coordinate diagram of CYP3A4 metabolic activity.
【Fig. 9】It is the extinction spectrum showing the CYP3A4 protein solution purifying from the pupa applying amino-laevulic acid
Figure.
【Figure 10】It is the extinction spectrum showing the cytochrome b5 protein solution purifying from the pupa applying hemin
Figure.
【Figure 11】It is the coordinate diagram showing the CYP3A4 metabolic activity of microsomal fraction adding cytochrome b5.
【Figure 12】It is the electrophoretogram of silkworm source microsomal fraction and commercially available microsomal fraction.
【Figure 13】It is the microsomal fraction showing from the pupa modulation applying hemin and amino-laevulic acid
The coordinate diagram of CYP1A2 metabolic activity.
【Figure 14】It is the microsomal fraction showing from the pupa modulation applying hemin and amino-laevulic acid
The coordinate diagram of CYP2C8 metabolic activity.
【Figure 15】It is the microsomal fraction showing from the pupa modulation applying hemin and amino-laevulic acid
The coordinate diagram of CYP2C9 metabolic activity.
【Figure 16】It is the microsomal fraction showing from the pupa modulation applying hemin and amino-laevulic acid
The coordinate diagram of CYP2C19 metabolic activity.
【Figure 17】It is the microsomal fraction showing from the pupa modulation applying hemin and amino-laevulic acid
The coordinate diagram of CYP2D6 metabolic activity.
【Embodiment】
The production method of the hemoprotein of the present invention is included to the amino acid sequence importing coding hemoprotein
Polynucleotides individual at least a kind applying selected from ferroheme precursor and ferroheme analog of lepidopterous insects, in Lepidoptera
The operation that hemoprotein produces is made in insect individuality.
As long as hemoprotein is combined with being referred to as the holoprotein of the porphyrin iron complex of ferroheme, just not special
Limit.As hemoprotein, such as Cytochrome P450, cytochromes, hemoglobin, myoglobins, peroxide can be enumerated
Compound enzyme, cytochrome c, cytochrome b, CooA, HemT etc..In the present embodiment, hemoprotein is preferably cell
Cytochrome p 450 and/or cytochromes.Cytochrome P450 be preferably CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19、CYP2D6、CYP2E1、CYP3A5、CYP2J2、CYP4F2、CYP2C9*2、CYP2C9*3、CYP2D6*10、
CYP2D6*39.Furthermore, " * " represents Gene polymorphism.Cytochromes are preferably cytochrome b, cytochrome c.Specifically, may be used
Illustrate cytochrome b5 (hereinafter also referred to " cytochrome b5 ").
In addition, the combination of ferroheme in hemoprotein and apolipoprotein is not particularly limited.For example, can be to load
Emptying aperture (sometimes referred to as heme pocket) coordination bonding of lipoprotein, also can be tied by hydrophobic interaction or electrostatic interaction
Close.Stereochemical structure parsing is carried out for a part of hemoprotein, thus it presents and wraps in apolipoprotein with ferroheme
The mode of emptying aperture combine, so in the art, sometimes the amino acid sequence part of hemoprotein (is carried fat
Albumen) and ferroheme combine and hemoproteinization be referred to as " interior bag ferroheme ".
Generally known, in hemoprotein, ferroheme becomes activated centre, is produced by the overall structure of hemoprotein
Bear various function.As such function, work(is carried-stored to the oxygen that can enumerate such as hemoglobin or myoglobins etc.
The electron transmission of the oxidasic function of energy, Cytochrome P450 or peroxidase etc., cytochrome c and cytochrome b etc.
Gas sensor function of function, CooA or HemT etc. etc..In the present embodiment, hemoprotein can have appointing in these
What function, also can have other any functions that known hemoprotein can have.
In the present embodiment, hemoprotein can as a example protein as shown in table 1 below.Encode these blood
As long as the isolated coding that the gene of heme proteins matter derives from the desired animal kind having those hemoproteins is blood red
The gene of cellulose protein, is just not particularly limited, but preferably people source coding hemoprotein gene.
【Table 1】
In the present invention, ferroheme refers to containing as the Cytoheme of the main ferroheme in vivo existing, ferroheme b
And heme c.That is, the hemoprotein being intended in the present invention is alternatively combined with the albumen of any ferroheme in these
Matter.In a preferred embodiment, hemoprotein be lepidopterous insects in vivo synthesize combine ferroheme
Holoprotein.
As long as lepidopterous insects express suitable known lepidopterous insects for recombinant protein, just especially do not limit
Fixed.Silkworm (Bombyx mori), the numb line moths attracted by lamplight (Spilosoma imparilis) of yellow neck, tussah (Antheraea for example can be enumerated
Pernyi), Spodopterafrugiperda (Spodoptera frugiperda), cabbage looper (Trichoplusia ni) etc..At them
Among also be particularly preferably silkworm.Lepidopterous insects are individual can be any form of adult, pupa and larva, from serine protease
Activity and sensitivity to baculoviral viewpoint, preferably use pupa.
In a preferred embodiment, due to the pupa using silkworm, come with the Escherichia coli as host or yeast or insect
The cultured cells strain in source is compared, can a small amount of co-factor manufacture with being not required to culture device containing purity is high and high concentration blood red
The microsomal fraction of the high specific acitivity of cellulose protein.Further, since being the method directly applying co-factor to silkworm chrysalis, with large intestine bar
Bacterium or insect cell etc. using culture medium expression system than the co-factor that can reduce high price usage amount.
The polynucleotides of the amino acid sequence of coding hemoprotein are imported in lepidopterous insects individuality.Polynucleotides
As long as expressing the amino acid sequence part (apolipoprotein) of hemoprotein in lepidopterous insects individuality it is possible to any
Form imports.It is preferably, polynucleotides are integrated into the startup making the gene expression in lepidopterous insects individuality be possibly realized
Son, can insert the carrier DNA of polynucleotides, more preferably, to can be by the homology weight with baculovirus DNA in the downstream of promoter
Group makes the transfer vector insertion of recombinant baculovirus.Such carrier DNA itself is known in the prior art, can enumerate for example
PM01, pM02, pYNG, pBM030, pBM050, pVL1392 etc..In a preferred embodiment, using pM01.The carrier of pM01
Collection of illustrative plates is shown in Fig. 1.Furthermore, described promoter can suitably select from known promoter in the prior art, can enumerate for example
Polyhedrin promoter, p10 promoter, silkworm actin promoter etc..In addition, code book also can be merged on polynucleotides
The polynucleotides of label known to skilled person, the polynucleotides of such as encoding D DDDK label, such as coding SEQ ID
NO:Polynucleotides of amino acid sequence shown in 1 etc..Such label can make the acquirement of hemoprotein become easy.
In the carrier DNA being incorporated with the polynucleotides of amino acid sequence of the described hemoprotein of coding, opening
The downstream of mover incorporates the polynucleotides of coding hemoprotein.Wherein, code tag is merged on this polynucleotides
During polynucleotides, the polynucleotides of code tag also can be upper to the polynucleotides of the amino acid sequence of coding hemoprotein
Trip or downstream insertion.Polynucleotides can be according to well known to a person skilled in the art method artificially synthesizes.
In lepidopterous insects individuality, in the production method of the present invention, make the amino acid sequence part of hemoprotein
(apolipoprotein) expresses.The means that apolipoprotein is expressed are made to be not particularly limited in lepidopterous insects individuality, can be by this area skill
Art personnel suitably determine.For example, can be by individual to lepidopterous insects by known electroinjection direct transfection by carrier DNA
Apolipoprotein is made to express.The present invention preferred embodiment in, can be by making by the shaft-like disease of described carrier DNA restructuring
Poison infection lepidopterous insects individuality makes apolipoprotein express.
The method itself that baculoviral is recombinated with the DNA having desired base sequence is known in the prior art.For example,
When carrier DNA is transfer vector, by the linearizing baculovirus DNA such as restriction enzyme and coding ferroheme will be incorporated
The carrier DNA of the polynucleotides of the amino acid sequence of protein, to the cultured cells cotransfection of lepidopterous insects, is felt by screening
Dye cell, can get recombinant baculovirus.
In embodiments of the present invention, as long as the species of baculoviral can infect described lepidopterous insects or its elder brother
The virus of the cultured cells of worm, is just not particularly limited, but preferably NPV (NPV) or its change virus.Specifically
For, BmNPV, HycuNPV, AnpeNPV, AcNPV, the silkworm (Bombyx mori) of Bombycidae or noctuid clover can be illustrated
The recombinant baculovirus of infection two host of three-spotted plusia (Autographa californica) etc. are (with reference to JP 2003-
No. 52371 publications) etc..In a preferred embodiment, using cysteine proteinase defect (CPd) baculoviral (with reference to special
Open flat No. 7-303488).
The means making recombinate shape virus infection lepidopterous insects individual are not particularly limited, can be known from the prior art
Method suitably select.For example, the side of the liquid containing recombinant baculovirus to the injection of this insect can be enumerated when infecting lepidopterous insects
Method etc..Raised after infecting virus in the cultured cells making lepidopterous insects or its insect or cultivate the period (such as 1 specified
~4 days), the amino acid sequence part (apolipoprotein) of hemoprotein can be made to express.
Also the multinuclear of the amino acid sequence of coenzyme of coding hemoprotein can be imported further in lepidopterous insects
Thuja acid.Introduction method is identical with for the method described in hemoprotein.As such coenzyme, such as cell color can be enumerated
Plain P450 reductase etc..Coenzyme can be made to express in the vicinity of hemoprotein.
In the embodiment of 1, coenzyme is cytochrome P450 reductase (CPR).As long as the gene of coding CPR comes
Come from the isolated CPR gene of the desired animal kind of CPR, be just not particularly limited, but the preferably gene of encoding human CPR.
Furthermore, the base sequence of the gene of amino acid sequence part of people CPR is known in itself, for example, by US National medical science
In the database that the state-run Bioinformatics Institute in library provides, with the registration of accession number NM_000941.In other embodiment
In, coenzyme is the cytochrome b5 as one of cytochromes.It is as described above for cytochrome b5.In other embodiment party again
In formula, coenzyme is both people CPR and cytochrome b5.
So that the means that coenzyme is expressed is not particularly limited in lepidopterous insects individuality, suitably can be determined by those skilled in the art
Fixed.For example, can be by carrier DNA be made coenzyme express by known electroinjection direct transfection to lepidopterous insects individuality.
The present invention preferred embodiment in, by making the baculovirus infection Lepidoptera elder brother using described carrier DNA restructuring
Worm is individual and so that apolipoprotein is expressed.
Now, coding coenzyme amino acid sequence polynucleotides can to coding hemoprotein amino acid many
Nucleotides identical carrier DNA inserts, also can be to each different carrier DNA insertions.In addition, the ammonia of coding hemoprotein
The polynucleotides of the polynucleotides of base acid sequence and coding coenzyme can be incorporated in identical baculoviral, also can be incorporated into each
In different baculovirals.
It is incorporated into not in the polynucleotides of the amino acid sequence of coding hemoprotein and the polynucleotides of coding coenzyme
When on same baculoviral, by making these baculovirals to 1 lepidopterous insects individuality coinfection, hemoprotein can be made
With coenzyme coexpression.In addition, also can be by infecting respective 2 lepidopterous insects individualities respectively with these baculoviral, not
Express in same lepidopterous insects individuality.
In the polynucleotides of the amino acid sequence making coding hemoprotein and the polynucleotides of coding coenzyme identical
Lepidopterous insects individuality in be co-expressed when, as long as their blending ratio can produce hemoprotein, be just not particularly limited.
Blending ratio is for example with virus titer 500:1~1:500 about, it is preferably 150:1~1:150 about.
As long as ferroheme precursor can in vivo be changed into the molecule of ferroheme, just it is not particularly limited, but be preferably and divide
Son amount is little.It is preferably, ferroheme precursor is the intermediate of ferroheme route of synthesis.As such intermediate, example can be enumerated
As former in amino-laevulic acid (hereinafter also referred to " ALA "), courage dyestuff, methylol bilane, uroporphyrinogen III, coproporphyrinogen
III, protoporphyrinogen, protogen IX etc..More preferably, ferroheme precursor is amino-laevulic acid.
As ferroheme analog, as long as there being the molecule of the structure similar with ferroheme and/or function, just especially do not limit
Fixed, but preferably molecular weight is little.As ferroheme analog it is intended to comprise so-called synthesis ferroheme, can enumerate to there being porphin
The compound of quinoline skeleton is coordinated the molecule of iron, such as hemin, protoferriheme etc..
Ferroheme precursor or ferroheme analog can be applied as solution, also can apply as solid.It is preferably, as molten
Liquid is applied.When applying as solution, as solvent, can enumerate dimethyl sulfoxide (DMSO) (DMSO), water, physiological saline, buffer solution and
Sodium hydrate aqueous solution and their mixture etc..It is preferably, solvent is dimethyl sulfoxide (DMSO).
Described solvent also can also contain dissolution accelerator.Such dissolution accelerator itself can be solid, liquid or gas
Any one, be dissolvable in water suitable solvent when being solid or gas and use.As dissolution accelerator, such as carbon can be enumerated former
The lower alcohol of subnumber 1~6.As the concrete example of the lower alcohol of carbon number 1~6, ethanol, propane diols, these mixed can be enumerated
Compound etc..When dissolution accelerator is liquid, also dissolution accelerator can be used as solvent itself.
In addition to ferroheme precursor or ferroheme analog, also can iron administration again.Iron can be divalent, alternatively trivalent.Iron can
By well known to a person skilled in the art method manufacture.Iron can be available commercially.For example, iron chloride (II) (Iron (II) can be enumerated
Chloride tetrahydrate and the pure medicine of light), ironic citrate (III) (Iron (III) citrate, Sigma-Aldrich) etc..Iron
Itself can exist as ion in arbitrary solvent, alternatively solid.In a preferred embodiment, with ferroheme precursor
And/or exist as ion in ferroheme analog identical solvent.As long as ion can be produced with hemoprotein, just not special
Do not limit.Preferably chlorion or citrate ion.
As long as the amount of application of described ferroheme precursor is to produce hemoprotein, just it is not particularly limited.Before ferroheme
The upper limit of the amount of application of body is preferably each individuality 0.7mg, more preferably each individuality 0.27mg, is further preferably each individuality
0.067mg.The lower limit of the amount of application of ferroheme precursor is preferably each individuality 0.001mg, more preferably each individuality
0.0034mg, be further preferably each individuality 0.017mg.In the present embodiment, for example, applied at 2 with the amount of 50 μ l at every 1
Amino-laevulic acid as the concentration of ferroheme precursor 2mM (0.34mg/ml).I.e., in the present embodiment, each individuality is applied
Amino-laevulic acid with 0.034mg.
As long as the amount of application of described ferroheme analog is to produce hemoprotein, just it is not particularly limited.Ferroheme
The upper limit of the amount of application of analog is preferably each individuality 3mg, more preferably each individuality 0.65mg, is further preferably each individuality
0.16mg.The lower limit of the amount of application of ferroheme analog is preferably each individuality 0.001mg, more preferably each individuality
0.02mg, be further preferably each individuality 0.04mg.In the present embodiment, for example, work is applied at 2 with the amount of 50 μ l at every 1
Hemin for the concentration of ferroheme analog 1.25mM (0.82mg/ml).I.e., in the present embodiment, each individuality
Apply the hemin of 0.082mg.
As long as the administration means of described ferroheme precursor and ferroheme analog are to produce hemoprotein, just not special
Do not limit.For example, can be applied by injection, oral, coating etc..It is preferably, applied by injection.In a preferred embodiment, will
As the hemin of ferroheme analog, the amino-laevulic acid as ferroheme precursor or their mixture to conduct
Apply between the individual abdominal segment of silkworm chrysalis of lepidopterous insects, more specifically abdominal segment 4-5 and between 6-7 2.But,
This embodiment is not excluded for applying ferroheme analog and ferroheme precursor to other individual places of silkworm chrysalis.As long as blood can be produced
Heme proteins matter is so that it may apply to any place.
As long as the Dressing date of described ferroheme precursor and ferroheme analog is to produce hemoprotein, just not special
Do not limit.It is preferably, the 2nd~5 day after the importing of polynucleotides, apply within more preferably the 3rd~4 day.
When applying material of more than two kinds as described ferroheme precursor and ferroheme analog, they can mix, and also may not be used
Mix and apply at the same time or separately, or can also arbitrarily order apply successively.
Ferroheme precursor and ferroheme analog are considered to be changed into ferroheme in internal decomposition of lepidopterous insects etc.,
Or combined with the amino acid sequence part (apolipoprotein) of the state of script and hemoprotein, produce hemoprotein
(holoprotein).In the past, in the hemoprotein using silkworm produces, the amino acid sequence part of hemoprotein (carries
Lipoprotein) and the combination of ferroheme do not occur well, the protein obtaining is in the state of apolipoprotein.The present inventor recognizes
For, this is because not enough in the endogenic ferroheme synthetic quantity in silkworm body.This, by silkworm chrysalis apply ferroheme precursor or
Ferroheme analog, supplements the deficiency of this endogenic ferroheme synthesis, also can synthesize hemoprotein in silkworm expression system
Matter is by the surprising achievement of the present inventor.The production method Escherichia coli of the present invention or needing as insect cell are cultivated
The expression system ratio of base, can suppress the usage amount of expensive co-factor.
Next, being related to preferred embodiment in the generation operation of the illustration present invention.But, the invention is not restricted to this
A little embodiments.The 1st preferred embodiment in, use Cytochrome P450 as hemoprotein.Cytochromes
P450 is the protein using the double film of lipid as support function, related to drug metabolism.Specifically, by-H the base of medicine
It is changed into-OH base, undertake the metabolism of medicine.The 1st preferred embodiment in, to the ammonia having imported Codocyte cytochrome p 450
The silkworm of the polynucleotides of base acid sequence applies hemin, amino-laevulic acid or their mixture, produces thin in silkworm
Born of the same parents' cytochrome p 450.
The 2nd preferred embodiment in, use cytochrome b5 as hemoprotein.Cytochrome b5 can conduct
Using the double film of lipid as the memebrane protein function of support.The 2nd preferred embodiment in, to having imported Codocyte color
The silkworm of the polynucleotides of the amino acid sequence of plain b5 applies hemin, amino-laevulic acid or their mixture, in silkworm
Middle generation cytochrome b5.
The 3rd preferred embodiment in, use Cytochrome P450 as hemoprotein and use as coenzyme
Cytochrome P450 reductase (CPR).Cytochrome P450 reductase in the vicinity function of Cytochrome P450, to cell
Cytochrome p 450 provides and for NADPH to be changed into NADP+The electronics that obtains of process.It is present in by cytochrome P450 reductase
The vicinity of Cytochrome P450, can play the metabolic function of Cytochrome P450, thus preferably.The 3rd preferred embodiment
In, many to the polynucleotides of the amino acid sequence having imported Codocyte cytochrome p 450 and Codocyte cytochrome p 450 reductase
The silkworm of nucleotides applies hemin, amino-laevulic acid or their mixture, produce in silkworm Cytochrome P450 and
Cytochrome P450 reductase.
The 4th preferred embodiment in, use Cytochrome P450 and thin as coenzyme as hemoprotein
Born of the same parents' cytochrome p 450 reductase and cytochrome b5.As described above, cytochrome b5 also can be used as using the double film of lipid as support
Memebrane protein function, but be added to by solubilising and/or purifying containing Cytochrome P450 and Cytochrome P450 reduction
The microsomal fraction of enzyme, also can be auxiliary by the drug metabolism activity of Cytochrome P450 as increasing in the state of dissociating from film
Factor function.The 4th preferred embodiment in, to the multinuclear of the amino acid sequence having imported Codocyte cytochrome p 450
1st silkworm of the polynucleotides of thuja acid and Codocyte cytochrome p 450 reductase and the many nucleosides having imported Codocyte pigment b5
2nd silkworm of acid each applies hemin, amino-laevulic acid or their mixture, produces cytochromes in these silkworms
P450, cytochrome P450 reductase and cytochrome b5.
The production method of the hemoprotein of the present invention is included from lepidopterous insects individual acquirement hemoprotein
Operation.Adquisitiones is not particularly limited, can be according to the species of the hemoprotein producing or property by those skilled in the art
Suitable selection.For example, when needing the support of double-layer of lipoid for hemoprotein function, can be by obtaining particulate
Body fraction and obtain, in hemoprotein function in mitochondria, can be obtained by obtaining mitochondrial fraction.
The period obtaining is not particularly limited, can be according to the individual kind of the hemoprotein producing or host's lepidopterous insects
Class or property are suitably selected by those skilled in the art.For example can enumerate, after the importing of polynucleotides plays 3~8 days, preferably 4
After~6 days etc..In a preferred embodiment, obtain hemoprotein after polynucleotides have imported 6 days.
In a preferred embodiment, hemoprotein is the cytochromes using double-layer of lipoid as support function
P450.Due to Cytochrome P450, in endoplasmic reticulum, volume exists, and obtains hemoprotein by obtaining microsomal fraction.
More specifically, first, by grinding, ultrasonic disruption or be dissolved in solution of cell lytic agent containing surfactant etc. etc. and break
The individual cell of broken lepidopterous insects and modulate homogenate, by making core fraction sink with the slow speed centrifugation of 1,000g degree
Form sediment, then pass through to make mitochondrial fraction precipitate with the slightly higher revolution centrifugation of 9,000g degree this supernatant, then by by this
The ultracentrifugation of the clear revolution by 100,000g degree separates makes microsomal fraction precipitate, and can obtain and be combined with the support of double-layer of lipoid
State Cytochrome P450 (hemoprotein).
As described above, when obtaining hemoprotein etc. in the state of the support of double-layer of lipoid combines, obtain is blood red
Cellulose protein also can be solubilized further using solubilizer.Double-layer of lipoid is removed by solubilising, hemoprotein can be made to dissociate.Solubilising
As long as agent brings impact to the structure of the hemoprotein obtaining and function, just it is not particularly limited.As solubilizer, example
As using well known to a person skilled in the art surfactant.More specifically sodium taurocholate, lauryl sodium sulfate etc. can be enumerated
Anion system surfactant, the cationic systems surfactant of hexadecyltrimethylammonium bromide etc., CHAPS etc.
Amphoteric surfactant or these mixture.The raw material of solubilising is not limited to microsomal fraction.Broken silkworm chrysalis, from classification point
From any fraction all can solubilising.Preferably microsomal fraction.
As described above, that hemoprotein of solubilising etc. also can use chromatography etc. well known to a person skilled in the art side
Method is further purified.In a preferred embodiment, using gel-filtration chromatography or cation exchange chromatography.Solubilising and obtain
Purifying haemachrome protein arriving etc. also can be using liposome or Nanodisc etc. well known to a person skilled in the art method exists
Build again on the double film of lipid.
Next, being related to preferred embodiment in the acquirement operation of the illustration present invention.But, the invention is not restricted to this
A little embodiments.The 1st preferred embodiment in, produce operation in produce hemoprotein be cytochromes
P450.As described above, Cytochrome P450 is the protein using the double film of lipid as support function, volume in endoplasmic reticulum
Exist.Thus, in obtaining operation, obtain microsomal fraction.Microsomal fraction can be according to people in the art as above
Known to member, centrifugal separation obtains.Cytochrome P450 is obtained preferably in the state of being combined with the support of double-layer of lipoid.
The 2nd preferred embodiment in, produce operation in produce hemoprotein be cytochrome b5.As above
Described, cytochrome b5 is the protein using the double film of lipid as support function, and in endoplasmic reticulum, volume exists.Thus,
In obtaining operation, obtain microsomal fraction.Microsomal fraction can according to as above well known to a person skilled in the art from
Centrifugal separation obtains.Cytochrome b5 is obtained preferably in the state of being combined with the support of double-layer of lipoid.
The 3rd preferred embodiment in, produce operation in produce hemoprotein be Cytochrome P450, auxiliary
Enzyme is cytochrome P450 reductase.Cytochrome P450 reductase is also the memebrane protein using double-layer of lipoid as support,
In endoplasmic reticulum, volume exists.Thus, in obtaining operation, obtain microsomal fraction.Microsomal fraction can be according to as above
Well known to a person skilled in the art centrifugal separation obtains.Cytochrome P450 reductase is preferably tied in the support with double-layer of lipoid
Obtain in the state of conjunction.
The 4th preferred embodiment in, produce operation in produce hemoprotein be Cytochrome P450, auxiliary
Enzyme is cytochrome P450 reductase and cytochrome b5.As described above, the cytochrome b5 as co-factor is preferably, pass through
Solubilising and/or purify and be added to the microsomal fraction containing Cytochrome P450 and cytochrome P450 reductase, swimming from film
From in the state of increase by the drug metabolism activity of Cytochrome P450.Thus, in this embodiment, for cytochromes
P450 and cytochrome P450 reductase, are obtained, a side by the lepidopterous insects individual acquirement microsomal fraction from 1 body
Face, for cytochrome b5, preferably from the silkworm importing cytochrome b5, obtains first containing the cell color existing as memebrane protein
The microsomal fraction of plain b5, by the microsomal fraction solubilising obtaining and/or purifying, obtaining the cell of free state from film
Pigment b5.Such purifying cells pigment b5 is according to as above well known to a person skilled in the art method obtains microsome level
After point, can using well known to a person skilled in the art purification process, be preferably affinity chromatography, gel-filtration chromatography or sun from
Sub- exchange chromatography obtains.
Include within the scope of the invention the hemoprotein that obtained by described production method itself.In addition,
Also the composition containing described hemoprotein and/or coenzyme and its production method is included in the scope of the present invention.Such
Composition is for example useful as drug metabolism test reagent.When hemoprotein is present in microsomal fraction, this
The composition of sample can according to well known to a person skilled in the art method, by the individual cell of broken lepidopterous insects, obtain micro-
Plastochondria fraction produces.The microsomal fraction obtaining also can pass through resolubilization, purifies and the hemoprotein as the product of purifying.
When hemoprotein is present in core and when being present in mitochondria, composition can be according to basis as above
Centrifugal separation known to skilled person, produced by obtaining core fraction or mitochondrial fraction.The core fraction obtaining or line
Plastochondria fraction also can pass through resolubilization, purify and make the hemoprotein of purifying product.
When hemoprotein is present in cytoplasm, described composition can according to well known to a person skilled in the art
Method, by the individual cell of broken lepidopterous insects, take supernatant to produce.The composition obtaining can concentrate and make more highly concentrated
The fluid composition of degree, also can make solvent evaporation or freeze-drying make solid composite.In addition, the composition obtaining also may be used
By purifying the hemoprotein making purifying product.In a preferred embodiment, composition can be microsomal fraction itself
Or by purifying product to its solubilising and obtained from purifying.In addition, also can be by their purifying product according to known in liposome etc.
Method be building up to again on the double film of lipid.
Next, being related to preferred embodiment in the composition of the illustration present invention.But, the invention is not restricted to these
Embodiment.The 1st preferred embodiment in, composition contain Cytochrome P450.Composition containing Cytochrome P450 can
By the lepidopterous insects individuality of the polynucleotides from the amino acid sequence having imported Codocyte cytochrome p 450, obtain microsome
Fraction and produce.As described above, Cytochrome P450 is the protein using the double film of lipid as support function.Thus, group
Compound preferably contains the Cytochrome P450 being combined with the double-layer of lipoid as support.That is, in composition when containing Cytochrome P450,
Composition is preferably microsomal fraction itself.
1st composition being preferred embodiment related to, for example, can be utilized the production of the composition containing Cytochrome P450
Method produces, and methods described includes:Lepidoptera to the polynucleotides of the amino acid sequence having imported Codocyte cytochrome p 450
Individual at least a kind applying selected from ferroheme precursor and ferroheme analog of insect, makes blood in described lepidopterous insects individuality
The operation that heme proteins matter produces;Individual from described lepidopterous insects, obtain the work of the microsomal fraction containing Cytochrome P450
Sequence.
The 2nd preferred embodiment in, composition contain cytochrome b5.Composition containing cytochrome b5 can by from
Import the lepidopterous insects individuality of the polynucleotides of amino acid sequence of Codocyte pigment b5, obtain microsomal fraction life
Produce.As described above, cytochrome b5 is the protein using the double film of lipid as support function.Thus, composition preferably contains
The cytochrome b5 being combined with the double-layer of lipoid as support.That is, in composition when containing cytochrome b5, composition is preferably micro-
Plastochondria fraction itself.
2nd composition being preferred embodiment related to, for example, can be by the production method of the composition containing cytochrome b5
Produce, methods described includes:Lepidopterous insects to the polynucleotides of the amino acid sequence having imported Codocyte pigment b5
Body applies at least a kind selected from ferroheme precursor and ferroheme analog, makes ferroheme egg in described lepidopterous insects individuality
The operation that white matter produces;Individual from described lepidopterous insects, obtain the operation of the microsomal fraction containing cytochrome b5.
The 3rd preferred embodiment in, composition contain Cytochrome P450 and cytochrome P450 reductase.Containing cell
The composition of cytochrome p 450 reductase can be by the multinuclear from the amino acid sequence having imported Codocyte cytochrome p 450 reductase
The lepidopterous insects of thuja acid are individual, obtain microsomal fraction produces.As described above, cytochrome P450 reductase is double with lipid
Film is as the protein of support function.Thus, composition preferably contains the cytochromes being combined with the support of double-layer of lipoid
P450 reductase.That is, in composition when containing cytochrome P450 reductase, composition is preferably microsomal fraction itself.
3rd composition being preferred embodiment related to, for example, can be by containing Cytochrome P450 and Cytochrome P450 also
The production method of the composition of protoenzyme produces, and methods described is included to the amino acid sequence having imported Codocyte cytochrome p 450
The individual administration of the lepidopterous insects of the polynucleotides of polynucleotides and Codocyte cytochrome p 450 reductase is selected from ferroheme precursor
And at least a kind of ferroheme analog, described lepidopterous insects individuality makes the operation that hemoprotein produces;From described
Lepidopterous insects are individual, obtain the operation of the microsomal fraction containing Cytochrome P450 and cytochrome P450 reductase.
The 4th preferred embodiment in, composition contains Cytochrome P450, cytochrome P450 reductase and cell color
Plain b5.As described above, the cytochrome b5 as co-factor is preferably, color containing cell is added to by solubilising and/or purifying
Plain P450 and the microsomal fraction of cytochrome P450 reductase, increase by Cytochrome P450 in the state of dissociating from film
Drug metabolism activity.Thus, in this embodiment, for Cytochrome P450 and cytochrome P450 reductase, by from 1
The lepidopterous insects of body are individual to be obtained microsomal fraction and obtains, on the one hand, for cytochrome b5, preferably from importing cell color
The silkworm of plain b5, obtains the microsomal fraction containing the cytochrome b5 existing as memebrane protein, first by the particulate obtaining
Body fraction solubilising and/or purifying, obtain the cytochrome b5 of free state, by containing Cytochrome P450 and cell from film
The microsomal fraction of cytochrome p 450 reductase and solubilized fraction mixing, obtain composition.
More specifically, such composition can be by containing Cytochrome P450, cytochrome P450 reductase and cell color
The production method of the composition of element produces, and it includes
Reduce to the polynucleotides of the amino acid sequence having imported Codocyte cytochrome p 450 and Codocyte cytochrome p 450
1st lepidopterous insects individuality of the polynucleotides of the amino acid sequence of enzyme and the polynucleotides having imported Codocyte pigment b5
Individual at least a kind each applying selected from ferroheme precursor and ferroheme analog of the 2nd lepidopterous insects, in described Lepidoptera
The operation that hemoprotein produces is made in insect individuality;
Reduce containing Cytochrome P450 and Cytochrome P450 from individual each acquirement of described 1st and the 2nd lepidopterous insects
1st microsomal fraction of enzyme and the operation of the 2nd microsomal fraction containing cytochromes;
Make described 2nd microsomal fraction solubilising, obtain the operation of solubilized fraction;
By described 1st microsomal fraction and described solubilising fraction mixing, obtain the operation of composition.
Next, the present invention is explained by embodiment, but the invention is not restricted to these embodiments.
【Embodiment】
【Embodiment 1:Produce pouch-type CYP3A4 in ferroheme by applying to the hemin of silkworm】
Completely long gene additional code to encoding human CYP3A4 is made from artificial synthesized (to the outsourcing of FASMAC company)
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of DYKDDDDK sequence label;SEQ ID NO:1) DNA piece
Section.This sequence label is configured to be attached to the C-terminal of people's CYP3A4 amino acid sequence.This DNA fragmentation is incorporated into pM01 carrier
The MCS (restriction enzyme BglII/XhoI) of (Sysmex Co., Ltd.).The Plasmid Constructs obtaining are referred to as
CYP3A4_pM01.
Method (the Invertebrate Cell System and being made through change Maeda etc. of recombinant baculovirus
Applications, Vol.1, p.167-181, CRC Press, Boca Raton (1989)) and carry out.Specifically, use
Lipofectin reagent (X-tremeGENE 9DNA transfection reagent:Roche company), by described Plasmid Constructs CYP3A4_pM01
(50ng) and straight chain CPd baculoviral (cysteine proteinase defective virus strain, Sysmex) DNA (20ng) to BmN
Cell (Maeda, 1989) cotransfection.After confirming infection sign, reclaim culture supernatant.Thus, obtain incorporating people's CYP3A4 base
The recombinant baculovirus of cause.
Recombinant baculovirus are inoculated into silkworm chrysalis (kind:Bright and beautiful autumn clock and, from Shang Tian silkworm egg, company buys silkworm seed, by
The artificial feeding of Sysmex Co., Ltd. is to pupa), infect after 3 days in virus, inoculated 50 at every 1 pupa 2, with injection
(in dimethyl sulfoxide (DMSO) (following DMSO), (Hemin is derived from ox, Sigma- to the hemin of 10mM to the hemin solution of μ L
Aldrich)).Inoculation position at 2 is set between abdominal segment 4-5 and between 6-7.Virus inoculation reclaim pupa after rising 6 days and in -80
DEG C freezing.
To every 5 freezing pupa add 25mL buffer solution (50mM Tris acetic acid, 250mM sucrose, protease inhibitors,
PH7.6), crush pupa and obtain suspension using pressure-even pulp crusher (paddle crushes for ELMEX, pattern SH-IIM).By by this suspension
Remove after the residue of pupa epidermis etc. with net filtration, and centrifugation (1850 × g, 4 DEG C, 10 minutes) and reclaim supernatant, by this supernatant
As the 1st supernatant.In addition, to the sediment after centrifugation add 25mL buffer solution (50mM Tris acetic acid, 250mM sucrose,
Protease inhibitors, pH7.6) and after suspension, crushed with micro-ultrasonic ripple pressure-even pulp crusher (QSonica, model Q55).To containing ultrasonic
The suspension centrifugation (1850 × g, 4 DEG C, 10 minutes) of ripple broken thing and reclaim supernatant, using this supernatant as the 2nd supernatant.Will
After 1st supernatant and the mixing of the 2nd supernatant, and centrifugation (9000 × g, 4 DEG C, 20 minutes) and reclaim supernatant.To this supernatant ultracentrifugation
Separate (100,000 × g, 4 DEG C, 60 minutes) after, remove supernatant, to sediment with buffer solution (50mM Tris acetic acid, 250mM sugarcane
Sugar, 0.25mM EDTA, pH7.6) 15mL and suspend.
Furthermore, crush by using teflon pressure-even pulp crusher (ASONE, revolution 5000rpm, under 10) and obtain thick film fraction suspension
Liquid.Add solubilizing solution (1.2% sodium taurocholate, 100mM potassium phosphate, 20% glycerine, 0.1mM DTT) to this thick film fraction suspension
15mL and after incubating 2 hours in 4 DEG C, centrifugation (100,000 × g, 4 DEG C, 60 minutes) and reclaim supernatant, obtain making label melt
Close the protein solution of CYP3A4 solubilising.Add resin (the DDDDK- labeling albumen combining anti-DDDDK peptide antibody to this solution
Matter purifies gel, MBL, 50% slurry) 4mL, so that tag fusion CYP3A4 is specifically combined with resin.By to it competitively
Reaction containing 0.1mg/mL DDDDK peptide dissolution fluid (100mM potassium phosphate, 0.1mM DTT, 0.1mM EDTA, 0.6% sodium taurocholate,
PH7.6), tag fusion CYP3A4 is made to dissociate from resin and reclaim.By reclaim protein solution with milipore filter (Apollo,
MWCO:20kDa) it is concentrated into 1mL (1.3mg/mL).
The tag fusion obtaining CYP3A4 protein solution is put into 96 orifice plates with 100 μ L/ holes, measures 280nm~600nm
Absorption spectrum.Meanwhile, measure buffer solution (100mM potassium phosphate, 0.1mM DTT, 0.1mM EDTA, 0.6% sodium taurocholate,
PH7.6 spectrum), by calculating the difference with protein solution, obtains the spectrum in tag fusion CYP3A4 source.Furthermore, former
This measures in the state of so that carbon monoxide (CO) is combined, and becomes and detects the peak of 450nm by taking CO difference spectra, but due to setting
It is not carried out for upper difficulty.
The spectrum of the CYP3A4 protein solution of modulation is shown in Fig. 2.By the pupa modulation applying hemin
Observe in CYP3A4 that there is near 420nm sharp great spectrum.This oxidized form P450 greatly with interior bag ferroheme
The spectrum of the Soret band of display is consistent.On the one hand, during the CYP3A4 purified with the silkworm chrysalis never applying hemin, confirm
Very big less than what the P450 of interior bag ferroheme originated.In addition, hemin is added to purifying CYP3A4 for 225 μM with final concentration
During protein solution it was observed that hemin source wide in range very big.With to solubilising-purge process buffer solution with
100 μM of final concentration adds during the CYP3A4 of modulation under conditions of hemins it was observed that the spectrum similar with oxidized form P450,
But very big wavelength is 408nm.Above discussion result prompting, by applying hemin to silkworm is individual, can obtain interior bag blood
The CYP3A4 of red pigment.
【Embodiment 2:Distribution in hemin lytic agent and silkworm chrysalis】
Inoculate hemin solution (10mM hemin, solvent to silkworm chrysalis similarly to Example 1:DMSO).In addition,
To at every 1 silkworm chrysalis 2, the chlorination with the injection inoculation solvent different from hemin solution used in embodiment 1 is blood red
Plain solution (10mM hemin, 37.5mM L-arginine, 2.5% ethanol, 10% propane diols, PBS).In hemin
Reclaim pupa after having inoculated 2 days and after 3 days, freeze in -80 DEG C.In addition, as comparison, by do not inoculate the pupa of hemin in-
80 DEG C of freezings.These are freezed pupa cutter backcut, pair cross-section feature shoots.The section not inoculating the pupa of hemin shows
In Fig. 3 (1), the section inoculating the pupa of hemin solution (10mM hemin in DMSO) is shown in Fig. 3 (2), inoculates chlorine
Change haemachrome solution (10mM hemin, 37.5mM L-arginine, 2.5% ethanol, 10% propane diols, PBS) pupa cut
Face is shown in Fig. 3 (3).Because hemin (10mM) is dense umbrinaceous solution, by the dyestuff that hemin is originated
As index, observe the distribution of the hemin in silkworm chrysalis body.Chlorine when inoculating hemin it was observed that inside pupa
Change the sign of ferroheme diffusion.It was observed that the sign integrally being spread by pupa when using dissolution accelerator.
【Embodiment 3:The mensure of the CYP3A4 metabolic activity when hemin of various concentration is applied】
The people making in embodiment 1 CYP3A4 expression virus and people's CPR expression virus are mixed to virus effect
Valency becomes 1:1 ratio, to silkworm chrysalis inoculation.Wherein, people CPR expresses with viral making as described below.First, artificial synthesized to coding
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of the gene additional code DYKDDDDK sequence label of people CPR;
SEQ ID NO:1) DNA fragmentation (to the outsourcing of FASMAC company).This sequence label is configured to be attached to people's CPR amino acid sequence
C-terminal.Made by this DNA fragmentation being incorporated into the MCS (restriction enzyme BglII/XhoI) of pM01 carrier
Plasmid Constructs CPR_pM01, with the described baculoviral being similarly obtained and integrating people's CPR gene.
After virus infection, according to trial zone A (table 2 below) and trial zone B (table 3 below), use at every 1 pupa 2
Injection inoculates the hemin solution of 50 μ L.Reclaim pupa after virus inoculation plays 6 days, freeze in -80 DEG C.Using freezing
Pupa and obtain thick film fraction suspension.Method according to described in embodiment 1 for the modulation of thick film fraction suspension.Using commercially available
The CYP3A4 activity of the thick film fraction that P450-Glo CYP3A4 screening system (Promega company) mensure obtains.Replace described survey
Determine the subsidiary CYP3A4 film fraction of reagent and use the thick film fraction of oneself modulation, other conditions defer to the hand measuring reagent
Volume.Furthermore, in the detection using plate reader (Thermo Fisher Scientific, VarioSkan), measure and CYP3A4
The related luminous intensity (RLU) of metabolic activity.
The result of the CYP3A4 metabolic activity of thick film fraction that evaluation test area A (table 2) originates, the 4th after virus infection
It and the 5th day are applied in any system of hemin, in the whole concentration of 0.63mM, 2.5mM, 10mM and 40mM, all compare
The occasion not applying hemin shows higher CYP3A4 metabolic activity (Fig. 4).
Next, in order to inquire into application concentration, the thick film fraction that evaluation test area B (table 3) originates in further detail.Knot
Really, in the condition applying 1.25mM hemin on the 4th day after virus infection, show highest activity.The 3rd after virus infection
It administration confirms less than significant difference (Fig. 5) with the administration of the 4th day.
【Table 2】Trial zone A
【Table 3】Trial zone B
【Embodiment 4:The mensure of the CYP3A4 metabolic activity when amino-laevulic acid of various concentration is applied】
The people's CPR expression disease making and in embodiment 3 the people making in embodiment 1 CYP3A4 expression virus
Poison mixes and becomes 2 to virus titer:1 ratio, to silkworm chrysalis inoculation.Infect after 3 days to every 1 silkworm chrysalis 2 in virus
Inoculate amino-laevulic acid (5-Aminolevulinic Acid hydrochloride and the pure medicine of the light) solution of 50 μ L with injection.Amino
Levulic acid solution is modulated according to trial zone A (table 4 below) and trial zone B (table 5 below).After virus inoculation plays 6 days
Reclaim silkworm chrysalis and freeze in -80 DEG C.
Pupa using freezing obtains the 1st supernatant and the 2nd supernatant.The modulation of the 1st supernatant and the 2nd supernatant is according in embodiment 1
The method recorded.After 1st supernatant and the 2nd supernatant are mixed, and centrifugation (9000 × g, 4 DEG C, 20 minutes) and reclaim supernatant.Super
This high speed centrifugation supernatant of centrifugation (105,000 × g, 4 DEG C, 90 minutes) and after removing supernatant, to sediment plus 154mM KCl
Solution 30mL.Will be (colourless from glycogen fraction for sedimentary microsomal fraction (umbrinaceous upper strata) by the operation of pipettor compressing
Transparent lower floor) separate, after only reclaiming microsomal fraction, with teflon pressure-even pulp crusher (ASONE, revolution 5000rpm, under 10) modulation
Microsomal fraction suspension.
After (105,000 × g, 4 DEG C, 60 minutes) being separated to its ultracentrifugation and removing supernatant, to sediment with buffer solution
(50mM Tris acetic acid, 250mM sucrose, 0.25mM EDTA, pH7.6) 3mL.By by this sediment Dounce type pressure-even pulp crusher
Broken (under 30) obtain microsomal fraction suspension.Surveyed using commercially available P450-Glo CYP3A4 screening system (Promega company)
The CYP3A4 activity of the microsomal fraction surely obtaining.The subsidiary CYP3A4 film fraction of described mensure reagent is replaced to use and oneself adjust
The microsomal fraction of system, other conditions defer to the handbook measuring reagent.
The result of the CYP3A4 metabolic activity of evaluation test area A (table 4) source microsomal fraction, in amino-laevulic acid
(ALA) concentration is that during 2mM, active ratio is high when concentration is 0.2mM.On the one hand, for the species of the iron applied simultaneously, in chlorination
Iron (II) (Iron (II) chloride tetrahydrate and the pure medicine of light) and ironic citrate (III) (Iron (III) citrate,
Sigma-Aldrich confirm less than significant difference between).In addition, the application concentration of iron is to confirm not between 0.2mM and 2mM
To significant difference (Fig. 6).Thus, using iron chloride (II) in discussion from now on.Next, for inquiring into ammonia in further detail
Base levulic acid application concentration, the microsomal fraction that evaluation test area B (table 5) originates.
Result is learnt, by applying amino-laevulic acid, activity raises.In addition, virus infection after the 2nd day administration and
Confirm less than significant difference (Fig. 7) in the administration of the 3rd day.Judge from above discussion result, applying amino-laevulic acid
In system, to after virus infection, the pupa of the 3rd day applies 2mM ALA, and the condition of 0.2mM iron chloride (II) solution is the most suitable.
【Table 4】Trial zone A
Condition | Amino-laevulic acid (ALA) concentration | The species of iron and concentration |
1 | 0.2mM ALA | 0.02mM ironic citrate (III) |
2 | 0.2mM ALA | 0.2mM ironic citrate (III) |
3 | 0.2mM ALA | 0.02mM iron chloride (II) |
4 | 0.2mM ALA | 0.2mM iron chloride (II) |
5 | 2mM ALA | 0.02mM ironic citrate (III) |
6 | 2mM ALA | 0.2mM ironic citrate (III) |
7 | 2mM ALA | 0.02mM iron chloride (II) |
8 | 2mM ALA | 0.2mM iron chloride (II) |
【Table 5】Trial zone B
Condition | ALA concentration | Concentration of iron | Apply day |
1 | 4mM ALA | 0.2mM iron chloride (II) | 2nd day |
2 | 8mM ALA | 0.2mM iron chloride (II) | 2nd day |
3 | 4mM ALA | 0.2mM iron chloride (II) | 3rd day |
4 | 8mM ALA | 0.2mM iron chloride (II) | 3rd day |
【Embodiment 5:The mixture of hemin, amino-laevulic acid and hemin and amino-laevulic acid is applied
The mensure of the CYP3A4 metabolic activity of used time】
The people's CPR expression disease making and in embodiment 3 the people making in embodiment 1 CYP3A4 expression virus
Poison mixes and becomes 2 to virus titer:1 ratio, to silkworm chrysalis inoculation.After virus infection, according to trial zone (table 6 below) to every
Inoculate the hemoprotein solution of 50 μ L with injection at 1 pupa 2.Reclaim pupa after virus inoculation plays 6 days, in -80 DEG C
Freezing.Pupa modulation microsomal fraction suspension using freezing.The modulation of microsomal fraction suspension is according to described in embodiment 4
Method.Measure the microsomal fraction obtaining using commercially available P450-Glo CYP3A4 screening system (Promega company)
CYP3A4 activity.The subsidiary CYP3A4 film fraction of described mensure reagent is replaced to use the microsomal fraction of oneself modulation, other
Condition defer to measure reagent handbook.
The result of the CYP3A4 metabolic activity of evaluation test area (table 6 below) source microsomal fraction, is applying chlorination
During ferroheme, when applying amino-laevulic acid, and when applying two side of hemin and amino-laevulic acid, all can obtain height
The microsomal fraction (Fig. 8) of activity.
【Table 6】Trial zone
【Embodiment 6:The production of pouch-type CYP3A4 in the ferroheme applied by amino-laevulic acid】
The people's CYP3A4 expression virus making in embodiment 1 to silkworm chrysalis inoculation.Virus infected after 3 days to
Inoculate hemoprotein solution (2mM amino-laevulic acid, the 0.2mM iron chloride of 50 μ L with injection at every 1 pupa 2
(II)).Reclaim pupa after virus inoculation plays 6 days, freeze in -80 DEG C.Obtain the 1st supernatant using the pupa of freezing.1st supernatant
Method according to described in embodiment 1 for the modulation.In addition, add buffer solution (the 50mM phosphoric acid of 25mL to the sediment after centrifugation
Potassium, 6mM magnesium acetate, 20% glycerine, 1mM DTT, 1mM EDTA, pH7.4) and after suspension, with micro-ultrasonic ripple pressure-even pulp crusher
(QSonica, model Q55) crushes.To the suspension centrifugation (1850 × g, 4 DEG C, 10 minutes) containing ultrasonic disruption thing and
Reclaim supernatant, using this supernatant as the 2nd supernatant.Obtain microsomal fraction suspension using the 1st supernatant and the 2nd supernatant.
Method according to described in embodiment 4 for the modulation of this microsomal fraction suspension.To microsomal fraction suspension
24mL adds solubilizing solution (50mM potassium phosphate, 20% glycerine, 1mM DTT, 1mM EDTA, 5%Tergitol NP-10, pH7.4)
6mL and after incubating 2 hours in 4 DEG C, centrifugation (105,000 × g, 4 DEG C, 60 minutes) and reclaim supernatant, made
The protein solution of DYKDDDDK tag fusion CYP3A4 solubilising.Add the resin combining anti-DDDDK peptide antibody to this solution
(DDDDK- labeling protein purification gel, MBL, 50% slurry) 8mL, makes tag fusion CYP3A4 and resin specifically tie
Close.By competitively react to it DDDDK peptide containing 0.1mg/mL dissolution fluid (100mM potassium phosphate, 0.1mM DTT,
0.1mM EDTA, 0.2%Tergitol NP-10, pH7.4), so that tag fusion CYP3A4 is dissociated from resin and reclaim.
Protein solution milipore filter (Millipore, AmiconUltra-15, the MWCO that will reclaim:3kDa) it is concentrated into
After 2mL, make experience gel permeation chromatography (post:GE HealthcareSuperdex 200Increase 10/300GL, movement
Phase:20mM Tris-HCl, 100mM NaCl, 5% glycerine, 0.1%Tergitol NP-10, pH7.5), molecular weight dependence ground
Separated-purified.Concentrate the solution of the fraction containing target protein, be concentrated into 2mL with milipore filter.By the tag fusion obtaining
CYP3A4 protein solution puts into 96 orifice plates with 100 μ L/ holes, measures the absorption spectrum of 280nm~600nm.Meanwhile, measure buffering
The spectrum of liquid (20mM Tris-HCl, 100mM NaCl, 5% glycerine, 0.1%Tergitol NP-10, pH7.5), calculates and egg
The difference of white matter solution and obtain tag fusion CYP3A4 source spectrum.
The spectrum of the CYP3A4 protein solution of modulation is shown in Fig. 9.As shown in embodiment 1, with never applying blood
During the CYP3A4 that the silkworm chrysalis of heme proteins matter solution purifies, confirm less than very big wavelength, but with by administration hemin
Pupa modulation CYP3A4 when it was observed that interior bag ferroheme oxidized form P450 source very big wavelength (420nm) (Fig. 2).?
In the present embodiment, similarly, in the pupa applying amino-laevulic acid, observe near 420nm greatly.Due to amino second
Acyl propionic acid is the compound of no absorption near 420nm it is believed that being from the interior bag of the amino-laevulic acid synthesis ferroheme applied
Result in CYP3A4.Above discussion result prompting, by applying amino-laevulic acid to silkworm is individual, can obtain interior bag blood red
The CYP3A4 of element.
【Embodiment 7:The production of pouch-type cytochrome b5 in the ferroheme applied by hemin】
Made from artificial synthesized (to the outsourcing of FASMAC company) and add to the completely long gene of encoding human cytochrome b5
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of encoding D YKDDDDK sequence label;SEQ ID NO:1)
DNA fragmentation.This sequence label is configured to be attached to the N-terminal of human-cytochrome b5 amino acid sequence.This DNA fragmentation is incorporated into
The MCS (restriction enzyme BglII/XhoI) of pM01 carrier (Sysmex Co., Ltd.).By the plasmid construction obtaining
Body is referred to as cytochrome b5 _ pM01.
Method (the Invertebrate Cell System and being made through change Maeda etc. of recombinant baculovirus
Applications, Vol.1, p.167-181, CRC Press, Boca Raton (1989)) and carry out.Specifically, use
Lipofectin reagent (X-tremeGENE 9DNA transfection reagent:Roche company), by described Plasmid Constructs CYP3A4_pM01
(50ng) and straight chain CPd baculoviral (cysteine proteinase defective virus strain, Sysmex) DNA (20ng) to BmN
Cell (Maeda, 1989) cotransfection.After confirming infection sign, reclaim culture supernatant.Thus, obtain integrator cell pigment b5 base
The recombinant baculovirus of cause.
Inoculate recombinant baculovirus to silkworm chrysalis, infected after 4 days to every 1 pupa 2 in virus and inoculated with injection
50 μ L hemin solution (1.25mM hemin, 4.7mM L-arginine, 0.31% ethanol, 1.25% propane diols,
PBS).Reclaim pupa after virus inoculation plays 6 days and freeze in -80 DEG C.Obtain the 1st supernatant using the pupa of freezing.1st supernatant
Method according to described in embodiment 4 for the modulation.In addition, add buffer solution (the 10mM phosphoric acid of 25mL to the sediment after centrifugation
Potassium, 1mM EDTA, pH7.4) and after suspension, crushed with micro-ultrasonic ripple pressure-even pulp crusher (QSonica, model Q55).To containing ultrasonic wave
The suspension centrifugation (1850 × g, 4 DEG C, 10 minutes) of broken thing and reclaim supernatant, using this supernatant as the 2nd supernatant.Use
1st supernatant and the 2nd supernatant, with method modulation microsomal fraction suspension similarly to Example 4.
Add solubilizing solution (10mM potassium phosphate, 1mM EDTA, 5%Tergitol to this microsomal fraction suspension 40mL
NP-10, pH7.4) 10mL and after incubating 2 hours in 4 DEG C, centrifugation (105,000 × g, 4 DEG C, 60 minutes) and reclaim supernatant.
This is made to contain the solution experience cation-exchange chromatography (post of the memebrane protein of cytochrome b5 solubilising:GE HealthcareHiTrap
Q HP, buffer solution (A):20mM Tris-HCl, 1mM EDTA, 0.2%Tergitol NP-10, pH8.0, buffer solution (B):
20mM Tris-HCl, 1mM EDTA, 0.2%Tergitol NP-10,0.4M NaCl, pH8.0), by linearly raising
The separated-purified target protein of salinity of dissolution buffer solution.Concentrate the solution of the fraction containing cytochrome b5, it is implemented
Gel permeation chromatography (post:GE HealthcareSuperdex 200Increase 10/300GL, mobile phase:20mM potassium phosphate,
0.1mM EDTA, pH7.4), molecular weight dependence ground is separated-purified.Concentrate the solution of the fraction containing target protein, with surpassing
Filter membrane (Millipore, AmiconUltra-15, MWCO:3kDa) it is concentrated into 2mL.
The cytochrome b5 obtaining protein solution is put into 96 orifice plates with 100 μ L/ holes, measures the suction of 280nm~600nm
Receive spectrum.Meanwhile, measure the spectrum of buffer solution (20mM potassium phosphate, 0.1mM EDTA, pH7.4), calculate and protein solution
Difference and obtain cytochrome b5 source spectrum.The spectrum of the cytochrome b5 protein solution of modulation is shown in Figure 10.According to
Figure 10, in the cytochrome b5 that the silkworm chrysalis with never applying hemin solution is purified, confirms less than very big wavelength, but
With by apply hemin pupa modulation cytochrome b5 when it was observed that ferroheme source very big wavelength (409nm~
426nm), bag ferroheme in prompting.Above discussion result proves, is made in ferroheme by applying hemin to silkworm chrysalis
This method being wrapped in recombinant protein is suitable for not only for P450 (CYP3A4), is also suitable for cytochrome b5, and then is recognized
The modulation overall for can be suitably used for hemoprotein.
【Embodiment 8:By the active elevating effect of cytochrome b5 confirmation and with commercially available product (escherichia expression system)
Expression activitiy】
By the purified of the cytochrome b5 of the coenzyme as P450 being added to the particulate with the modulation of silkworm expression system
Whether body fraction research CYP3A4 metabolic activity raises.Specifically, similarly to Example 5 to silkworm chrysalis inoculation CYP3A4 expression
With virus and people's CPR expression virus.Through the time point of 3 days after virus infection, inoculated with injection at every 1 pupa 2
Hemoprotein solution (1mM hemin, 5mM L-arginine, 0.3% ethanol, 1mM propane diols, PBS, 2mM
ALA, 0.2mM iron chloride (II)) 50 μ L.Pupa is reclaimed after virus inoculation plays 6 days.Tune for the microsomal fraction from pupa
System, the method according to described in embodiment 6.On the one hand, the purifying of cytochrome b5 is modulated according to embodiment 7.
For the microsomal fraction of total protein concentration 4.4mg/mL, add purifying cells pigment b5 protein solution to end
Concentration becomes 16.3~326 μ g/mL (1.0~20nmol/mL).After making it react more than 4 hours on ice, using commercially available P450-
Glo CYP3A4 screening system (Promega company) measures the CYP3A4 activity that cytochrome b5 adds microsomal fraction.Replace
Described measure the subsidiary CYP3A4 film fraction of reagent and use the microsomal fraction of oneself modulation, other conditions defer to mensure examination
The handbook of agent.In addition, as comparison other, using the city to the CYP3A4 microsomal fraction modulated with escherichia expression system
Product (the cytochrome b5 sold product and add cytochrome b5 with product:5nmol/mL).The CYP3A4 metabolic activity value obtaining
It is used in the total protein concentration correction of respective microsomal fraction used in measuring and calculate as specific activity.
The result evaluating the CYP3A4 metabolic activity of microsomal fraction shows, by the interpolation of cytochrome b5, activity raises
(Figure 11).In addition confirm, with the cytochrome b5 of silkworm expression system modulation, as the coenzyme of P450, possess and make CYP3A4 metabolism
The function that activity raises.Confirm, the CYP3A4 microsomal fraction with the modulation of silkworm expression system is widely used bigger than market simultaneously
The product in enterobacteria source shows higher specific activity.
【Embodiment 9:Compare with the expression of commercially available microsomal fraction (escherichia expression system)】
Modulate for the silkworm expression system CYP3A4 microsomal fraction of modulation in embodiment 8 and escherichia expression system
Microsomal fraction commercially available product, each personal Bradford method measures total protein concentration.On the basis of the concentration obtaining, make every 1
The microsomal fraction experience polyacrylamide gel of swimming lane 2 μ g total protein quality, by electrophoretic separation-analysing protein (Figure 12).
Show the detailed content of the sample to the application of each swimming lane in table 7 below.
As shown in figure 12, confirm that display is by virus infection enforced table from the microsomal fraction that silkworm expression system obtains
The CYP3A4 reaching and the clear and definite band of people CPR.On the one hand, in the Escherichia coli microsomal fraction as commercially available product,
Confirm less than clear and definite band near the theoretical molecular of CYP3A4 and people CPR.Above result prompting, uses silkworm expression system
The expression of the CYP3A4 in the microsomal fraction of modulation and people CPR is significantly higher than the commercially available product of Escherichia coli.
【Table 7】
【Embodiment 10:The usage amount of hemoprotein reagent compares (silkworm expression system vs Bacillus coli expression system
System)】
When with escherichia expression system modulation CYP3A4 microsomal fraction, the hemoprotein reagent of use
(5-ALA hydrochloride) becomes every 1mg microsomal fraction 280~340 μ g (Pritchard MP, McLaughlin
L,Friedberg T.(2006)Establishment of functional human Cytochrome
P450monooxygenase systems in Escherichia coli.Methods Mol Biol.320,19-29).One
Aspect, in the microsomal fraction same with the modulation of silkworm chrysalis expression system, the amino-laevulic acid of use is every 1mg microsome level
Divide 5.7~8.5 μ g (table 8).It follows that when with silkworm expression system, as long as using blood red in escherichia expression system
The 1/40~1/50 of the usage amount of cellulose protein reagent is so that it may obtain the microsome of isodose.
【Table 8】The usage amount of hemoprotein reagent compares
Escherichia expression system | Silkworm expression system | |
Beginning condition | 100mL cultivates | Silkworm chrysalis 5 |
Amino-laevulic acid concentration | 0.5mM | 2.0mM |
Amino-laevulic acid amount of solution | 100mL | 100 μ L/ × 5=500 μ L |
Amino-laevulic acid usage amount | 8.5mg | 0.17mg |
The receipts amount of microsomal fraction | 25~30mg | 20~30mg |
The amino-laevulic acid necessary amount of the every 1mg of microsomal fraction | 280~340 μ g | 5.7~8.5 μ g |
【Embodiment 11:The comparison with escherichia expression system of CYP1A2 metabolic activity】
Completely long gene additional code to encoding human CYP1A2 is made from artificial synthesized (to the outsourcing of FASMAC company)
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of DYKDDDDK sequence label;SEQ ID NO:1) DNA piece
Section.The configuration of this sequence label is attached to the C-terminal of people's CYP1A2 amino acid sequence.This DNA fragmentation is incorporated into pM01 carrier
The MCS (restriction enzyme BglII/XhoI) of (Sysmex Co., Ltd.).The Plasmid Constructs obtaining are referred to as
CYP1A2_pM01.
Producer CYP1A2 expression virus similarly to Example 1, by this virus and the people making in embodiment 3
CPR expression virus mixes and becomes 1 to virus titer:0.125 ratio, to silkworm chrysalis inoculation.After virus infection, to every 1 pupa 2
Place's injection inoculates the hemoprotein solution of 50 μ L, and ((Hemin is derived from ox, Sigma- to the hemin containing 1mM
Aldrich), the 5-ALA hydrochloride (5-Aminolevulinic Acid hydrochloride and the pure medicine of light) of 2mM,
The iron chloride (II) (Iron (II) chloride tetrahydrate and the pure medicine of light) of 0.2mM, 5mM L-arginine, 0.3% ethanol,
The PBS of 1mM propane diols).Modulate thick film fraction suspension similarly to Example 1, obtain CYP1A2 microsomal fraction.For such as
The CYP1A2 microsomal fraction of upper described use silkworm expression system modulation and the microsomal fraction with escherichia expression system modulation
Commercially available product, measures CYP1A2 activity using commercially available P450-Glo CYP1A2 measurement system (Promega company).Replace described survey
Determine the subsidiary CYP1A2 film fraction of reagent and use the microsomal fraction of oneself modulation, other conditions defer to the hand measuring reagent
Volume.Result is shown in Figure 13.As shown in figure 13, the CYP1A2 metabolic activity of the microsomal fraction obtaining from silkworm expression system be higher than from
The activity of the commercially available microsomal fraction of escherichia expression system modulation.
【Embodiment 12:CYP2C8 metabolic activity and the comparison of escherichia expression system】
Completely long gene additional code to encoding human CYP2C8 is made from artificial synthesized (to the outsourcing of FASMAC company)
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of DYKDDDDK sequence label;SEQ ID NO:1) DNA piece
Section.This sequence label is configured to add to the C-terminal of people's CYP2C8 amino acid sequence.This DNA fragmentation is incorporated into pM01 carrier
The MCS (restriction enzyme BglII/XhoI) of (Sysmex Co., Ltd.).The Plasmid Constructs obtaining are referred to as
CYP2C8_pM01.
Producer CYP2C8 expression virus similarly to Example 1, by this virus and the people making in embodiment 3
CPR expression virus mixes and becomes 1 to virus titer:0.0156 ratio, to silkworm chrysalis inoculation.After virus infection, to every 1 pupa
At 2, with the hemoprotein solution of injection inoculation 50 μ L, ((Hemin is derived from ox, Sigma- to the hemin containing 1mM
Aldrich), the 5-ALA hydrochloride (5-Aminolevulinic Acid hydrochloride and the pure medicine of light) of 2mM,
The iron chloride (II) (Iron (II) chloride tetrahydrate and the pure medicine of light) of 0.2mM, 5mM L-arginine, 0.3% ethanol,
The PBS of 1mM propane diols).Modulate thick film fraction suspension similarly to Example 1, obtain CYP2C8 microsomal fraction.For such as
The CYP2C8 microsomal fraction of upper described use silkworm expression system modulation and the microsomal fraction with escherichia expression system modulation
Commercially available product, measures CYP2C8 activity using commercially available P450-Glo CYP2C8 measurement system (Promega company).Replace described survey
Determine the subsidiary CYP2C8 film fraction of reagent and use the microsomal fraction of oneself modulation, other conditions defer to the hand measuring reagent
Volume.Result is shown in Figure 14.As shown in figure 14, the CYP2C8 metabolic activity of the microsomal fraction obtaining from silkworm expression system be higher than from
The activity of the commercially available microsomal fraction of escherichia expression system modulation.
【Embodiment 13:CYP2C9 metabolic activity and the comparison of escherichia expression system】
Completely long gene additional code to encoding human CYP2C9 is made from artificial synthesized (to the outsourcing of FASMAC company)
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of DYKDDDDK sequence label;SEQ ID NO:1) DNA piece
Section.This sequence label is configured to be attached to the C-terminal of people's CYP2C9 amino acid sequence.This DNA fragmentation is incorporated into pM01 carrier
The MCS (restriction enzyme BglII/XhoI) of (Sysmex Co., Ltd.).The Plasmid Constructs obtaining are referred to as
CYP2C9_pM01.
Producer CYP2C9 expression virus similarly to Example 1, by this virus and the people making in embodiment 3
CPR expression virus mixes and becomes 1 to virus titer:0.0313 ratio, to silkworm chrysalis inoculation.After virus infection, to every 1 pupa 2
Place's injection inoculates the hemoprotein solution of 50 μ L, and ((Hemin is derived from ox, Sigma- to the hemin containing 1mM
Aldrich), the 5-ALA hydrochloride (5-Aminolevulinic Acid hydrochloride and the pure medicine of light) of 2mM,
The iron chloride (II) (Iron (II) chloride tetrahydrate and the pure medicine of light) of 0.2mM, 5mM L-arginine, 0.3% ethanol,
The PBS of 1mM propane diols).Modulate thick film fraction suspension similarly to Example 1, obtain CYP2C9 microsomal fraction.For such as
The CYP2C9 microsomal fraction of upper described use silkworm expression system modulation and the microsomal fraction with escherichia expression system modulation
Commercially available product, measures CYP2C8 activity using commercially available P450-Glo CYP2C9 measurement system (Promega company).Replace described survey
Determine the subsidiary CYP2C9 film fraction of reagent and use the microsomal fraction of oneself modulation, other conditions defer to the hand measuring reagent
Volume.Result is shown in Figure 15.As shown in figure 15, the CYP2C9 metabolic activity of the microsomal fraction obtaining from silkworm expression system be higher than from
The activity of the commercially available microsomal fraction of escherichia expression system modulation.
【Embodiment 14:CYP2C19 metabolic activity and the comparison of escherichia expression system】
Completely long gene additional code to encoding human CYP2C19 is made from artificial synthesized (to the outsourcing of FASMAC company)
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of DYKDDDDK sequence label;SEQ ID NO:1) DNA piece
Section.This sequence label is configured to be attached to the C-terminal of people's CYP2C19 amino acid sequence.This DNA fragmentation is incorporated into pM01 carrier
The MCS (restriction enzyme BglII/XhoI) of (Sysmex Co., Ltd.).The Plasmid Constructs obtaining are referred to as
CYP2C19_pM01.
Producer CYP2C19 expression virus similarly to Example 1, by this virus and the people making in embodiment 3
CPR expression virus mixes and becomes 1 to virus titer:0.0313 ratio, to silkworm chrysalis inoculation.After virus infection, to every 1 pupa
At 2, with the hemoprotein solution of injection inoculation 50 μ L, ((Hemin is derived from ox, Sigma- to the hemin containing 1mM
Aldrich), the 5-ALA hydrochloride (5-Aminolevulinic Acid hydrochloride and the pure medicine of light) of 2mM,
The iron chloride (II) (Iron (II) chloride tetrahydrate and the pure medicine of light) of 0.2mM, 5mM L-arginine, 0.3% ethanol,
The PBS of 1mM propane diols).Modulate thick film fraction suspension similarly to Example 1, obtain CYP2C19 microsomal fraction.For
CYP2C19 microsomal fraction as said above with the modulation of silkworm expression system and the microsome level with escherichia expression system modulation
Divide commercially available product, measure CYP2C8 activity using commercially available P450-Glo CYP2C19 measurement system (Promega company).Replace described
Measure the subsidiary CYP2C19 film fraction of reagent and use the microsomal fraction of oneself modulation, other conditions defer to mensure reagent
Handbook.Result is shown in Figure 16.As shown in figure 16, the CYP2C19 metabolic activity of the microsomal fraction obtaining from silkworm expression system
Activity higher than the commercially available microsomal fraction from escherichia expression system modulation.
【Embodiment 15:CYP2D6 metabolic activity and the comparison of escherichia expression system】
Completely long gene additional code to encoding human CYP2D6 is made from artificial synthesized (to the outsourcing of FASMAC company)
Polynucleotides (the 5 '-GACTATAAAGACGACGATGATAAA-3 ' of DYKDDDDK sequence label;SEQ ID NO:1) DNA piece
Section.This sequence label is configured to be attached to the C-terminal of people's CYP2D6 amino acid sequence.This DNA fragmentation is incorporated into pM01 carrier
The MCS (restriction enzyme BglII/XhoI) of (Sysmex Co., Ltd.).The Plasmid Constructs obtaining are referred to as
CYP2D6_pM01.
Producer CYP2D6 expression virus similarly to Example 1, by this virus and the people making in embodiment 3
CPR expression virus mixes and becomes 1 to virus titer:0.0078 ratio, to silkworm chrysalis inoculation.After virus infection, to every 1 pupa
At 2, with the hemoprotein solution of injection inoculation 50 μ L, ((Hemin is derived from ox, Sigma- to the hemin containing 1mM
Aldrich), the 5-ALA hydrochloride (5-Aminolevulinic Acid hydrochloride and the pure medicine of light) of 2mM,
The iron chloride (II) (Iron (II) chloride tetrahydrate and the pure medicine of light) of 0.2mM, 5mM L-arginine, 0.3% ethanol,
The PBS of 1mM propane diols).Modulate thick film fraction suspension similarly to Example 1, obtain CYP2D6 microsomal fraction.For such as
The CYP2D6 microsomal fraction of upper described use silkworm expression system modulation and the microsomal fraction with escherichia expression system modulation
Commercially available product, measures CYP2D6 activity using commercially available P450-Glo CYP2D6 measurement system (Promega company).Replace described survey
Determine the subsidiary CYP2D6 film fraction of reagent and use the microsomal fraction of oneself modulation, other conditions defer to the hand measuring reagent
Volume.Result is shown in Figure 17.As shown in figure 17, the CYP2D6 metabolic activity of the microsomal fraction obtaining from silkworm expression system be higher than from
The activity of the commercially available microsomal fraction of escherichia expression system modulation.
Claims (16)
1. the production method of hemoprotein, it includes:
It is selected from blood to individual administration of lepidopterous insects of the polynucleotides of the amino acid sequence having imported coding hemoprotein
Red pigment precursor and at least a kind of ferroheme analog, produce the operation of hemoprotein in described lepidopterous insects individuality;
And
From the individual operation obtaining described hemoprotein of described lepidopterous insects.
2. the method described in claim 1, wherein said lepidopterous insects are silkworms.
3. the method described in claim 1, wherein said hemoprotein is selected from Cytochrome P450 and cytochromes
At least a kind.
4. the method described in claim 1, wherein said hemoprotein is Cytochrome P450 and cytochromes.
5. the method described in claim 1, wherein said hemoprotein is Cytochrome P450,
In described generation operation, import, to described lepidopterous insects are individual, the coenzyme encoding described Cytochrome P450 further
Amino acid sequence polynucleotides.
6. the method described in claim 5, wherein said coenzyme is cytochrome P450 reductase.
7. the method described in claim 1, wherein in described generation operation, by whole to the individual infection of described lepidopterous insects
Close the baculoviral of described polynucleotides, import described polynucleotides.
8. the method described in claim 1, wherein said ferroheme precursor is hemin, and described ferroheme analog is ammonia
Base levulic acid.
9. the method described in claim 1, wherein in described generation operation, applies containing selected from ferroheme precursor and ferroheme class
Like at least a kind of thing of solution.
10. the method described in claim 9, wherein said solution also contains to be made selected from ferroheme precursor and ferroheme analog extremely
The dissolution accelerator that few a kind of dissolubility raises.
Method described in 11. claims 10, wherein said dissolution accelerator is the lower alcohol of carbon number 1~6.
Method described in 12. claims 1, wherein in described generation operation, each individual apply more than 0.001mg 3mg with
Under hemin.
Method described in any one of 13. claims 1~12, wherein in described generation operation, each individual administration
The amino-laevulic acid of more than 0.001mg below 0.7mg.
Method described in 14. claims 9, wherein said solution contains iron ion.
15. hemoproteins, it passes through the squama of the polynucleotides to the amino acid sequence having imported coding hemoprotein
Individual at least a kind applying selected from ferroheme precursor and ferroheme analog of homopterous insect, in described lepidopterous insects individuality
Hemoprotein is made to produce and obtain.
The production method of 16. compositions containing Cytochrome P450, cytochrome P450 reductase and cytochromes, it includes:
To the polynucleotides of the amino acid sequence having imported Codocyte cytochrome p 450 and Codocyte cytochrome p 450 reductase
1st lepidopterous insects of the polynucleotides of amino acid sequence are individual to be applied selected from ferroheme precursor and ferroheme analog at least
1 kind, described 1st lepidopterous insects individuality produces the operation of Cytochrome P450 and cytochrome P450 reductase;
Individual apply selected from ferroheme precursor and blood red to the 2nd lepidopterous insects of the polynucleotides having imported Codocyte pigment
At least a kind of plain analog, produces the operation of cytochromes in described 2nd lepidopterous insects individuality;
From individual the 1st microsome obtaining containing Cytochrome P450 and cytochrome P450 reductase of described 1st lepidopterous insects
The operation of fraction;
From the individual operation obtaining the 2nd microsomal fraction containing cytochromes of described 2nd lepidopterous insects;
Make described 2nd microsomal fraction solubilising, obtain the operation of solubilized fraction;And
By described 1st microsomal fraction and described solubilising fraction mixing, obtain the operation of composition.
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