CN1280417C - Human tyrosinase expression carrier and its use - Google Patents
Human tyrosinase expression carrier and its use Download PDFInfo
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- CN1280417C CN1280417C CN 200410088717 CN200410088717A CN1280417C CN 1280417 C CN1280417 C CN 1280417C CN 200410088717 CN200410088717 CN 200410088717 CN 200410088717 A CN200410088717 A CN 200410088717A CN 1280417 C CN1280417 C CN 1280417C
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Abstract
The present invention discloses a human tyrosinase expression carrier and the application thereof, which has the purpose to provide a human tyrosinase expression carrier, a recombinant Pichia pastoris cell for inducing in the carrier, and a method for generating human tyrosinase by the recombinant cell. The human tyrosinase expression carrier is an expression carrier with a human tyrosinase gene and His-tag gene Pichia pastoris, the method for expressing human tyrosinase comprises the following steps that a recombinant Pichia pastoris cell is cultivated; human tyrosinase is expressed by methanol induction, and the human tyrosinase is separated and purified by metal affinity chromatography. The human tyrosinase gene expression carrier of the present invention enables human tyrosinase gene to be integrated into a gene group of the Pichia pastoris cell by homologous recombination, and then the human tyrosinase is stably expressed. Furthermore, an expensive antibiotic and IPTG are unnecessarily used during the fermentation process so that the production cost is greatly reduced, and in addition, the high density growth of the Pichia pastoris is also beneficial to the expression of the human tyrosinase, and the expression quantity can reach 0.39 g/L.
Description
Technical field
The present invention relates to a kind of human tyrosinase expression carrier and application thereof, particularly relate to a kind of human tyrosinase expression carrier and import the reorganization pichia pastoris phaff cell of this carrier and utilize this reconstitution cell to produce the method for human tyrosinase.
Background technology
Human tyrosinase (tyrosinase, EC 1.14.18.1) is the key enzyme of synthesis of melanin in the human body, and it can form the first two steps reaction of reacting by catalysis melanochrome, and promptly the first step tyrosine hydroxylation forms DOPA, and the second step DOPA is oxidized to the DOPA quinone.Then, take off in the melanosome melanocytic, the DOPA quinone further reacts and generates halfcystine acyl group melanocyte, and melanocyte is taken off in polymerization formation; And in melanophore eumelanin corpusculum, the DOPA quinone forms eumelanin under the effect of tyrosine oxidase and associated protein thereof.
Human tyrosinase cdna was cloned successfully (Kwon by Kwon the earliest in 1987, B.S., Haq, A.K., Pomerantz, S.H., Halaban, R.Isolation and sequence of a cDNA clone for humantyrosinase that maps at the mouse c-albino locus.Proc.Natl.Acad.Sci.USA.1987,84,7473-7477), called after Pme134,566 amino-acid residues of encoding wherein, hold 1-12 amino acids residue may be signal peptide sequence (it is hydrophobic amino acids that 10 amino acid are arranged) from N.There is a polyadenylic acid signal sequence AATAAA at about 180 base places behind the terminator codon TAA of Pme134 nucleotide sequence.Also encode among the Pme134 5 glycosylation signal Asn-X-Thr/Ser are arranged.Shibahara (Shibahara, S., Tomita in 1988, Y., Tagami, H., Muller, R.M., Cohen, T..Molecular basis for theheterogeneity of human tyrosinase.Tohoku J.Exp.Med.1988,156,403-414) wait the mRNA that obtains human tyrosine oxidase, 529 amino-acid residues of encoding.It is signal peptide sequence that this amino acid residue sequence is held 1-18 amino acids residue from N.The amino acid residue sequence of human tyrosine oxidase maturation protein is made of 511 amino-acid residues.The human tyrosinase gene is positioned at chromosomal 11q14-q21 district No. 11, and this gene contains 5 exons and 4 introns.5 exon length is respectively 918,135,150,183 and 219bp.Also have a GATA tumor-necrosis factor glycoproteins at 713bp place, translation initiation site upstream, supposition may be a DNA hinge arrangement.The promoter region of human tyrosinase gene 5` end upstream has 4 transcription initiation sites, mainly be positioned at apart from 79 base places, initiator codon ATG upstream, 5 ' end of this gene has 2 TATA box spline structures, lays respectively at transcription initiation site upstream 32 and 6 base places.120 base places also have a GCCAATAC sequence in the transcription initiation site upstream, may play the effect of CAT box.3 cAMP binding site sequence TGACGTCA occupy the 627th, 444 and 247 bit base places respectively.On No. 11 karyomit(e)s of people, also find to have a tyrosine oxidase pseudogene, it is the tyrosinase cdna of a brachymemma, it only contains the 4th, 5 exons of tyrosinase cdna and the 4th complete intron (Sun Renshan, human albinic molecule genetics research [Y], foreign medical science genetics fascicle, 1994, (5): 250-253).
Studies show that, the sudden change of karyomit(e) tyrosinase cdna is to cause recessive inheritance disease eye-skin albinism (major cause of OCA, the molecular mechanism research that carries out to I type OCA, show that tyrosinase cdna contains 4 important function districts, two combining sites that functional zone are two copper atoms wherein, the sudden change in these two zones can weaken itself and the combining of copper atom, or disturbs copper atom to combine with oxygen.Two other sudden change bunch Ji Qu is positioned at the 1st, 4 exons respectively, and these two zones also are the important function districts, are likely the binding site of DOPA or tyrosinase inhibitor.In addition, local pigment related pathologies such as vitiligo, chloasma and cutaneous melanoma are relevant unusually with the local expression of tyrosine oxidase.Therefore, the human tyrosine zymoprotein is had many uses preclinical medicine and clinicing aspect, has good market outlook.
In the past, mostly the tyrosinase protein vivoexpression is to carry out in intestinal bacteria, but expression in escherichia coli exist easily form insoluble inclusion body, can not glycosylation etc. problem, and owing to adopt plasmid expression, fermenting process need add expensive microbiotic and IPTG etc., has improved fermentation costs greatly.Also the someone attempts to adopt other eukaryotic expression system to express tyrosine oxidase, yeast saccharomyces cerevisiae for example, but also had glycosylation or problems such as glycosylation deficiency, culture condition harshness.
Pichia pastoris phaff (Pichia pastoris) is can be with the saccharomyces neoformans expression system (Ogata of methyl alcohol as sole carbon source, K., Nishikawa, H.and Ohsugi, M.A yeast capable of utilizingmethanol.Agric.B finishes red pasteur ol.Chem.1969,33,1519-1520).It has two genes of the alcohol oxidase of encoding, and is respectively AOX1 and AOX2.The expression product overwhelming majority of AOX1 has alcohol oxidase activity (Tschopp in the cell, J.F., Brust, P.F., Cregg, J.M., Stillman, C.A.andGingeras, T.R.Expression of the LacZ gene from twomethanol-regulatedpromoters in Pichia pastoris.Nucleic Acids Res.1987,15,3859-3876).Typical pichia pastoris phaff expression vector contains alcohol oxidase gene 5 ' AOX1 promotor, 3 ' AOX1 terminator, is the multiple clone site of inserting for foreign gene between these two sections sequences.Usually (Histidinol dehydrogenase, HIS) gene HIS4 screens sign or Zeocin and kanamycin gene (kanamycin) as the nutrition complement type and gives zymic Geneticin (G418) resistance as the screening sign with histidinol dehydrogenase.As the shuttle plasmid that an energy is bred in intestinal bacteria, increased, it also contains the partial sequence and the penbritin (Amp of pBR322 plasmid
R) the screening sign of resistant gene.Expression vector is incorporated in the chromosomal DNA of yeast cell by homologous recombination.The pichia pastoris phaff expression system has following distinct advantages: (1) high expression level.It can utilize very strong promotor, has the cell speed of growth that is exceedingly fast, so expression amount is relatively very high.(2) high stable.The carrier energy and the cellular genome of this system are integrated, so the bacterial strain stabilization characteristics of genetics that makes up generally the phenomenon that foreign gene is lost with growth and breeding can not occur.(3) high secretion.In some examples of having studied, it can utilize multiple signal peptide guiding foreign gene secreting, expressing, and its foreign protein exocytosis expression amount reaches as high as 10g/L.
In addition, finish red saccharomyces pastorianus the foreign protein of expressing is had glycosylation (Monteino R, GarciaR, Quintero O, et all Variation in N-linked oligosaccharide structureson heterologous proteins secreted by the methylotrophic yeast Pichia pastorislProtein Expr Purif, 1998,14 (2): 197-207), can connect nitrogen-atoms (N) covalent attachment that makes on oligonucleotide chain and foreign protein peptide chain glycosylation recognition site (Asn-X-Ser-Thr) l-asparagine by N-; Also can connect hydroxyl (OH) covalent attachment that makes on oligonucleotide chain and the foreign protein peptide chain Serine P Threonine by O-.
Summary of the invention
The purpose of this invention is to provide a kind of human tyrosinase expression carrier.
Human tyrosinase expression carrier provided by the present invention is the pichia pastoris phaff expression vector that contains human tyrosinase gene (tyr) and His-tag gene.
Described human tyrosinase genes encoding has the amino acid residue sequence of sequence 3 in the sequence table.
Described human tyrosinase gene can have the nucleotide sequence of sequence 2 in the sequence table, or with sequence table in sequence 2 from 5 ' the 11st nucleotide sequence that limits to 1546 bit bases of end have the nucleotide sequence of the amino acid residue sequence of 90% above homology and encoding sequence 3.
6 histidine residues of described His-tag genes encoding.
Described His-tag gene has the nucleotide sequence of sequence 1 in the sequence table.
Described pichia pastoris phaff expression vector can be pPIC3.5K, pPIC3, pPIC9, pHIL-D1, pA0804, pA0815, pPSC3K or pPIC9K.
Described pichia pastoris phaff expression vector is preferably pPIC3.5K.Described human tyrosinase gene is inserted between the EcoRI and NotI recognition site in the multiple clone site of pPIC3.5K, described His-tag gene is inserted into to make up between SnaBI in the multiple clone site and the EcoRI recognition site obtains human tyrosinase expression carrier pPIC3.5KHis-tyr.
The reconstitution cell that above-mentioned human tyrosinase expression carrier importing pichia pastoris phaff cell is obtained also belongs to protection scope of the present invention.
Described pichia pastoris phaff cell can be pichia pastoris phaff GS115, KM71 or SMD1168.
Can utilize electrotransformation that above-mentioned human tyrosinase expression carrier is imported described pichia pastoris phaff cell.
Another object of the present invention provides a kind of method of expressing human tyrosine oxidase.
The method of expressing human tyrosine oxidase provided by the present invention comprises and cultivates above-mentioned reorganization pichia pastoris phaff cell, the step of methanol induction expressing human tyrosine oxidase.
In the method for above-mentioned expressing human tyrosine oxidase, also comprise the step of utilizing the described human tyrosinase of metal affinity column chromatography separation and purification.
Described metal affinity chromatography can be the nickel ion affinity column chromatography.
Human tyrosinase expression vector of the present invention makes up easily, and the human tyrosinase gene integration is gone in the genome of pichia pastoris phaff cell by homologous recombination, the stably express human tyrosinase, avoided in this proteic plasmid loss phenomenon of expression in escherichia coli, and need not to use expensive microbiotic and IPTG (isopropyl-) during the fermentation, production cost is reduced greatly, in addition, the high-density growth of pichia pastoris phaff, also help the expression of human tyrosinase, expression amount can reach 0.39g/L, and the present invention has important application value.
Description of drawings
Fig. 1 is the building process synoptic diagram of human tyrosinase yeast expression vector pPIC3.5KHis-tyr
Fig. 2 is the SDS-PAGE electrophoretogram of reorganization pichia pastoris phaff intracellular protein
Fig. 3 is reorganization pichia pastoris phaff intracellular protein western-blot qualification result
Fig. 4 is through the proteic SDS-PAGE electrophoretogram of the human tyrosinase of nickel ion affinity column chromatography purifying
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and used enzyme reagent is TaKaRa company product.
The structure of embodiment 1, human tyrosinase yeast expression vector pPIC3.5KHis-tyr
As shown in Figure 1, the building process of carrier pPIC3.5KHis-tyr may further comprise the steps:
One, the clone of His-tag encoding gene and contain the structure of the recombinant vectors pPIC3.5Khis of this gene
Primer sequence is as follows:
Primer 1:(upstream primer) 5 '-gaagtacgtaatgggcagcagccatcatcatcatc-3 '
Primer 2: (downstream primer) 5 '-gagagaattcacccatttgctgtccaccagt-3 '
1, be template with plasmid pET-28a (+), under the guiding of primer 1 and primer 2, carry out pcr amplification His-tag gene, the PCR reaction conditions is: 94 ℃ 3 minutes; 94 ℃ 1 minute, 57 ℃ 1 minute, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 4 ℃ of insulations 10 minutes.The PCR product is carried out 1% agarose gel electrophoresis to be detected, show that having obtained length is about the 113bp DNA cloning fragment of (the structure gene length that comprises His-tag is about 90bp), through order-checking, show the nucleotide sequence that this DNA cloning fragment has sequence 1 in the sequence table, comprising: nt5-10 SnaBI recognition site; Nt23-40 His-tag; Nt50-67 zymoplasm encoding gene; Nt64-69 EcoRI recognition site has obtained the correct dna fragmentation that contains the His-tag encoding gene.
2, after the DNA cloning fragment that includes the His-tag gene that step 1 is increased is cut with restriction enzyme SnaBI and EcoRI enzyme, use T with the plasmid vector pPIC3.5K that cuts through the same enzyme enzyme (Invitrogen company)
1Dna ligase connects, to connect product electrotransformation transformed into escherichia coli JM109 (TaKaRa company), screen with the resistance LB substratum that contains 100 μ g/L penbritins and 50 μ g/L kantlex, positive colony is inoculated in the LB liquid nutrient medium 37 ℃ to be cultivated 12-24 hour, with alkaline lysis upgrading grain, obtain recombinant vectors pPIC3.5KHis.
Two, the structure of human tyrosinase yeast expression vector pPIC3.5KHis-tyr
Primer sequence is as follows:
Primer 3:(upstream primer) 5 '-aggtgaattccatttccctagagcctgtgtctc-3 '
Primer 4:(downstream primer) 5 '-attagcggccgcttataaatggctctgatacaa-3 '
1, with plasmid pRHOHT2 (Shibahara, S., Tomita, Y., Tagami, H., Muller, R.M., Cohen, T..Molecular basis for the heterogeneity of human tyrosinase.Tohoku J.Exp.Med.1988,156,403-414) be template, under the guiding of primer 3 and primer 4, pcr amplification human tyrosinase gene, the PCR reaction conditions is: 94 ℃ 3 minutes; 94 ℃ 1 minute, 57 ℃ 1 minute, 72 ℃ 2 minutes, 30 circulations; 72 ℃ were extended 4 ℃ of insulations 10 minutes.The PCR product is carried out 1% agarose gel electrophoresis to be detected, show that having obtained length is about the 1558bp DNA cloning fragment of (length that contains the structure gene of tyr is about 1536bp), through order-checking, show the nucleotide sequence that this DNA cloning fragment has sequence 2 in the sequence table, comprising: nt 4-10 EcoRI recognition site; Nt11-1546 tyr; Nt 1547-1554 NotI recognition site has obtained the correct dna fragmentation that contains the human tyrosinase gene.
2, after the DNA cloning fragment that comprises the human tyrosinase gene that step 1 is increased is cut with restriction enzyme NotI and EcoRI enzyme, with the recombinant vectors pPIC3.5KHis T of the step 1 structure of cutting through the same enzyme enzyme
1Dna ligase connects, to connect product electrotransformation transformed into escherichia coli JM109 (TaKaRa company), screen with the resistance LB substratum that contains 100 μ g/L penbritins and 50 μ g/L kantlex, positive colony is inoculated in the LB liquid nutrient medium 37 ℃ to be cultivated 12-24 hour, with alkaline lysis upgrading grain, obtain human tyrosinase yeast expression vector pPIC3.5KHis-tyr.
The abduction delivering of embodiment 2, human tyrosinase and Western-blot identify
One, the abduction delivering of human tyrosinase
1, the linearizing of plasmid pPIC3.5KHis-tyr
Human tyrosinase yeast expression vector pPIC3.5KHis-tyr with SacI digestion with restriction enzyme embodiment 1 obtains reclaims behind 1% agarose gel electrophoresis and obtains electrophoretically pure linearization plasmid pPIC3.5KHis-tyr.
2, linearizing plasmid pPIC3.5KHis-tyr is transformed pichia pastoris phaff
Detailed process may further comprise the steps:
1) get glycerine and guarantee the pichia pastoris phaff GS115 that deposits, line on the YPD Agar flat board, cultivate for 30 ℃ and made its activation in 12-24 hour, picking list colony inoculation is in the YPD liquid nutrient medium, and 30 ℃ are cultured to OD
600Value is 1.4-1.5.
2) pipette the nutrient solution (parallel two pipes of doing) in the EP centrifuge tube of 1mL step 1), 4 ℃ of 5000rpm collected yeast cell in centrifugal 5 minutes, abandon supernatant, with the YPD liquid nutrient medium of 1mLYPD/HEPES (containing 20%HEPES among the YPD) and the 37.5 μ L1M DTT above-mentioned yeast cell precipitation that suspends, 30 ℃ cultivate 15 minutes after, 4 ℃ of 5000rpm collected yeast cell in centrifugal 5 minutes, abandoned supernatant.
3) get 1.5mL 1M sorbyl alcohol suspension step 2) yeast cell precipitation, centrifugal 5 minutes of 4 ℃ of 5000rpm abandon supernatant, repeat 3 times, use 20 μ L 1M sorbyl alcohol suspension yeast cell then, merge 2 and manage, and obtain the electroreception attitude yeast cell of 40 μ L.
4) after the electroreception attitude yeast cell GS115 that gets 5 μ L step 1 neutral line plasmid pPIC3.5KHis-tyr and 40 μ L mixes, transfer in the 2mm electric shock cup, carry out electricity conversion at voltage 1500V.
5) take out the electric shock cup, add the YPD liquid nutrient medium that 1mL contains the 182mg/mL sorbyl alcohol rapidly, then it is transferred in the Eppendorf pipe of sterilization, cultivated 1.5 hours for 30 ℃.
6) nutrient solution of step 5) is coated on the YNB resistant panel that contains 50mg/mL G418 (Geneticin), cultivated 2-5 days for 30 ℃, obtain transforming positive single bacterium colony of human tyrosinase yeast expression vector pPIC3.5KHis-tyr.
3, the expression of human tyrosinase
Substratum:
Growth medium (g/L): yeast extract 10g, peptone 20g, glycerine 20mL, phosphoric acid buffer (pH7.0) 100mL, 13.4%YNB 100mL, 0.02% vitamin H 20mL, distilled water 780mL.
Inducing culture (g/L): yeast extract 10g, peptone 20g, glycerine 20mL, phosphoric acid buffer (pH7.0) 100mL, 13.4%YNB 100mL, 0.02% vitamin H 20mL, methyl alcohol 20mL, distilled water 760mL.
Picking grows preferably that positive colony is inoculated in growth medium, cultivates about 48h for 30 ℃, and centrifugal and change the methanol induction substratum under aseptic condition, 30 ℃ are continued cultivations and carried out abduction delivering in 48-96 hour.
Two, the evaluation of expression product
The Western-blot detection system:
One is anti-: mouse anti tyrosine oxidase monoclonal antibody (Tyrosinase Ab-1, Clone T311), NeoMarkers, USA;
Two is anti-: the anti-mouse IgG of alkali phosphorus enzyme labelling horse, Bioisystech Co., Ltd of China fir Golden Bridge in Beijing;
BCIP/NBT: Bioisystech Co., Ltd of China fir Golden Bridge in Beijing.
After cultivating end, get the centrifugal collection thalline of bacterium liquid of 1mL step 1, with somatic cells granulated glass sphere succusion lysing cell, get 25 μ L cell pyrolysis liquids, add the SDS-PAGE electrophoresis, the result is (the saccharomyces pastorianus pichia spp transformant of swimming lane 1-2 for expressing as shown in Figure 2, swimming lane 3 is wild-type pichia pastoris phaff GS115, swimming lane M is albumen Marker (the molecular weight ranges 20-200KD of Marker), (position shown in the arrow) has a protein band at the 75KD place, electrophoresis discards spacer gel after finishing, successively with negative plate, fender, filter paper, glue, the NC film, filter paper, fender, positive plate is folded, (Frederick M.Ausubel et al. face grain husk, Wang Hailin is translated according to a conventional method.Fine works molecular biology guide, Science Press, 1998:366) carrying out Western-blot identifies, (swimming lane 1 is albumen Marker (the molecular weight ranges 20-200KD of Marker) to the result as shown in Figure 3, the saccharomyces pastorianus pichia spp transformant of swimming lane 2 for expressing), show that having obtained molecular weight is that (human tyrosinase not glycosylation molecular weight is 55KD for the human tyrosinase of 75KD (position shown in the arrow), be 75KD after the glycosylation), consistent with the glycosylated human tyrosinase size of bibliographical information.
Sample-loading buffer (MCAC-0): Tris-HCl 20mM pH7.9, NaCl 500mM, glycerine 10%, PMSF (phenylmethylsulfonyl fluoride) 1mM.
Elution buffer (MCAC-500): imidazoles 500mM, Tris-HCl 20mM, NaCl 500mM, glycerine 10%, PMSF 1mM.
The target protein of expressing among the embodiment 2 is carried out purifying with nickel ion affinity column chromatography method, and concrete steps are as follows:
1) collect among the embodiment 2 through the yeast cell of abduction delivering, it is dissolved among the sample-loading buffer MCAC-0, low-temperature homogenate 15, the centrifugal 10min of 000rpm collects crude protein solution;
2) crude protein solution is added nickel ion affinity chromatograph post (available from QIAGEN company) upper strata, keep 0.01ml/ second flow speed, all adsorbed to target protein;
3) add elution buffer MCAC-500 wash-out, collect 5ml albumen elutriant, promptly obtain the human tyrosinase of purifying, can-20 ℃ of preservations standby.
The purified human tyrosinase that obtains is carried out SDS-PAGE, and (swimming lane 1 is the human tyrosinase of purifying to the result, and swimming lane 2 is albumen Marker (the molecular weight ranges 20-200KD of Marker), shows to have obtained highly purified human tyrosinase as shown in Figure 4.The result shows that the human tyrosinase expression amount among the embodiment 2 is 0.39g/L.
Sequence table
<160>3
<210>1
<211>113
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gaagtacgta?atgggcagca?gccatcatca?tcatcatcac?agcagcggcc?tggtgccgcg 60
cggcagccat?atggctagca?tgactggtgg?acagcaaatg?ggtgaattct?ctc 113
<210>2
<211>1558
<212>DNA
＜213〉Genus Homo people (Homo sapiens)
<400>2
aggtgaattc?catttcccta?gagcctgtgt?ctcctctaag?aacctgatgg?agaaggaatg 60
ctgtccaccg?tggagcgggg?acaggagtcc?ctgtggccag?ctttcaggca?gaggttcctg 120
tcagaatatc?cttctgtcca?atgcaccact?tgggcctcaa?tttcccttca?caggggtgga 180
ggagtcgtgg?ccttccgtct?tttataatag?gacctgccag?tgctctggtg?accgcaactt 240
catgggattc?aactgtggaa?actgcaagtt?tggcttttgg?ggaccaaact?gcacagagag 300
acgactcttg?gtgagaagaa?acatcttcga?tttgagtgcc?ccagagaagg?acaaattttt 360
tgcctacctc?actttagcaa?agcataccat?cagctcagac?tatgtcatcc?ccatagggac 420
ctatggccaa?atgaaaaatg?gatcaacacc?catgtttaac?gacatcaata?tttatgacct 480
ctttgtctgg?atgcattatt?atgtgtcaat?ggatgcactg?cttgggggat?ctgaaatctg 540
gagagacatt?gattttgccc?atgaagcacc?agcttttctg?ccttggcata?gactcttctt 600
gttgcggtgg?gaacaagaaa?tccagaagct?gacaggagat?gaaaacttca?ctattccata 660
ttgggactgg?cgggatgcag?aaaagtgtga?catttgcaca?gatgagtaca?tgggaggtca 720
gcaccccaca?aatcctaact?tactcagccc?agcatcattc?ttctcctctt?ggcagattgt 780
ctgtagccga?ttggaggagt?acaacagcca?tcagtcttta?tgcaatggaa?cgcccgaggg 840
acctttacgg?cgtaatcctg?gaaaccatga?caaatccaga?accccaaggc?tcccctcttc 900
agctgatgta?gaattttgcc?tgagtttgac?ccaatatgaa?tctggttcca?tggataaagc 960
tgccaatttc?agctttagaa?atacactgga?aggatttgct?agtccactta?ctgggatagc 1020
ggatgcctct?caaagcagca?tgcacaatgc?cttgcacatc?tatatgaatg?gaacaatgtc 1080
ccaggtacag?ggatctgcca?acgatcctat?cttccttctt?caccatgcat?ttgttgacag 1140
tatttttgag?cagtggctcc?gaaggcaccg?tcctcttcaa?gaagtttatc?cagaagccaa 1200
tgcacccatt?ggacataacc?gggaatccta?catggttcct?tttataccac?tgtacagaaa 1260
tggtgatttc?tttatttcat?ccaaagatct?gggctatgac?tatagctatc?tacaagattc 1320
agacccagac?tcttttcaag?actacattaa?gtcctatttg?gaacaagcga?gtcggatctg 1380
gtcatggctc?cttggggcgg?cgatggtagg?ggccgtcctc?actgccctgc?tggcagggct 1440
tgtgagcttg?ctgtgtcgtc?acaagagaaa?gcagcttcct?gaagaaaagc?agccactcct 1500
catggagaaa?gaggattacc?acagcttgta?tcagagccat?ttataagcgg?ccgctaat 1558
<210>3
<211>511
<212>PRT
＜213〉Genus Homo people (Homo sapiens)
<400>3
His?Phe?Pro?Arg?Ala?Cys?Val?Ser?Ser?Lys?Asn?Leu?Met?Glu?Lys?Glu
1 5 10 15
Cys?Cys?Pro?Pro?Trp?Ser?Gly?Asp?Arg?Ser?Pro?Cys?Gly?Gln?Leu?Ser
20 25 30
Gly?Arg?Gly?Ser?Cys?Gln?Asn?Ile?Leu?Leu?Ser?Asn?Ala?Pro?Leu?Gly
35 40 45
Pro?Gln?Phe?Pro?Phe?Thr?Gly?Val?Glu?Glu?Ser?Trp?Pro?Ser?Val?Phe
50 55 60
Tyr?Asn?Arg?Thr?Cys?Gln?Cys?Ser?Gly?Asp?Arg?Asn?Phe?Met?Gly?Phe
65 70 75 80
Asn?Cys?Gly?Asn?Cys?Lys?Phe?Gly?Phe?Trp?Gly?Pro?Asn?Cys?Thr?Glu
85 90 95
Arg?Arg?Leu?Leu?Val?Arg?Arg?Asn?Ile?Phe?Asp?Leu?Ser?Ala?Pro?Glu
100 105 110
Lys?Asp?Lys?Phe?Phe?Ala?Tyr?Leu?Thr?Leu?Ala?Lys?His?Thr?Ile?Ser
115 120 125
Ser?Asp?Tyr?Val?Ile?Pro?Ile?Gly?Thr?Tyr?Gly?Gln?Met?Lys?Asn?Gly
130 135 140
Ser?Thr?Pro?Met?Phe?Asn?Asp?Ile?Asn?Ile?Tyr?Asp?Leu?Phe?Val?Trp
145 150 155 160
Met?His?Tyr?Tyr?Val?Ser?Met?Asp?Ala?Leu?Leu?Gly?Gly?Ser?Glu?Ile
165 170 175
Trp?Arg?Asp?Ile?Asp?Phe?Ala?His?Glu?Ala?Pro?Ala?Phe?Leu?Pro?Trp
180 185 190
His?Arg?Leu?Phe?Leu?Leu?Arg?Trp?Glu?Gln?Glu?Ile?Gln?Lys?Leu?Thr
195 200 205
Gly?Asp?Glu?Asn?Phe?Thr?Ile?Pro?Tyr?Trp?Asp?Trp?Arg?Asp?Ala?Glu
210 215 220
Lys?Cys?Asp?Ile?Cys?Thr?Asp?Glu?Tyr?Met?Gly?Gly?Gln?His?Pro?Thr
225 230 235 240
Asn?Pro?Asn?Leu?Leu?Ser?Pro?Ala?Ser?Phe?Phe?Ser?Ser?Trp?Gln?Ile
245 250 255
Val?Cys?Ser?Arg?Leu?Glu?Glu?Tyr?Asn?Ser?His?Gln?Ser?Leu?Cys?Asn
260 265 270
Gly?Thr?Pro?Glu?Gly?Pro?Leu?Arg?Arg?Asn?Pro?Gly?Asn?His?Asp?Lys
275 280 285
Ser?Arg?Thr?Pro?Arg?Leu?Pro?Ser?Ser?Ala?Asp?Val?Glu?Phe?Cys?Leu
290 295 300
Ser?Leu?Thr?Gln?Tyr?Glu?Ser?Gly?Ser?Met?Asp?Lys?Ala?Ala?Asn?Phe
305 310 315 320
Ser?Phe?Arg?Asn?Thr?Leu?Glu?Gly?Phe?Ala?Ser?Pro?Leu?Thr?Gly?Ile
325 330 335
Ala?Asp?Ala?Ser?Gln?Ser?Ser?Met?His?Asn?Ala?Leu?His?Ile?Tyr?Met
340 345 350
Asn?Gly?Thr?Met?Ser?Gln?Val?Gln?Gly?Ser?Ala?Asn?Asp?Pro?Ile?Phe
355 360 365
Leu?Leu?His?His?Ala?Phe?Val?Asp?Ser?Ile?Phe?Glu?Gln?Trp?Leu?Arg
370 375 380
Arg?His?Arg?Pro?Leu?Gln?Glu?Val?Tyr?Pro?Glu?Ala?Asn?Ala?Pro?Ile
385 390 395 400
Gly?His?Asn?Arg?Glu?Ser?Tyr?Met?Val?Pro?Phe?Ile?Pro?Leu?Tyr?Arg
405 410 415
Asn?Gly?Asp?Phe?Phe?Ile?Ser?Ser?Lys?Asp?Leu?Gly?Tyr?Asp?Tyr?Ser
420 425 430
Tyr?Leu?Gln?Asp?Ser?Asp?Pro?Asp?Ser?Phe?Gln?Asp?Tyr?Ile?Lys?Ser
435 440 445
Tyr?Leu?Glu?Gln?Ala?Ser?Arg?ILe?Trp?Ser?Trp?Leu?Leu?Gly?Ala?Ala
450 455 460
Met?Val?Gly?Ala?Val?Leu?Thr?Ala?Leu?Leu?Ala?Gly?Leu?Val?Ser?Leu
465 470 475 480
Leu?Cys?Arg?His?Lys?Arg?Lys?Gln?Leu?Pro?Glu?Glu?Lys?Gln?Pro?Leu
485 490 495
Leu?Met?Glu?Lys?Glu?Asp?Tyr?His?Ser?Leu?Tyr?Gln?Ser?His?Leu
500 505 510
Claims (9)
1, a kind of human tyrosinase expression carrier is the pichia pastoris phaff expression vector that has transformed the human tyrosinase gene and had the His-tag gene of sequence 1 nucleotide sequence in the sequence table; Described pichia pastoris phaff expression vector is pPIC3.5K, pPIC3, pPIC9, pHIL-D1, pA0804, pA0815, pPSC3K or pPIC9K.
2, human tyrosinase expression carrier according to claim 1 is characterized in that: the protein of sequence 3 amino acid residue sequences in the described human tyrosinase gene coded sequence table.
3, human tyrosinase expression carrier according to claim 2 is characterized in that: described human tyrosinase gene is the nucleotide sequence of sequence 2 in the sequence table.
4, human tyrosinase expression carrier according to claim 1 is characterized in that: described human tyrosinase expression carrier is pPIC3.5KHis-tyr shown in Figure 1.
5, arbitrary described human tyrosinase expression carrier among the claim 1-4 is imported the reconstitution cell that the pichia pastoris phaff cell obtains.
6, reconstitution cell according to claim 5 is characterized in that: described pichia pastoris phaff cell is pichia pastoris phaff GS115, KM71 or SMD1168.
7, a kind of method of expressing human tyrosine oxidase comprises and cultivates claim 5 or 6 described reorganization pichia pastoris phaff cells, the step of methanol induction expressing human tyrosine oxidase.
8, the method for expressing human tyrosine oxidase according to claim 7 is characterized in that: also comprise the step of utilizing metal affinity column chromatography separation and purification human tyrosinase in the described method.
9, the method for expressing human tyrosine oxidase according to claim 8 is characterized in that: described metal affinity column chromatography is the nickel ion affinity column chromatography.
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| CN111218432B (en) * | 2018-11-27 | 2023-03-31 | 中国科学院大连化学物理研究所 | Tyrosinase precursor, encoding gene, preparation and application thereof |
| CN111004785A (en) * | 2019-12-16 | 2020-04-14 | 华中科技大学 | Tyrosinase protein sequence and application thereof in preparation of tyrosinase |
| KR102269740B1 (en) * | 2020-04-22 | 2021-06-25 | 중앙대학교 산학협력단 | Recombinant strain for tyrosinase production and tyrosinase purificationmethod using same |
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