CN104946609A - Preparation method for glutaminyl cyclase - Google Patents

Preparation method for glutaminyl cyclase Download PDF

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CN104946609A
CN104946609A CN201510319520.3A CN201510319520A CN104946609A CN 104946609 A CN104946609 A CN 104946609A CN 201510319520 A CN201510319520 A CN 201510319520A CN 104946609 A CN104946609 A CN 104946609A
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preparation
glutaminyl cyclase
carry out
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enzyme
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吴海强
李满满
郑易之
邹永东
贺震旦
刘志刚
董瑶
徐迪宗
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Shenzhen University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02005—Glutaminyl-peptide cyclotransferase (2.3.2.5)

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Abstract

The invention discloses a preparation method for glutaminyl cyclase. The preparation method comprises the following steps: transforming a recombinant vector which can be used for expressing QC<33-361> into competent escherichia coli cells so as to obtain gene engineering bacteria capable of synthesizing QC<33-361>; inoculating the gene engineering bacteria into an LB culture medium containing resistant markers and carrying out induction expression; after completion of induction expression, collecting fusion protein containing QC<33-361>; desalting, carrying out enzyme digestion on the fusion protein and carrying out molecular sieve column chromatography purification so as to separate out QC<33-361> protein. The synthesized QC<33-361> protein has the obvious activity of catalyzing glutaminyl cyclization reaction in bstrate molecules. The synthesized QC<33-361> can be used for screening and finding of QC inhibitors, agonists, antagonists and the like, QC related action mechanism research, QC detection related kit development, detection and diagnosis of QC high expression related diseases and the like.

Description

A kind of preparation method of glutaminyl cyclase
Technical field
The present invention relates to gene engineering technology field, particularly relate to a kind of preparation method of glutaminyl cyclase.
Background technology
Glutaminyl cyclase (Glutaminyl cyclase; QC; EC2.3.2.5) be that one can hold glutamine residue intramolecular cyclization reaction to generate the enzyme of Pyrrolidonecarboxylic acid (pGlu) by the N such as catalytic polypeptide, albumen, there is N such as changing albumen and hold chemical structure, regulate the important biomolecule functions such as activity, enhanced stability.
1963, QC found first in tropical plants papaya (Carica papaya) latex, and rear research confirms, QC all has distribution in plant, animal, microorganism.But in plant, the physiological function of QC is very not clear and definite, may have certain effect in plant defense pathogenic micro-organism process.QC enzyme kinetics behavior in plant, animal all meets Michaelis-Menton equation (Mechaelis-Menten), and has strict specificity to position, substrate N end L-glutaminate.Mammals QC has well-conserved, and without any sequence homology between papaya QC, and with bacterium aminopeptidase, there is significant sequence homology, therefore animal and plant QC has different evolutionary source.
Along with the development of human society age structure aging, senile dementia sickness rate raises rapidly.Alzheimer's disease (Alzheimer ' s disease, AD) be a kind of common nerve degenerative diseases, be the principal mode of senile dementia, epidemiology survey shows, and AD patient exceedes more than 65% of old dementia patients sum.A few days ago, AD sickness rate raises increasingly, has become the third-largest worldwide health problem being only second to cardiovascular and cerebrovascular diseases and tumour.
AD main clinic symptoms comprises Progressive symmetric erythrokeratodermia memory and cognition dysfunction etc., there is no specific treatment medicine at present.AD pathogenesis is complicated, through 100 years of researches, there has been proposed the hypothesis that some are different, as AD related protein entanglement and gathering, oxidative stress, biological metal ionic homeostasis is unbalance, mitochondrial function exhaustion etc., but exact mechanism does not understand.The Hyperphosphorylationof Tau albumen entanglement etc. of the AD pathological characteristics amyloid-beta precipitation that mainly brain neuroblastoma unit is outside and inside neurons.Amyloid-beta (A β) is the main component of senile plaque, and according to A β cascade theory, the excessive generation of A β and gathering, precipitation are considered to fall ill with AD and be in progress have direct relation.A β is that beta amyloid precursor protein (APP) is hydrolyzed gained fragment products, as A β 42/A β 40 through beta-secretase and gamma-secretase.The displays such as in vitro study, A β overexpression and gathering, precipitation directly can cause mitochondrial disorders, oxidative stress, nerve synapse transmission interruption, axonal transport interrupt, the impaired multiple AD related pathologies change such as to break of film integrality.
In recent years, jointly confirm with clinical study in multinomial body, be different from the senile plaque precipitation of Elderly people human brain, the main component of AD patient's brain senile plaque precipitation is not aβ protein, but the aβ protein of multiple variation, particularly N holds the pGlu-A β of 3-position glutamine residue molecule inner ring condensation, and comprise pGlu-A β 42/pGlu-A β 40, content is more than 50%.PGlu-A β has stronger stability, faster aggregate and precipitate speed, stronger neurotoxicity, and its generation is directly related with the generation of AD clinical symptom.This provides new clue for AD correlative study.Deeply showing further of correlative study, pGlu-A β is the product of QC effect.QC specificity overexpression is that AD falls ill early stage specific lesions, it is the crucial promoting factor of AD morbidity, development, Selective depression QC activity significantly can suppress the generation of pGlu-A β and the formation of senile plaque precipitation, and obviously improves the AD such as cognition, impaired memory function symptom.Therefore, QC is that AD pathology and causal innovation anti-AD drug research open new research direction.
QC zymoprotein is the necessary starting material of correlative study, and its product confrontation QC mechanism of action and inhibitor research have material impact.The acquisition of QC is prepared by the separation of QC active in natural goods and adopts genetic engineering technique preparation restructuring QC two kinds of methods to realize.The deficiencies such as extraction and separation method is lower by QC content in natural goods, extraction cost is high, lot stability is poor, loss of enzyme activity is serious, have larger restriction in applying.By contrast, genetic engineering technique preparation restructuring QC is the active QC preparation method of one being easier to realize.At present, have been reported and claim to express preparation restructuring QC by E.coli, but method exists certain defect, prepare that expression amount is low, enzymic activity is poor, step is various.Visible, a kind of newly, efficiently, fast can prepare the foundation of the method for high activity recombinant human QC, to QC Study on Correlative Mechanisms, the anti-AD new drug research of QC inhibitor class and the clinical diagnosis of QC abnormal expression relative disease etc., all there is very important scientific meaning and using value.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of preparation method of glutaminyl cyclase, provide a kind of newly efficiently, fast can prepare high reactivity human glutamine acyl group cyclase prepare approach.
Technical scheme of the present invention is as follows:
A preparation method for glutaminyl cyclase, wherein, comprises the following steps:
A, will can be used for express QC 33-361recombinant vectors be transformed in competent escherichia coli cell, acquisition can synthesize QC 33-361genetic engineering bacterium;
B, be inoculated into genetic engineering bacterium containing resistance marker LB substratum, abduction delivering;
After c, abduction delivering terminate, collect thalline, the broken thalline of bacteriolyze, high pressure, centrifugation, get supernatant liquor and carry out affinity chromatography and carry out purifying, collect containing QC 33-361fusion rotein;
D, to containing QC 33-361fusion rotein carry out PPE enzyme, desalination, molecular sieve column chromatography purifying, isolates QC 33-361albumen.
The preparation method of described glutaminyl cyclase, wherein, in step b, comprises the following steps:
Picking genetic engineering bacterium list colony inoculation is to being inoculated into containing in antibiotic LB liquid nutrient medium, and jolting is cultivated, and obtains seed culture fluid;
Be inoculated into by seed culture fluid containing in antibiotic LB nutrient solution, 37 DEG C, jolting is cultivated, and is cultured to OD600 value and reaches 0.6-0.8, with IPTG inducing culture.
The preparation method of described glutaminyl cyclase, wherein, in step c, comprises the following steps:
By bacterium liquid collected by centrifugation thalline, use high pressure smudge cells instrument to split abundant cracking thalline, centrifugal, supernatant liquor Ni ion affinity chromatography carries out separation and purification, collects containing QC 33-361fusion rotein.
The preparation method of described glutaminyl cyclase, wherein, in steps d, comprises the following steps:
With desalting column to the desalination of collection gained fusion rotein, with PPE enzyme, enzyme is carried out to fusion rotein and cut, with molecular sieve S-200Column, mixed system is cut to enzyme and carry out molecular sieve column chromatography and purify, collect target protein QC 33-361.
The preparation method of described glutaminyl cyclase, wherein, in step a, comprises the following steps:
Add in competent escherichia coli cell and can be used for expressing QC 33-361recombinant vectors, leave standstill on ice, water-soluble pot heat shock 60-70s, leaves standstill on ice, adds appropriate LB liquid nutrient medium, and be applied on the LB solid plate with resistance marker, overnight incubation, obtains genetic engineering bacterium.
The preparation method of described glutaminyl cyclase, wherein, in step a, described competent escherichia coli cell is bacillus coli DH 5 alpha or e. coli bl21 (DE3).
The preparation method of described glutaminyl cyclase, wherein, in step a, before conversion recombinant vectors, carries out activation treatment to competent escherichia coli cell:
By the actication of culture preserved, in LB liquid nutrient medium, 37 DEG C are cultured to OD600 value is 0.6 ~ 0.8, makes bacterium liquid be cooled to 0 DEG C on ice, collected by centrifugation thalline, with the 0.1mol/L CaCl of precooling 2solution prepares competent cell.
The preparation method of described glutaminyl cyclase, wherein, described for expressing QC 33-361the preparation method of recombinant vectors, comprise the following steps:
Adopt round pcr, with upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2 for primer pair, amplification obtains QC 33-361gene fragment; Wherein, upstream primer is 5 '-ACCTGGATCCGCTTCTGCTTGGCCGG-3 '; Downstream primer is 5 '-TATCCTCGAGTTACAGGTGCAGGTATTC-3 ';
To carrier pET28a and pET32a, carry out EcoRI, XhoI double digestion respectively; Linearized vector two ends being existed equally EcoRI, XhoI site connects, and obtains improved pET32a carrier;
To QC 33-361gene fragment and improved pET32a carrier carry out BamHI and XhoI double digestion, two ends are existed equally the QC in BamHI, XhoI site 33-361gene fragment is connected with linearized vector, obtains pET32a/QC 33-361recombinant vectors.
Beneficial effect: the invention provides a kind of recombinant vectors for expressing human glutamine acyl group cyclase, can be used for efficiently, fast prepare high activity recombinant human QC 33-361enzyme.By this vector in recipient cell, can be used for synthesis human glutamine acyl group cyclase.Synthesize the human glutamine acyl group cyclase QC obtained 33-361for having the activity of glutaminyl cyclase reaction in remarkable catalytic substrate molecule, can the intramolecular cyclisation of glutaminyl be held to generate the product of Pyrrolidonecarboxylic acid by efficient catalytic albumen (polypeptide) N.Synthesize the human glutamine acyl group cyclase QC obtained 33-361can be used for the screening discovery of QC inhibitor, agonist, antagonist etc., QC dependent interaction Mechanism Study, QC detect the exploitation of related kit, the checkout and diagnosis etc. of QC high expression level relative disease.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of QC recombinant expression vector of the present invention.
Fig. 2 is QC in the embodiment of the present invention 1 33-361gene PCR amplification.
Fig. 3 is recombinant plasmid pET32a/QC in the embodiment of the present invention 1 33-361double digestion qualification result.
Fig. 4 is QC in the embodiment of the present invention 4 33-361sDS-PAGE analytical results in the expression and purification process of albumen.
Fig. 5 is gained QC in the embodiment of the present invention 4 33-361protein spectrum analyses and comparison result.
Fig. 6 is QC in the embodiment of the present invention 5 33-361enzymic activity test philosophy schematic diagram
Fig. 7 is QC in the embodiment of the present invention 5 33-361two matched curve figure reciprocal.
Embodiment
The invention provides a kind of preparation method of glutaminyl cyclase, for making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The present invention utilizes human glutamine acyl group cyclase QC 33-361it is a fragment of EC2.3.2.5 full-length proteins; the cDNA sequence of human pituitary QC is retrieved from GenBank; in conjunction with the feature of pET32a carrier; a pair Auele Specific Primer is designed with software Oligo; amplification obtains the gene order of QC enzyme, and is inserted on pET32a carrier, builds the recombinant vectors obtaining may be used for expressing human glutamine acyl group cyclase.Further, by this vector in recipient cell, can be used for synthesis human glutamine acyl group cyclase.Synthesize the human glutamine acyl group cyclase QC obtained 33-361for having the activity of glutaminyl cyclase reaction in remarkable catalytic substrate molecule, can the intramolecular cyclisation of glutaminyl be held to generate the product of Pyrrolidonecarboxylic acid by efficient catalytic albumen (polypeptide) N.Synthesize the human glutamine acyl group cyclase QC obtained 33-361can be used for the screening discovery of QC inhibitor, agonist, antagonist etc., QC dependent interaction Mechanism Study, QC detect the exploitation of related kit, the checkout and diagnosis etc. of QC high expression level relative disease.Wherein, described human glutamine acyl group cyclase QC 33-361aminoacid sequence be:
ASAWPEEKNYHQPAILNSSALRQIAEGTSISEMWQNDLQPLLIERYPGSPGSYAARQHIMQRIQRLQADWVLEIDTFLSQTPYGYRSFSNIISTLNPTAKRHLVLACHYDSKYFSHWNNRVFVGATDSAVPCAMMLELARALDKKLLSLKTVSDSKPDLSLQLIFFDGEEAFLHWSPQDSLYGSRHLAAKMASTPHPPGARGTSQLHGMDLLVLLDLIGAPNPTFPNFFPNSARWFERLQAIEHELHELGLLKDHSLEGRYFQNYSYGGVIQDDHIPFLRRGVPVLHLIPSPFPEVWHTMDDNEENLDESTIDNLNKILQVFVLEYLHL。
Particularly, provide a kind of recombinant vectors for expressing human glutamine acyl group cyclase and preparation method thereof in the present invention, wherein, the preparation method of the described recombinant vectors for expressing human glutamine acyl group cyclase, comprises the following steps:
1, adopt round pcr, with upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2 for primer pair, amplification obtains QC 33-361gene fragment SEQ ID NO.4, QC 33-361the nucleotide sequence of gene fragment is as follows:
GCTTCTGCTTGGCCGGAAGAAAAAAACTACCACCAGCCGGCTATCCTGAACTCTTCTGCTCTGCGTCAGATCGCTGAAGGTACCTCTATCTCTGAAATGTGGCAGAACGACCTGCAGCCGCTGCTGATCGAACGTTACCCGGGTTCTCCGGGTTCTTACGCTGCTCGTCAGCACATCATGCAGCGTATCCAGCGTCTGCAGGCTGACTGGGTTCTGGAAATCGACACCTTCCTGTCTCAGACCCCGTACGGTTACCGTTCTTTCTCTAACATCATCTCTACCCTGAACCCGACCGCTAAACGTCACCTGGTTCTGGCTTGCCACTACGACTCTAAATACTTCTCTCACTGGAACAACCGTGTTTTCGTTGGTGCTACCGACTCTGCTGTTCCGTGCGCTATGATGCTGGAACTGGCTCGTGCTCTGGACAAAAAACTGCTGTCTCTGAAAACCGTTTCTGACTCTAAACCGGACCTGTCTCTGCAGCTGATCTTCTTCGACGGTGAAGAAGCGTTCCTGCACTGGTCTCCGCAGGACTCTCTGTACGGTTCTCGTCACCTGGCTGCTAAAATGGCTTCTACCCCGCACCCGCCGGGTGCTCGTGGTACCTCTCAGCTGCACGGTATGGACCTGCTGGTTCTGCTGGACCTGATCGGTGCTCCGAACCCGACCTTCCCGAACTTCTTCCCGAACTCTGCTCGTTGGTTCGAACGTCTGCAGGCTATCGAACACGAACTGCACGAACTGGGTCTGCTGAAAGACCACTCTCTGGAAGGTCGTTACTTCCAGAACTACTCTTACGGTGGTGTTATCCAGGACGACCACATCCCGTTCCTGCGTCGTGGTGTTCCGGTTCTGCACCTGATCCCGTCTCCGTTCCCGGAAGTTTGGCACACCATGGACGACAACGAAGAAAACCTGGACGAATCTACCATCGACAACCTGAACAAAATCCTGCAGGTTTTCGTTCTGGAATACCTGCACCTGTAA。
Wherein, upstream primer SEQ ID NO.1:5 '-ACCTGGATCCGCTTCTGCTTGGCCGG-3 '; Downstream primer SEQ ID NO.2:5 '-TATCCTCGAGTTACAGGTGCAGGTATTC-3 '.In upstream primer, " GGATCC " is nucleotide sequence corresponding to BamHI restriction enzyme site, and downstream primer " CTCGAG " is nucleotide sequence corresponding to XhoI restriction enzyme site.
2, respectively EcoRI, XhoI double digestion is carried out to existing carrier pET28a (28a-EcoRI-His6-PPE-BamHI-XhoI) and pET32a (32a-Trx-tag-s.tag-EcoRI-Hind-XhoI); Linearized vector two ends being existed equally EcoRI, XhoI connects, and obtain improved pET32a carrier (32a-Trx-tag-s.tag-EcoRI-His6-PPE-BamHI-XhoI), PPE is protease cutting site.
3, to QC 33-361gene fragment and improved pET32a carrier carry out BamHI and XhoI double digestion, two ends are existed equally the QC in BamHI, XhoI site 33-361gene fragment is connected with linearized vector, obtains pET32a/QC 33-361recombinant vectors.
A kind of recombinant vectors for expressing human glutamine acyl group cyclase provided in the present invention, described recombinant vectors is pET32a/QC 33-361recombinant vectors, adopt aforesaid method to prepare, the structure schematic diagram of described recombinant vectors as shown in Figure 1.A kind of genetic engineering bacterium also provided in the present invention, containing the above-mentioned recombinant vectors for expressing human glutamine acyl group cyclase in described genetic engineering bacterium.The bacterial classification of described genetic engineering bacterium can be bacillus coli DH 5 alpha or e. coli bl21 (DE3).
A kind of preparation method of glutaminyl cyclase is also provided in the present invention, comprises the following steps:
1, pET32a/QC is built 33-361recombinant vectors;
2, by pET32a/QC 33-361recombinant vectors is transformed in competent escherichia coli cell, and acquisition can synthesize QC 33-361genetic engineering bacterium.
3, genetic engineering bacterium is inoculated in the LB substratum containing resistance marker, abduction delivering.
4, after abduction delivering terminates, collect thalline, the broken thalline of bacteriolyze, high pressure, centrifugation, get supernatant liquor and carry out affinity chromatography and carry out purifying, collect containing QC 33-361fusion rotein.
5, to containing QC 33-361fusion rotein carry out PPE enzyme and cut, desalination, molecular sieve column chromatography purifying, isolates QC 33-361albumen.
Wherein, described competent escherichia coli cell is bacillus coli DH 5 alpha or e. coli bl21 (DE3).The described LB substratum containing resistance marker is the LB substratum containing Cam (clarithromycin) resistance, or the LB substratum containing Amp (ammonia benzyl mycin) resistance.
Before conversion recombinant vectors, activation treatment can be carried out: by the actication of culture preserved, 37 DEG C are cultured to OD600 value in LB liquid medium is 0.6 ~ 0.8, makes bacterium liquid be cooled to 0 DEG C on ice to competent escherichia coli cell, collected by centrifugation thalline, with the 0.1mol/L CaCl of precooling 2solution prepares competent cell.
In step 2, detailed process can be, in competent escherichia coli cell, add pET32a/QC 33-361recombinant plasmid, leaves standstill 30min, 42 DEG C of water-soluble pot heat shock 60-70s on ice, leaves standstill 2min on ice, add appropriate LB liquid nutrient medium, 37 DEG C, 200rpm cultivates 50min, be applied on the LB solid plate with resistance marker, 37 DEG C of overnight incubation, obtain genetic engineering bacterium.
In step 3, detailed process can be, picking genetic engineering bacterium list colony inoculation is to being inoculated into containing in 1 ‰ antibiotic LB liquid nutrient mediums, and 37 DEG C, 200rpm shaking table jolting about 10h, obtains seed culture fluid; Be inoculated into containing in 1 ‰ antibiotic LB nutrient solutions by seed culture fluid with the ratio of 1:200,37 DEG C, 200rpm, is cultured to OD600 value and reaches 0.6-0.8, induces 48 hours, 20 DEG C, 160rpm with the IPTG of 1mM (isopropylthiogalactoside).
In step 4, detailed process can be, centrifugal for bacterium liquid 8000rpm 30min is collected thalline, and use high pressure smudge cells instrument to split abundant cracking thalline, the centrifugal 50min of 25000rpm, supernatant liquor Ni ion affinity chromatography carries out separation and purification, collects containing QC 33-361fusion rotein.
In step 5, detailed process can be, with desalting column to the desalination of collection gained fusion rotein, carry out enzyme with PPE enzyme to fusion rotein to cut, cut mixed system with molecular sieve S-200Column to enzyme to carry out molecular sieve column chromatography and purify, carry out SDS-PAGE analysis, collect target protein QC 33-361.
Below by way of specific embodiment, the present invention will be further described.
Materials and methods:
People's pituitary body glutaminyl cyclase and QC 1-361gene is synthesized by Takara company.
Molecular cloning pET32a expression plasmid, Host Strains intestinal bacteria Ecoli DH5 α, expressive host bacterium Ecoli BL21 DE3 etc. is our unit and preserves genetically engineered research material.
1 enzyme activity unit represents 25 DEG C, per minute internal consumption 1 micromole (μm ol) enzyme amount required for NADH, and unit is μM/min.
PrimeSTAR HS DNA Polymerase, Upstream primer, Downstream primer, dNTP Mixture equimolecular biological reagent are purchased from Takara company; The purchased from American NEB companies such as DNA restriction enzyme (BamHI, XhoI), T4 DNA ligase; E.Z.N.A glue reclaims the purchased from American Omega company such as test kit, E.Z.N.A plasmid extraction kit; Albumen Marker, determination of protein concentration test kit etc. are purchased from TIANGEN Bioisystech Co., Ltd; The available from Sigma such as NADH, glutamate dehydrogenase, DMSO; N-Boc-QQ-2 is purchased from the biochemical company limited of Shanghai gill, and other common drugs, reagent, substratum etc. are purchased from Shanghai biotechnology company limited.
In the present invention, involved plasmid extraction, PCR, enzyme are cut, are linked the routine operation that conversion, abduction delivering, purification etc. are genetically engineered field
Embodiment 1 is containing the structure of people QC expression vector
According to QC enzyme number EC 2.3.2.5, retrieve the cDNA sequence of mankind QC from GenBank, according to pET32a carrier feature, with Oligo software design a pair Auele Specific Primer:
Upstream primer: 5-3:ACCTGGATCCGCTTCTGCTTGGCCGG;
Downstream primer: 5-3:TATCCTCGAGTTACAGGTGCAGGTATTC.
The restriction enzyme site of primer is cohesive terminus, and ensures except having corresponding restriction enzyme site in designed primer, and not containing the restriction enzyme site design primer in primer in the QC gene order of amplification, primer is synthesized by Takara company.With above-mentioned primer pair, carry out pcr amplification, goal gene (QC 33-361gene) pcr amplification result as shown in Figure 2, wherein, 1 is Marker, gene QC for the purpose of 2 33-361pCR primer.Adopt Omega E.Z.N.A glue to reclaim test kit and reclaim the goal gene (QC containing restriction enzyme site 33-361gene) fragment, BamHI-XhoI double digestion is carried out to goal gene fragment.
To existing carrier pET28a (28a-EcoRI-His6-PPE-BamHI-XhoI) and pET32a (32a-Trx-tag-s.tag-EcoRI-Hind-XhoI), carry out EcoRI, XhoI double digestion respectively; Linearized vector two ends being existed equally EcoRI, XhoI connects, and obtains improved pET32a carrier (32a-Trx-tag-s.tag-EcoRI-His6-PPE-BamHI-XhoI), wherein inserts PPE restriction enzyme site.
Also BamHI-XhoI double digestion is carried out to transformed pET32a carrier, obtains linearized vector, then two ends are existed equally the QC in BamHI, XhoI site 33-361object fragment is connected with linearized vector, obtains pET32a/QC 33-361positive recombinant plasmid.Carry out the qualification of BamHI-XhoI double digestion to recombinant plasmid, as shown in Figure 3, wherein, 1 is Marker to result, and 2 is the QC after EcoR Ι and Xho Ι double digestion 33-361gene fragment and plasmid fragments.
By pET32a/QC 33-361positive recombinant plasmid transformed, in Ecoli.DH5 α competent cell, is cultivated with Amp resistance LB solid medium, and PCR identifies primary dcreening operation positive colony.After positive colony is cultivated, extract plasmid, through Shanghai, raw work is identified correctly further, completes for the construction of recombinant vector of expressing human glutamine acyl group cyclase.
Embodiment 2 is recombinated the structure of QC expressing gene engineering bacteria
By the Ecoli.DH5 α actication of culture preserved, in LB liquid medium, 37 DEG C are cultured to OD600 value is 0.6 ~ 0.8, makes bacterium liquid be cooled to 0 DEG C on ice, collected by centrifugation thalline, with the 0.1mol/L CaCl of precooling 2solution prepares competent cell.Then, in competent cell, pET32a/QC is added 33-361positive recombinant plasmid, leave standstill 30min on ice, 42 DEG C of water-soluble pot heat shock 60-70s, leave standstill 2min on ice, add 700 μ l LB liquid nutrient mediums, 37 DEG C, 188 × g cultivates 50min, is applied on the LB solid plate with Amp resistance, 37 DEG C of overnight incubation, build recombinant human QC expressing gene engineering bacteria, can directly carry out next step abduction delivering operation.
The efficiently purifying preparation of embodiment 3 recombinant human QC
On the LB solid plate with ammonia benzyl (Amp) resistance marker, the positive single bacterium colony of picking, is inoculated in the 5ml LB liquid medium containing 1 ‰ Amp, 37 DEG C, 188 × g jolting about 10h, and expand by 1:200 and connect, 37 DEG C are cultured to OD 600value reaches 0.6-0.8, adds the IPTG of 1mM, 20 DEG C, 150 × g abduction delivering two days; Can regularly sample with the expression amount of SDS-PAGE analysis purposes albumen.
Abduction delivering terminates, and the centrifugal 30min of bacterium liquid 7530 × g collects thalline, and use the abundant cracking thalline of high pressure smudge cells instrument, the centrifugal 50min of 35000 × g, cellular lysate supernatant liquor carries out Ni affinitive layer purification.Get 250ul Ni filler in 1.5mlEP pipe, centrifugal, abandoning supernatant, then balances pillar 2 times with lysate, supernatant discarded, the supernatant liquor of cellular lysate is transferred to the EP pipe of 15ml, Ni-bead is transferred in the protein liquid of 15ml, be placed on refrigerator rocker and shake 45min, 800-900rpm, 4 DEG C, 2min, supernatant discarded; With 20mM imidazole elution by Ni filler-protein delivery in 1.5ml EP pipe, and wash three times, then wash two times with 40mM imidazole elution, finally wash with 250mM imidazole elution, collect the fusion rotein containing target protein.
Preparation 150mM NaCl, 50mM Tris damping fluid, with desalting column to the desalination of collection gained fusion rotein, PPE:QC=1:100 (concentration of PPE is 2mg/ml) adds PPE enzyme by volume, 4 DEG C of enzymes cut through night, cut rear mixed system and carry out molecular sieve column chromatography and purify, and carry out 15%SDS-PAGE analysis with molecular sieve S-200Column to enzyme, collect target protein, Coomassie brilliant blue G250 solution surveys protein concentration, protein concentrate, directly use or frozen for subsequent use in-80 DEG C.Gained QC recombinant protein is identified as people QC, so far successful expression QC through MS.
QC 33-361as shown in Figure 4, wherein, band 1 is Marker to SDS-PAGE analytical results in the expression and purification process of albumen, and band 2 is the cellular lysate liquid supernatant liquor of IPTG abduction delivering 48h, and band 3 is the pET32a/QC after Ni affinitive layer purification 33-361warm albumen, band 4 cuts the mixed system of 12-16h for PPE enzyme, and band 5 is molecular sieve column chromatography purification QC 33-361.
Gained QC 33-361as shown in Figure 5, retrieve expressed albumen is Human QC to protein spectrum analyses and comparison result really.
Embodiment 4 recombinant human QC enzymatic activity is tested
Now join TFA in proportion: tri isopropyl silane: phenol: water=88:5:5:2 mixed solution; prepare fresh QC substrate Gln-Gln for enzyme test alive to N-Boc-Gln (Trt)-Gln (Trt) deprotection, QC enzymic activity test philosophy schematic diagram as shown in Figure 6.Enzyme is lived in testing and is carried out in 96 hole enzyme plates, adopt 200ul pH8.0 Tris buffer system: 0.3mM NADH, different concns substrate Gln-Gln (0mM, 0.12mM, 0.24mM, 0.96mM, 1.2mM, 1.92mM, 2.4mM, 3.84mM), 14mM α-ketoglutaric acid, 30U/ml glutamate dehydrogenase, 50mM Tris, pH8.0 damping fluid, finally adds the QC of different concns 33-361(0.028 μM, 0.056 μM, 0.11 μM, 0.14 μM, 0.28 μM), with the absorption value change in 340nm wavelength place 15min of NADH during microplate reader detection of dynamic 25 DEG C, carries out a data gathering every 30s.Test result as shown in Figure 7, according to test result, analyzes Vmax=333.33mM/min, Km=0.86mM, Kcat=12.68s of recombinant human QC with Michaelis-Menton equation, two reciprocal equation -1.
Should be understood that, application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.

Claims (8)

1. a preparation method for glutaminyl cyclase, is characterized in that, comprises the following steps:
A, will can be used for express QC 33-361recombinant vectors be transformed in competent escherichia coli cell, acquisition can synthesize QC 33-361genetic engineering bacterium;
B, be inoculated into genetic engineering bacterium containing resistance marker LB substratum, abduction delivering;
After c, abduction delivering terminate, collect thalline, the broken thalline of bacteriolyze, high pressure, centrifugation, get supernatant liquor and carry out affinity chromatography and carry out purifying, collect containing QC 33-361fusion rotein;
D, to containing QC 33-361fusion rotein carry out PPE enzyme, desalination, molecular sieve column chromatography purifying, isolates QC 33-361albumen.
2. the preparation method of glutaminyl cyclase according to claim 1, is characterized in that, in step b, comprises the following steps:
Picking genetic engineering bacterium list colony inoculation is to being inoculated into containing in antibiotic LB liquid nutrient medium, and jolting is cultivated, and obtains seed culture fluid;
Be inoculated into by seed culture fluid containing in antibiotic LB nutrient solution, 37 DEG C, jolting is cultivated, and is cultured to OD600 value and reaches 0.6-0.8, with IPTG inducing culture.
3. the preparation method of glutaminyl cyclase according to claim 1, is characterized in that, in step c, comprises the following steps:
By bacterium liquid collected by centrifugation thalline, use high pressure smudge cells instrument to split abundant cracking thalline, centrifugal, supernatant liquor Ni ion affinity chromatography carries out separation and purification, collects containing QC 33-361fusion rotein.
4. the preparation method of glutaminyl cyclase according to claim 1, is characterized in that, in steps d, comprises the following steps:
With desalting column to the desalination of collection gained fusion rotein, with PPE enzyme, enzyme is carried out to fusion rotein and cut, with molecular sieve S-200 Column, mixed system is cut to enzyme and carry out molecular sieve column chromatography and purify, collect target protein QC 33-361.
5. the preparation method of glutaminyl cyclase according to claim 1, is characterized in that, in step a, comprises the following steps:
Add in competent escherichia coli cell and can be used for expressing QC 33-361recombinant vectors, leave standstill on ice, water-soluble pot heat shock 60-70s, leaves standstill on ice, adds appropriate LB liquid nutrient medium, and be applied on the LB solid plate with resistance marker, overnight incubation, obtains genetic engineering bacterium.
6. the preparation method of glutaminyl cyclase according to claim 1, is characterized in that, in step a, described competent escherichia coli cell is bacillus coli DH 5 alpha or e. coli bl21 (DE3).
7. the preparation method of glutaminyl cyclase according to claim 1, is characterized in that, in step a, before conversion recombinant vectors, carries out activation treatment to competent escherichia coli cell:
By the actication of culture preserved, in LB liquid nutrient medium, 37 DEG C are cultured to OD600 value is 0.6 ~ 0.8, makes bacterium liquid be cooled to 0 DEG C on ice, collected by centrifugation thalline, with the 0.1mol/L CaCl of precooling 2solution prepares competent cell.
8., according to the preparation method of the arbitrary described glutaminyl cyclase of claim 1 ~ 7, it is characterized in that, described for expressing QC 33-361the preparation method of recombinant vectors, comprise the following steps:
Adopt round pcr, with upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2 for primer pair, amplification obtains QC 33-361gene fragment; Wherein, upstream primer is 5 '-ACCTGGATCCGCTTCTGCTTGGCCGG-3 '; Downstream primer is 5 '-TATCCTCGAGTTACAGGTGCAGGTATTC-3 ';
To carrier pET28a and pET32a, carry out EcoRI, XhoI double digestion respectively; Linearized vector two ends being existed equally EcoRI, XhoI site connects, and obtains improved pET32a carrier;
To QC 33-361gene fragment and improved pET32a carrier carry out BamHI and XhoI double digestion, two ends are existed equally the QC in BamHI, XhoI site 33-361gene fragment is connected with linearized vector, obtains pET32a/QC 33-361recombinant vectors.
CN201510319520.3A 2015-06-11 2015-06-11 Preparation method for glutaminyl cyclase Pending CN104946609A (en)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CRISTIANA CASTALDO，ET AL: "Soluble Variants of Human Recombinant Glutaminyl Cyclase", 《PLOS》 *
KAI-FA HUANG，ET AL: "Crystal structures of human glutaminyl cyclase,an enzyme responsible for protein N-terminal pyroglutamate formation", 《PNAS》 *
KAI-FA HUANG，ET AL: "Snake venom glutaminyl cyclases: Purification, cloning, kinetic study, recombinant expression, and comparison with the human enzyme", 《TOXICON》 *
NCBI: "Chain A,Crystal Structure of Huamn Glutaminyl Cyclase At Ph 6.5;PDB:2AFM_A", 《NCBI》 *

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Application publication date: 20150930