CN105062987B - Recombined human/baboon is fitted into uricase protein and preparation method thereof - Google Patents
Recombined human/baboon is fitted into uricase protein and preparation method thereof Download PDFInfo
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- C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
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- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
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Abstract
The invention discloses a kind of recombined human/baboons to be fitted into uricase protein sequence, preparation process and its application.The uricase protein is referred to as JBUO, is originated from baboon uricase protein sequence, and has carried out mutation and the active mutation of enhancing of humanization, makes it while having the characteristics that immunogenicity is low and catalytic activity is high.It after JBUO codon optimizations, is cloned into prokaryotic expression carrier, converts Escherichia coli, harvest recombinant protein amount is up to every gram of dry bacterium of 10mg/, while the biological activity of JBUO is more than 6 IU/mg.The present invention can efficiently, quickly obtain the novel uricase sterling of the low immunogenicity homologous with human height, be widely used in and prepare the products such as treatment and drug, the food of human body high lithemia relevant disease, have high use value.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of to be transformed rear and human height based on baboon uricase
The very high uricase chimeric protein of homologous and biological activity and its drug that gout or hyperuricemia are treated as preparation
In application.
Background technology
Gout and hyperuricemia are to endanger the common disease of human health at present, according to statistics the incidence of hyperuricemia
The 10% of crowd is had reached.Gout is to cause urate crystal to be deposited in joint since serum uric acid level is excessively high and lead
A kind of acute arthritis caused.Uric acid is product of human body during being metabolized purine, and disorders of purine metabolism can cause uric acid to accumulate
It is tired excessive, cause uric acid in blood concentration to increase, causes high lithemia disease.Long-term uric acid is excessively high easily to cause gout, and increases more
The onset risk, including atherosclerosis, hypertension, brain soldier, diabetes etc. of kind cardiovascular and cerebrovascular disease.In addition, malignant tumour
Patient in chemotherapy process, the dead dissolvings of a large amount of cells can bring the tumor lysis syndrome TLS, most important symptom to be exactly
Hyperuricemia, and easily cause kidney failure.
Currently, controlling gout and treating the excessively high drug of uric acid has many defects.Colchicin can inhibit gout to send out
Acute inflammatory reaction when making, but toxicity is huge, and uric acid content cannot be reduced.The drugs such as probenecid, Benzbromarone can be with
Reabsorption of the renal tubule to uric acid is interfered, the excretion of uric acid is promoted, but it is huge to the damage of human kidney, it is easy to cause kidney knot
The generation of the nephrosis such as stone.The non-steroidal anti-inflammatory drugs such as Indomethacin can alleviate the symptoms such as the arthritis for improving that gout causes, and eliminate
Pain, but can not take for a long time, side effect is huge.The drugs such as allopurinol are blocked by inhibiting xanthine oxidase activity
The generation of uric acid reduces uric acid, but increases the load of kidney excretion uric acid precursor again, and is easy to cause xanthine kidney
Disease and calculus.Currently, hyperuricemia has been cited as the independent risk factor of angiocardiopathy.Therefore, effective treatment is found
Means are imperative.
In animal body, the level of uric acid is only the 1/10 of the mankind.Therefore gout and hyperuricemia are also considered as
The exclusive disease of the mankind.The low reason of uric acid level is since there are uric acid can be further decomposed in vivo for it in animal body
For the uricase of very soluble allantoin.It being found by comparing, there is also uricase genes in the genome of the mankind, but
During human evolution, due to the mutation of the gene, cause it that cannot express functional protein, to become a vacation
Gene.It can be seen that uricase is the ideal medicament for treating gout and hyperuricemia.Compared with said medicine, urate oxidase
The direct drug target that UOX is directed to is exactly uric acid, can quickly reduce the uric acid concentration in blood of human body, and can also be catalyzed kidney
Uric acid in dirty and joint precipitates fast decoupled, can fundamentally treat hyperuricemia, side effect is extremely low.
Urate oxidase UOX is a kind of enzyme in purine degradation metabolic pathway in organism, and energy efficient oxidation uric acid becomes
Allantoin and hydrogen peroxide.UOX molecules are homotetramers, and each protein protomer contains 301 amino acid, single molecular weight subunit
For 34kDa.Each subunit of UOX is made of two identical structural domains, is referred to as T- folded domains.4 subunits of UOX are logical
It crosses complicated hydrogen bond network and forms non-covalent linking, form symmetrical tetramer albumen.The only UOX of the tetramer just has biology
Enzymatic activity.In the uricase in all sources known today, activity is highest to derive from aspergillus flavus, reaches 27 IU/mg；Its
Secondary to be derived from Bacillus fastidiosus, its activity is maintained at 13 IU/mg.；From the uricase of mammal, through overweight
After group expression, the uricase activity of pig can reach 5IU/mg, and the uricase enzymatic activity of baboon only has 1IU/mg, and people source is urinated
Sour kinase inactive.
To uricase is applied to human body, the high activity for taking into account microorganism uricase and mammal uric acid enzyme are needed
Low immunogenicity is.Currently, the uricase in aspergillus flavus source（Uricozyme）In France and Italian approved, but its sequence
The homology for the people source uricase for arranging and deducing is less than 40%；The saccharomycete uricase of genetic recombination（Rasburicase）
USA and EU or approval.Since both uricases are both from bacterium, there is a very strong immunogenicity, Uricozyme and
There is the ratio of severe allergic reaction up to 5% or more in use in Rasburicase, and the patient of also nearly half sends out
Heat, the adverse reactions such as nausea and vomiting.At present in the U.S. for clinical uricase protein（Pegloticase）It is by PEGization
The recombination uricase chimeric protein of the pig of modification, trade name KRYSTEXXA.But it is anti-due to rejecting caused by the difference with the mankind
It answers, in addition the influence of PEGization, immunogenicity is still relatively strong.Once every two weeks in the patient of intravenous injection KRYSTEXXA
92% produces the antibody of anti-pegloticase.
Invention content
The purpose of the present invention is further improve the immunogenicity of recombination uricase, selection and the more close baboon of the mankind
Method as the source of uricase gene, and based on structure biology and albumen zymetology has carried out it to improve enzyme activity
Mutation transformation, to obtain with Uric Acid enzyme very high homology but with the chimeric uricase protein of people/baboon of high enzyme activity,
There is important application value for the prevention of gout and high lithemia disease.The present invention includes the excellent of this Novel chimeric uricase JBUO
Change gene order, protein sequence, the construction method of recombinant expression carrier, be fitted into uricase purifying technique, Property Identification and
Activity calibration etc..
The present invention adopts the following technical scheme that realization：
One, recombined human/baboon is fitted into the sequence design of uricase protein JBUO
Uricase in baboon body（Papio hamadryas Urate Oxidase, are abbreviated as PUO）With deduce human body
Uricase（Predicted Human Urate Oxidase, are abbreviated as pHUO）Similar, the homogeneity 95.4% of sequence height,
Far above the uricase in pig source（86.6%）, the uricase in mouse source（85.2%）（Rattus norvegicus Urate
Oxidase is abbreviated as RUO）With the uricase in dog source（88.2%）（Canis lupus familiaris Urate Oxidase,
It is abbreviated as CUO）.Therefore, the applicant carries out mutation transformation based on baboon uricase PUO, can be with the reduction human body of high degree
The immunogenicity of middle recombinant protein.However, the uricase activity in baboon body is not high（About 1U/mg）, come than other mammals
The uricase in source is all low（5 U/mg of pig uricase, 7 U/ml of Kynureninase）.Therefore, how to pass through replacement raising enzymatic activity
Uricase amino acid sequence, significantly improve the catalytic activity of baboon uricase PUO, be exactly the main object of the present invention.
The present inventor passes through the research of long-term system, is carried out especially in conjunction with the crystal structure of uricase tetramer compound
Analysis finds the amino acid substitution for carrying out specific site, and the recombined human/baboon that can form particular form is fitted into uricase protein
JBUO, amino acid sequence are as follows：
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHV
LAKFKGIKSIEAFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQMKSGPPVIH
SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLKKFAGPYDKGEY
SPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL。
Recombined human/the baboon is fitted into uricase protein JBUO and people source uricase pHUO amino acid sequence similarities are very high
（94.8%）, and activity is obviously improved（More than 6U/mg）, while low and active high two hang-up of immunogenicity has been taken into account, have
Extremely important application value.
Further, the sequence of JBUO comes from baboon uricase PUO sequences, is made of 304 amino acid.Such as Fig. 1 institutes
Show, compared with natural PUO, mutational site includes following 12：D3H, N7D, G91A, V92M, Y97H, I105V, L120F,
L146M, R147K, C202G, G212D, E220K.The mutation in which part site be in order to enhance uric acid enzymatic activity, it is other
The mutation in site is to be further reduced immunogenicity.
Two, recombined human/baboon is fitted into the recombinant expression of uricase protein JBUO
Recombined human/baboon is fitted into the corresponding nucleotide sequence of uricase protein and is inserted into prokaryotic expression carrier or eukaryotic expression
Carrier is expressed.Preferably, the corresponding nucleotide sequence of the albumen is inserted into coli expression carrier to express, is used
The alkaline buffer soluble protein of pH10-11 is rapidly purified.
According to the preferred codons table of the amino acid sequence of JBUO and Escherichia coli, design is conducive to the JBUO bases recombinantly expressed
Cause, nucleotide sequence is as shown in sequence table 1；Clone obtains JBUO gene fragment clones and enters escherichia coli high-level expression load
Body.JBUO expression vectors convert Escherichia coli, then induce recombination high efficient expression, can obtain the bacterium rich in JBUO albumen
Body.
Thalline, ultrasonication is resuspended with the neutral buffer of pH7.5, high speed centrifugation collects sediment fraction.Then it uses
The alkaline buffer solution dissolving precipitation of pH11, again high speed centrifugation collect supernatant to get having arrived the highly concentrated solution of JBUO albumen,
It is used directly for polishing purification, Activity determination or other purposes.
Solution containing JBUO albumen can be further subjected to purification process, including ion-exchange purification and molecular sieve it is pure
Change, obtain the JBUO albumen of high-purity, assay and Activity determination can be carried out, or be lyophilized spare.
A variety of expression vectors commonly used in the art may be used in heretofore described efficient expression vector, including but not
It is limited to pET-21, pET-26, pET-28 etc..
Heretofore described purification process may be used conventional use of multiple means in this field and carry out, such as affine
Chromatography, ion exchange etc..
Three, recombined human/baboon is fitted into the determination of activity of uricase protein JBUO
JBUO and other control uricases are all made of international enzyme activity scaling method test activity.Uric acid is in detection wave
There is the absorption peak of feature under long 292nM, it is allantoin that urate oxidase, which aoxidizes uric acid, and the absorption of oxidation product allantoin is maximum
Value is not in 292nM.So uric acid A292nM absorption values are directly proportional to its concentration in a certain range, so uric acid is aoxidized in uric acid
The reduction of absorption value can reflect enzymatic activity height under the action of enzyme.According to 1 μM to 500 μM of concentration range before reaction, urine is measured
The A292nM standard curves of acid solution.Reaction buffer solution is triethanolamine hydrochloride buffer, pH8.9.When detection, uric acid concentration
It is 100 μM, the protease that total amount is 50 μ g is added in the reaction system of 3ml, then reacts 5min under the conditions of 30 DEG C, then uses
1M KOH terminate reaction, measure the absorbance under 292nM.The activity definition of urate oxidase is, at the standard conditions 30 DEG C when,
Enzyme amount needed for 1 μm of ol uric acid oxidation of catalysis per minute is 1 international unit IU.
As shown in figure 4, experiment measures, the enzyme activity of baboon PUO is 1.9 U/mg, and the enzyme activity of dog CUO is 5.2 U/mg, with text
It is almost the same to offer report, and the enzyme activity of the measurement of JBUO is 6.0 U/mg, is significantly higher than the enzyme activity of original baboon PUO.
Reasonable design of the present invention provides a kind of people/baboon for having high activity completely newly and is fitted into uricase protein JBUO, fits
Cooperation is the drug of the relevant diseases such as treatment hyperuricemia.Disclosed recombined human/baboon is fitted into uricase JBUO, both retains
The part for predicting uricase high homology in baboon uricase with people source, reduces immunogenicity, further through point mutation
Method significantly improves enzyme activity, and entire preparation process is simple, and product can be used as drug or health treatment, be widely used in high lithemia
The treatment of associated disease, is widely popularized using to market.
Description of the drawings
Fig. 1 shows recombined human/baboon be fitted into uricase protein JBUO and prediction people's uricase pHUO, baboon uricase PUO,
The amino acid alignment figure of mouse uricase RUO, Kynureninase CUO；Several sites of JBUO transformations are shown in figure.
Fig. 2 indicates that recombined human/baboon is fitted into the SDS-PAGE of uricase JBUO, Kynureninase CUO, baboon uricase PUO
Electrophoretogram；The monomer band of 35kDa or so and the dimer band of 70kDa or so are mainly shown in figure.
Fig. 3 indicates to measure the standard curve of uric acid UA concentration.
Fig. 4 indicates to measure recombined human/baboon and is fitted into uricase protein JBUO, Kynureninase CUO, baboon uricase PUO
Catalytic activity.
Specific implementation mode
Specific embodiments of the present invention are described in detail below.
Embodiment 1
A kind of recombined human/baboon is fitted into the recombinant expression method of uricase protein JBUO, includes the following steps：
（1）, according to the nucleotide sequence of the JBUO after optimization（Sequence table 1）Design primer needs to include restriction enzyme site, from
5`-3` arrangements are as follows：
JBP1：GGAATTCCATATGGCCCATTATCACAACGACTATAAGAA
JBP2：CCGCTCGAGttaCAGGCGGCTACTCAGTTTGCGT
Primer is diluted to 50pmol/ μ l, then carries out PCR step.
（2）, using Tag enzymes carry out PCR step, agarose gel electrophoresis detection JBUO bands be located at 1000bp or so, glue
Target gene is recycled, gene enzyme is cut into cohesive end with Nde I and Xho I, it is same to handle pET-26b carriers, T4 is added
DNA ligase, 4 DEG C connect 12 hours, convert bacillus coli DH 5 alpha.Choose hickie gram with kanamycin sulfate-LB plate screenings
It is grand, plasmid is extracted using the alkaline lysis or boiling method of molecular cloning, is identified correctly, then using the method for digestion and PCR
Nucleotides sequence sequence analysis contains correct gene order reading frame, builds successfully pET-26b-JBUO carriers.
（3）, by correct pET-26b-JBUO plasmids convert e. coli bl21, with kanamycin sulfate-LB flat screens
Select single spot clone, the overnight incubation in 5ml LB culture mediums, according to 1:100 switchings, in shaking flask 37 DEG C of cultures be to OD600
0.4-0.6, according to 1:5000 are added IPTG liquid storages, and 20 DEG C are cultivated 16 hours（Time lengthening can significantly improve yield）, centrifugation receipts
Collect thalline, be stored in -20 DEG C or immediately enter lower step purifying.
（4）, with the Tris buffer solutions of pH 7.5 wash mixed bacterial sediment, be resuspended about with 20-40ml volumes
500ml bacterium solutions precipitate, and bacterium are cracked with lysozyme cooperation Triton X-100 helps, with carrying out ultrasonic bacteria breaking under mixture of ice and water environment
（4s ultrasounds, 8s intervals, overall length 30min protect 30 DEG C of temperature）, 16000rpm/min centrifugation 20min, collection precipitation.
（5）, with the Na of pH10-pH112CO3Buffer solution fully dissolves precipitation, and the PMSF that 1mM is added prevents protease
Solution.16000rpm/min centrifuges 20min again, collects supernatant.Acquired solution contains the molten of JBUO albumen for high concentration at this time
Liquid.It can carry out assay and Activity determination.
Embodiment 2
A kind of recombined human/baboon is fitted into the purification process of uricase protein JBUO, includes the following steps：
（1）, selection suitable load anion-exchange column, with the Na of the pH11 of 0.1M2CO3Buffer solution balance columns material, will
The slow upper prop of solution containing JBUO albumen prepared by embodiment 1.
（2）, with the NaHCO of the pH8.5 of 0.1M3Buffer solution elutes foreign protein, at least cleans 5 times of column material volumes；Then by
Gradually the NaCl salinity of buffer solution is promoted to 1.5M, and cleans 5 times of column material volumes again, fully elutes other impurity.
（3）, with the Na of pH112CO3Buffer solution flows through column material, and gradually promotes the NaCl salinity of buffer solution to 1M,
Collect the JBUO albumen eluted.
（4）, by JBUO albumen after purification molecular sieve carry out polymerized form identification, it is about 140kD to go out peak position, be four
Dimer form.
The activity test method that above-mentioned recombined human/baboon is fitted into uricase protein JBUO is as follows：
（1）, configuration various concentration uric acid solution, concentration measures the A292nM standards of uric acid solution from 1 μM to 500 μM
Curve, as shown in Figure 3.
（2）, configuration reaction buffer solution be triethanolamine hydrochloride buffer, pH8.9.It is added 100 in the reaction system of 3ml
μM uric acid is added JBUO protease or reference protein enzyme that total amount is 50 μ g, then reacts 5min under the conditions of 30 DEG C, finally use
1M KOH terminate reaction, measure the absorbance under 292nM.
（3）, urate oxidase activity definition be, at the standard conditions 30 DEG C when, catalysis per minute 1 μm of ol uric acid oxidation
Required enzyme amount is 1 international unit IU.Calculate each sample enzyme activity.
Above-mentioned recombined human/baboon after purification is fitted into uricase protein as preparation treatment gout or hyperuricemia
Drug in application.The drug is the active uricase protein given expression to through PEGylated prokaryotic expression carrier, the activity uric acid
The amino acid sequence of zymoprotein is the same as sequence table 2.
It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although ginseng
It is described in detail according to the embodiment of the present invention, it will be understood by those of ordinary skill in the art that, to technical scheme of the present invention
It is modified or replaced equivalently, without departure from the spirit and scope of technical scheme of the present invention, should all cover the present invention's
In claims.
1, it is fitted into the nucleotide sequence of uricase JBUO（After e. coli codon optimization）
ATGGCCCATTATCACAACGACTATAAGAAAAATGATGAAGTGGAGTTTGTGCGCACCGGCTATGGCAAG
GACATGGTGAAAGTGCTGCACATTCAGCGCGATGGTAAGTACCACAGCATCAAAGAAGTGGCCACCAGTGTGCAGCT
GACCCTGAGCAGCAAAAAGGATTATCTGCACGGCGACAACAGCGATATCATTCCGACCGATACCATTAAAAATACCG
TGCATGTTCTGGCCAAATTCAAGGGCATTAAGAGCATCGAGGCCTTTGCCATGAACATCTGCGAACACTTCCTGAGC
AGCTTCAACCACGTGATCCGTGCCCAGGTGTATGTGGAGGAAGTGCCGTGGAAACGCTTCGAGAAAAACGGCGTGAA
GCATGTTCACGCCTTCATTCACACCCCTACCGGCACCCACTTCTGCGAGGTGGAACAGATGAAAAGCGGCCCGCCGG
TTATTCACAGCGGTATCAAAGATTTAAAAGTTCTGAAGACAACCCAGAGCGGCTTTGAAGGCTTCATCAAGGACCAG
TTTACCACCCTGCCGGAAGTTAAGGACCGCTGTTTCGCCACCCAGGTTTACTGCAAGTGGCGTTATCACCAGGGTCG
CGATGTTGACTTTGAAGCCACCTGGGATACCGTGCGCGACATCGTGCTGAAAAAGTTCGCCGGTCCGTATGACAAGG
GTGAGTATAGCCCGAGTGTGCAGAAAACCCTGTACGATATTCAGGTGCTGAGCCTGAGCCGCGTGCCGGAGATTGAA
GACATGGAGATCAGCCTGCCGAACATCCATTATTTTAACATCGATATGAGCAAAATGGGCCTGATCAACAAGGAAGA
GGTGCTGCTGCCGTTAGATAACCCGTATGGCAAGATCACCGGCACCGTTAAACGCAAACTGAGTAGCCGCCTG
912 bases of overall length
2, it is fitted into the amino acid sequence of uricase JBUO
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTD
TIKNTVHVLAKFKGIKSIEAFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQM
KSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLKKFA
GPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKL
SSRL
304 amino acid of overall length
Claims (2)
1. a kind of recombined human/baboon is fitted into the preparation method of uricase protein：It is characterized in that：It is as follows：
（1）, according to the nucleotide sequence design primer of the JBUO after optimization, need include restriction enzyme site, from 5`-3` arrange it is as follows
It is shown：
JBP1：GGAATTCCATATGGCCCATTATCACAACGACTATAAGAA
JBP2：CCGCTCGAGTTACAGGCGGCTACTCAGTTTGCGT
Primer is diluted to 50pmol/ μ l, then carries out PCR step；
（2）, using Tag enzymes carry out PCR step, agarose gel electrophoresis detect JBUO bands, glue recycle target gene, use Nde
Gene enzyme is cut into cohesive end by I and Xho I, same to handle pET-26b carriers, and T4 DNA ligases are added, are ligated and transformed into
Bacillus coli DH 5 alpha；With kanamycin sulfate-LB plate screenings choose hickie clone, using molecular cloning alkaline lysis or
Boiling method extracts plasmid, identifies that correctly then nucleotides sequence sequence analysis contains correct gene using the method for digestion and PCR
Sequence reading frame builds successfully pET-26b-JBUO carriers；
（3）, by correct pET-26b-JBUO plasmids convert e. coli bl21, selected with kanamycin sulfate-LB flat screens
Single spot clone, the overnight incubation in LB culture mediums, according to 1:100 switchings, culture is 0.4-0.6 to OD600 in shaking flask, is added
IPTG liquid storages, thalline were collected by centrifugation after culture, is stored in -20 DEG C or immediately enters lower step purifying；
（4）, with Tris buffer solutions wash mixed bacterial sediment, cracked with lysozyme cooperation Triton X-100 helps thin
Precipitation is collected after centrifugation with carrying out ultrasonic bacteria breaking under mixture of ice and water environment in bacterium；
（5）, with the Na of pH 10-pH 112CO3Buffer solution fully dissolves precipitation, and PMSF is added prevents proteolysis；Again
Supernatant is collected after centrifugation, acquired solution is the solution containing JBUO albumen of high concentration at this time；
The amino acid sequence that the recombined human/baboon is fitted into uricase protein is as follows：
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKN
TVHVLAKFKGIKSIEAFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQMKSGP
PVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLKKFAGPYD
KGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL
。
2. a kind of recombined human/baboon is fitted into the purification process of uricase protein solution, it is characterised in that：Include the following steps：
（1）, selection suitable load anion-exchange column, with the Na of the pH 11 of 0.1M2CO3Buffer solution balance columns material, will contain
There is the slow upper prop of the solution of JBUO albumen；
（2）, with the NaHCO of the pH8.5 of 0.1M3Buffer solution elutes foreign protein, at least cleans 5 times of column material volumes；Then will gradually delay
The NaCl salinity of fliud flushing is promoted to 1.5M, and cleans 5 times of column material volumes again, fully elutes other impurity；
（3）, with the Na of pH112CO3Buffer solution flows through column material, and gradually promotes the NaCl salinity of buffer solution to 1M, collects
The JBUO albumen eluted；
（4）, by JBUO albumen after purification molecular sieve carry out polymerized form identification, it is about 140kD to go out peak position, be the tetramer
Form；
The amino acid sequence that the recombined human/baboon is fitted into uricase protein is as follows：
MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKN
TVHVLAKFKGIKSIEAFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQMKSGP
PVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLKKFAGPYD
KGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL
。
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| CN201510555258.2A CN105062987B (en) | 2015-09-01 | 2015-09-01 | Recombined human/baboon is fitted into uricase protein and preparation method thereof |
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| Chain A, Evolutionary History And Metabolic Insights Of Ancient Mammalian Uricases;Kratzer,J.T et al;《PDB: 4MB8_A》;20140702;1-2 * |
| 产朊圆酵母尿酸酶的分离纯化及其部分性质研究;辛渝等;《四川大学学报(自然科学版)》;20051231;第42卷(第6期);1246-1251 * |
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