CN104342415A

CN104342415A - Preparation method of recombinant uricase

Info

Publication number: CN104342415A
Application number: CN201410535887.4A
Authority: CN
Inventors: 李慎涛; 庞海; 刘爽
Original assignee: JILIN JINZIYUAN BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Current assignee: JILIN JINZIYUAN BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Priority date: 2014-07-08
Filing date: 2014-10-12
Publication date: 2015-02-11

Abstract

The invention provides a nucleotide sequence suitable for secretory expression of recombinant candida utilis uricase (uricase, EC1.7.3.3) by virtue of escherichia coli, a carrier containing the nucleotide sequence, an engineering cell containing the carrier, and a method for purifying the recombinant candida utilis uricase from the engineering cell. On the basis of a natural sequence of a uricase gene, a codon of the uricase gene is optimized, the transformed uricase gene is cloned to a prokaryotic expression vector pET42a(+); an escherichia coli Rossetta strain is transformed; the expression level can reach 50% of soluble protein of the escherichia coli by IPTG induction; the expressed protein is soluble protein; and the biological activity of the expressed protein can be up to that of 37.89U/mg recombinant protein. The recombinant candida utilis uricase product can be efficiently and simply obtained at low cost. The invention also comprises an application of the recombinant candida utilis uricase prepared by the method in preparation of medicines, foods, health care products and cosmetics for treating hyperuricemia-related diseases or pathological symptoms of a human body.

Description

A kind of preparation method of uriKoxidase of recombinating

Technical field

The present invention relates to and use DNA recombinant technology in E. coli Candida utilis uriKoxidase recombinant protein and the method adopting related protein purification technique separation and purification restructuring uriKoxidase, containing uriKoxidase recombinant protein prepared by the method and uses thereof, belong to genetically engineered and art of protein biochemistry.

Background technology

Along with the raising of living standards of the people, the intake of high protein group food increases year by year, the sickness rate of hyperuricemia and gout also rises gradually, hyperuricemia and the disease such as gout and obesity, hyperlipidaemia, essential hypertension, diabetes, atherosclerosis are remarkable positive correlation, and gout not only can invade bone and joint, also easily involve kidney and cardiovascular systems, very harmful.

UriKoxidase is a kind of enzyme in organism in purine degradation pathways metabolism, and the decomposition of energy catalysis uric acid, also can dissolve the uric acid crystal precipitated.People and most primate (as gorilla, ape etc.) are during evolution through the natural selection of transgenation, the gene of coding uriKoxidase is undergone mutation, can not the uriKoxidase of synthetic bioactive, and using uric acid as the end product of purine metabolism, easily cause hyperuricemia and gout.

The major measure of day preclinical therapy has control acute attack; Correct hyperuricemia, prevent sacroiliitis from recurring; The destruction of joint that prevention urate deposition causes, kidney damage and tophaceous formation.And the use of uncase preparation makes result for the treatment of be greatly improved, uncase preparation is a kind of from producing the non-recombinant uriKoxidase extracted fungi (as the flavus) culture of uriKoxidase, the effect of degraded uric acid is better than Zyloric, but because of the immunogenicity that it is potential, limit its clinical application.In recent years, along with the application of genetic engineering technique in pharmacy, abroad gone on the market genetically engineered uriKoxidase (rasburicase), and its cDNA derives from flavus, expresses in yeast saccharomyces cerevisiae, the respond well of uric acid of degrading.But there is the shortcoming of consumption large (0.2mg/kg body weight), and have many untoward reactions, as heating, Nausea and vomiting, fash, headache and allergy etc.

In Europe, listing uses the history having 20 years to oneself warp of native uricase extracted from flavus, and administrated by injection is used for the treatment of the serious hyperuricemia relevant with leukemia chemotherapy.The U.S. also started this product to be used for leukaemic in 1997.Compared with allopurinol, it is rapid and single-minded that uriKoxidase reduces serum uric acid level.Gout patients gives uriKoxidase acute attack capable of blocking and reduces tophaceous volume.Not only culture cycle is long, productive rate is low to extract uriKoxidase from the flavus cultivated, and easily by pathogen contamination.

(the Laboureur P such as Laboureur, Langlois C.Urate oxidase of Aspergillus flavus.I.Isolation, purification, properties.Bull Soc Chim Biol (Paris) .50 (4): 811-825 (1968)) from flavus culture, be extracted uriKoxidase first, and for reducing the concentration of uric acid in human body, for the application of uriKoxidase lays the foundation.But directly propose uriKoxidase from microorganism, output is extremely low, this greatly limits its clinical application, and the protein of this heterology has certain immunogenicity, and life-time service can produce immune response.(the Montalbini P such as Montalbini, Elstner EF.Ethylene evolution by rust-infected, detached bean (Phaseolus vulgaris L.) leaves susceptible and hypersensitive to Uromyces phaseoli (Pers.) Wint.Planta 135 (3): 301-306 (1977)) from the leaf of garbanzo, broad bean and wheat, purifying obtains the uriKoxidase of the even property of electrophoresis.

Genetic engineering technique is prepare uriKoxidase in a large number to open new road.1992, (the Legoux R such as Legoux, Delpech B, Dumont X, Guillemot JC, Ramond P, Shire D, Caput D, Ferrara P, Loison G 1992.Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.J Biol Chem 267 (12): 8565-8570 (1992)) clone aspergillus flavus urate enzyme gene, and attempt expressing in intestinal bacteria, product with soluble form at intracellular expression, and there is enzymic activity, but expression amount is low, only have 4% of thalline whole protein, not there is industrialization development be worth.Natural aspergillus flavus urate enzyme N holds acetylize to be conducive to the stable of enzyme and opposing proteasome degradation, and this is very important to the expression system that culture cycle is long, but is not necessary to activity.The same year, (the Chevalet L such as Chevalet, Tiraby G, Cabane B, Loison G.Transformation of Aspergillus flavus:construction of urate oxidase-deficient mutants by gene disruption.Curr Genet 21 (6): 447-453 (1992)) express with the aspergillus flavus strain of uriKoxidase defect, it can not be only nitrogen source with uric acid that result transforms bacterial strain, and easily reverts to wild-type.And (the Leplatois P such as LePlatois, Le Douarin B, Loison G.High-level production of a peroxisomal enzyme:Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae.Gene 122 (1): 139-145. (1992)) have made great progress hoc anno, they express aspergillus flavus urate enzyme gene in yeast saccharomyces cerevisiae, and expression amount reaches 13%.Other 2000, (the Hongoh Y such as Hongoh, Sasaki T, Ishikawa H 2000.Cloning, sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata fugens uricase gene intestinal bacteria is carried out amalgamation and expression Nilaparvata lugens.Insect Biochem Mol Biol30 (2): 173-182.), the target protein of 90% exists with occlusion body form, expression amount estimates at 20%.

Although uriKoxidase drug development becomes the focus of high lithemia disease medicament exploitation, to up to the present, only have the product of French Sanofi-Synthelabo company to get permission listing, but enter Chinese market not yet.We began one's study from 2010 restructuring uriKoxidase, and up to the present, we are the uriKoxidase at E. coli successfully, and establishes stable production technique.And certain achievement is achieved in the fundamental research of uriKoxidase.

According to the knowledge of contriver, prior art does not also solve the method at the efficient amalgamation and expression uriKoxidase of intestinal bacteria, provides a kind of method preparing the solvable uriKoxidase of purification of Recombinant uriKoxidase of high purity, high reactivity and high-recovery simultaneously.

Summary of the invention

Based on the shortcoming that prior art exists, the first object of the present invention is to provide a kind of by after the nucleotide sequence of coding Candida utilis uriKoxidase, the optimization of its codon, be cloned in expression vector, this expression vector is transformed and enters Bacillus coli cells, this Bacillus coli cells of fermentation culture and melt nuclear expression Candida utilis uriKoxidase recombinant protein, method that the uriKoxidase soluble protein of recombinating prepare in separation and purification.

The present inventor is surprised to find, by the native nucleotide sequence shown in the SEQ ID NO.3 of natural Candida utilis uriKoxidase recombinant protein shown in the aminoacid sequence obtaining coding SEQ ID NO:4 and to have with it more than 85% identity or the nucleotide sequence with it with more than 50% identity, or the nucleotide sequence of the codon optimization shown in SEQ ID NO.5 of coding SEQ ID NO:6 or the Candida utilis uriKoxidase recombinant protein shown in aminoacid sequence with it with more than 85% identity is cloned in pET expression vector, unexpectedly can obtain the Candida utilis uriKoxidase recombinant protein of high amalgamation and expression, and use the Candida utilis uriKoxidase recombinant protein of this vector expression to be easy to purifying, purity is high, receipts amount is large, active high.

Of the present invention in another, provide a kind of native nucleotide sequence shown in SEQ ID NO.3 of aminoacid sequence of SEQ ID NO:4 natural Candida utilis uriKoxidase recombinant protein of encoding, or the nucleotide sequence that the codon shown in SEQ ID NO.5 of the aminoacid sequence of coding SEQ ID NO:6 Candida utilis uriKoxidase recombinant protein is optimized, above-mentioned nucleotide sequence can in pET expression vector high amalgamation and expression.

In another aspect of the invention, provide a kind of expression vector, it contains the above-mentioned nucleotide sequence of the present invention, and preferably, described expression vector is pET42a (+).

In another aspect of the invention, provide a kind of engineering cell, it is obtained by the Sequence Transformed host cell of described expression vector, and is integrated with the nucleotide sequence of coding Candida utilis uriKoxidase recombinant protein in expression vector.

Preferably, described engineering cell is coli strain is DH5 α or Rossetta, is more preferably Rossetta.

Of the present invention further in, provide a kind of expression method preparing Candida utilis uriKoxidase recombinant protein, comprise the following steps: under applicable expression condition, cultivate the host cell that the present invention is above-mentioned, thus secreting, expressing Candida utilis uriKoxidase recombinant protein; Preferably, described host cell is coli strain is Rossetta.Described cultivation comprises: cultivate and be divided into cultivation stage and induction period, and cultivation stage is cultivated bacterial concentration and reached OD600, and time inductive phase is 8-12hrs, and fermentation and inducing temperature remain on 37 DEG C, and IPTG consumption is 0.1-1mM/L, and the pH value of inductive phase is 6-8.Described e. coli host cell comprises can use IPTG Induced synthesis Candida utilis uriKoxidase recombinant protein, and can be secreted into the Bacillus coli cells in cell walls and plasmalemma gap.

In another aspect of the present invention, carry out slightly carrying and being further purified to Candida utilis uriKoxidase recombinant protein after expression.It comprises the steps: (1) collects thalline to fermented sample by centrifugal and/or filter type, with the ultrasonic or broken thalline of N,O-Diacetylmuramidase, supernatant liquor is obtained again by centrifugal method, (2) by after sulphur ammonium fractionation precipitation preliminary purification, with further by anionresin, hydrophobic chromatography method, obtain the sterling that purity reaches more than 95% (as 95-99.9%), the Candida utilis uriKoxidase recombinant protein prepared by the present invention is active in more than 30U.

In another aspect of the present invention, provide the purposes of Candida utilis uriKoxidase recombinant protein in the medicine of preparation treatment and human body high lithemia relative disease or pathological symptom, food, healthcare products and makeup thereof.Described uriKoxidase recombinant protein comprises arbitrary following aminoacid sequence or has the aminoacid sequence of more than 85% identity with it:

A) aminoacid sequence shown in SEQ ID NO.4; Or

B) aminoacid sequence shown in SEQ ID NO.6.

Accompanying drawing explanation

Fig. 1 is for cloning the forward primer of the nucleotide sequence of code book invention signal peptide of the present invention.

Fig. 2 is for cloning the reverse primer of the nucleotide sequence of code book invention signal peptide of the present invention.

Fig. 3 display is for the nucleotide sequence of Candida utilis uriKoxidase of encoding.

The aminoacid sequence of the natural Candida utilis uriKoxidase of Fig. 4 the present invention.

Fig. 5 display is used for the nucleotide sequence of code book invention restructuring Candida utilis uriKoxidase.

Fig. 6 the present invention recombinates the aminoacid sequence of Candida utilis uriKoxidase.

Fig. 7 pET42a (+) Vector map

The pcr amplification product of Fig. 8 uricase gene

1 road: DNA Marker;

2 roads: the gene of wild-type Candida utilis uriKoxidase;

3 roads: the PCR primer of the uriKoxidase encoding sequence after sequence optimisation.

The SDS-PAGE qualification of Fig. 9 expression product

A wild-type Candida utilis uriKoxidase 1 road: Marker2 road: induce front 3 roads: after induction

UriKoxidase 1 road after B sequence optimisation: induce front 2 roads: 3 roads: Marker after induction

Figure 10 cation exchange chromatography collection of illustrative plates

A wild-type Candida utilis uriKoxidase

UriKoxidase after B sequence optimisation

Figure 11 gel filtration chromatography collection of illustrative plates

A wild-type Candida utilis uriKoxidase

UriKoxidase after B sequence optimisation

Figure 12 recombinates the expression and purification of uriKoxidase

UriKoxidase after A sequence optimisation

1 road: protein Marker

2 roads: before induction

3 roads: after induction

4 roads: induction postprecipitation

5 roads: supernatant after induction

6 roads: ammonium sulfate precipitation

7 roads: after desalination

8 roads: after ion-exchange

9 roads: after molecular sieve

B wild-type Candida utilis uriKoxidase

1 road: protein Marker

2 roads: supernatant after induction

3 roads: induction postprecipitation

4 roads: ammonium sulfate precipitation

5 roads: ammonium sulfate precipitation supernatant

6 roads: after ion-exchange

7 roads: component 2 after ion-exchange

8 roads: after molecular sieve

To recombinate after Figure 13 purifying the Mass Spectrometric Identification of uriKoxidase

Embodiment

In order to provide, substance of the present invention be understood, describe some aspect of the present invention, pattern, embodiment, modification and feature with different the level of details hereinafter.

In the practice of the invention, employ molecular biology, genetic engineering, genetics, Cell. Mol, biological chemistry, protein biochemistry, protein biochemistry engineering, pharmaceutical technology, medical science, physiology, pathology, experimentation on animals, (the Molecular Cloning:A Laboratory Manual such as Sambrook, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) etc. a lot of conventional art and basic theory.These technology are known.

In an embodiment of the invention, provide a kind of Candida utilis uriKoxidase recombinant protein, it is characterized in that, described uriKoxidase recombinant protein comprises arbitrary following aminoacid sequence or has the aminoacid sequence of more than 85% identity with it:

A) aminoacid sequence shown in SEQ ID NO.4; Or

B) aminoacid sequence shown in SEQ ID NO.6.

In another embodiment of the present invention, provided the nucleotide sequence of described Candida utilis uriKoxidase recombinant protein of encoding by molecular cloning.

Term of the present invention " nucleotide sequence " comprises DNA and RNA of genomic dna, cDNA, synthesis.Preferably, it refers to DNA, is more preferably the cDNA of encoding sequence.The present invention is also contained coding and is had the enzyme of special properties as herein defined or the nucleotide sequence of albumen.Term used herein " nucleotide sequence " refers to oligonucleotide sequence or polynucleotide sequence and its variant, homologue, fragment and derivative (as its part).Described nucleotide sequence can be derived from genome or synthetics or thing of recombinating, and it can be (no matter it represents positive-sense strand or antisense strand) nucleic acid molecule of double-strand or strand.

In a specific embodiment of the present invention, provide a kind of nucleic acid molecule of separation, wherein, the nucleic acid molecule of described separation comprises arbitrary following nucleotide sequence or has with it:

A) nucleotide sequence shown in SEQ ID NO.1; Or

B) with the antisense base sequences shown in SEQ ID NO.2; Or

C) nucleotide sequence shown in SEQ ID NO.3 of the aminoacid sequence of coding SEQ ID NO:4; Or

D) nucleotide sequence shown in SEQ ID NO.5 of the aminoacid sequence of coding SEQ ID NO:6.The nucleotide sequence that coding has enzyme as herein defined or albumen can obtain from producing any cell of described enzyme or albumen or biology to be separated.Various methods for separating of nucleotide sequence are all well known in the art.Be only citing character herein, such as utilize strong denaturant to extract total serum IgE, utilize and transcribe and reverse transcription method, the chromosomal DNA or the messenger RNA(mRNA) (mRNA) that produce the biology of described enzyme or albumen build genomic dna and/or cDNA library.

Further, also can be prepared the nucleotide sequence of the described enzyme of coding or albumen by the standard method of maturation by synthesis, for another example, utilize synthetic oligonucleotide on automatic dna synthesizer, be then purified, anneal, connect and be cloned in suitable carrier.

Therefore, described nucleotide sequence can be derived from the genome of mixing and synthesis source, the synthesis of mixing and the genome of cDNA source or mixing and cDNA source, its according to standard technique by connect be derived from synthesis, fragment that is genomic or cDNA obtains as required.The fragment of each connection corresponds to the different piece of whole nucleotide sequence.Described DNA sequence dna also can use specific primer to pass through polymerase chain reaction (PCR) to prepare.

Term " PCR " is referred to as polymerase chain reaction (polymerase chain reaction herein, PCR) refer under the catalysis of archaeal dna polymerase, by sex change, withdraw from a secret society or underworld gang, polymerization amplification waits iterative cycles to reach the region of DNA section of geometry order of magnitude amplification between two sections of known arrays.

Polynucleotide of the present invention (nucleotide sequence) can be used to prepare primer, as PCR primer, for the primer of optional amplified reaction; Maybe polynucleotide can be cloned in carrier.The length of described primer and other fragment can be at least 12, as at least 15, be preferably at least 20, as at least 25,30,35 or 40 Nucleotide, and it is also encompassed in as the term is employed herein within polynucleotide.

Usually, recombinant DNA technology (DNA namely recombinated) preparation coding is used to have the enzyme of special properties as herein defined or the nucleotide sequence of albumen.Therefore, in an Alternate embodiments of the present invention, chemical process known in the art can be used to synthesize all or part of nucleotide sequence.

Usually, prepare primer by synthetic method, the method comprises progressively prepares required nucleotide sequence in the mode of next Nucleotide every.Be easy in this area obtain and use automatic technology to realize the technology of aforesaid method.Design of primers can need to consider that the nucleotide sequence held at target protein N end or C adds the nucleotide sequence of code tag albumen (Tagged-protein expression system) according to improving purity, common label protein includes but not limited to His-tag albumen, Flag-tag albumen, GST-tag albumen etc.

Can recombinate, synthesize or be prepared according to polynucleotide of the present invention (as DNA polynucleotide) by the available any method of those skilled in the art.They also can be cloned by standard technique.Usual use recombination method, prepares longer polynucleotide as used PCR (polymerase chain reaction) clone technology.This comprises the pair of primers (according to appointment 12 to 40 Nucleotide) that preparation is positioned at the lipid targeted sequence area flank of required clone, primer is made to contact mRNA or cDNA obtained from ginseng, polymerase chain reaction is carried out under the condition that desired zone can be made to increase, be separated the fragment (as by purification reaction mixture on sepharose) of amplification, and reclaim the DNA of amplification.Can design to introduce makes it comprise applicable Restriction Enzyme recognition site, so that can by the DNA clone of amplification in the cloning vector be applicable to.

As mentioned above, can recombinate, synthesize or be prepared according to polynucleotide of the present invention (as DNA polynucleotide) by the available any method of those skilled in the art.They also can be cloned by standard technique.Usual use recombination method, prepares longer polynucleotide as used PCR (polymerase chain reaction) clone technology.This comprises the pair of primers (according to appointment 15 to 30 Nucleotide) that preparation is positioned at the lipid targeted sequence area flank of required clone, primer is made to contact mRNA or cDNA obtained from ginseng, polymerase chain reaction is carried out under the condition that desired zone can be made to increase, be separated the fragment (as by purification reaction mixture on sepharose) of amplification, and reclaim the DNA of amplification.Can design to introduce makes it comprise applicable Restriction Enzyme recognition site, so that can by the DNA clone of amplification in the cloning vector be applicable to.The design of restriction enzyme site can set according to the nucleotide sequence of vector cloning sites and the encode enzyme or albumen with special properties defined herein; often add several protection bases above, in detail with reference to the protection base setting reference manual that PROMEGA company provides.

In a specific embodiment of the present invention, described Candida utilis uriKoxidase recombinant protein is by expressing arbitrary nucleotide sequence shown below or having the nucleotide sequence of more than 50% identity with it and obtain:

A) nucleotide sequence shown in SEQ ID NO.3 of the aminoacid sequence of coding SEQ ID NO:4; Or

B) nucleotide sequence shown in SEQ ID NO.5 of the aminoacid sequence of coding SEQ ID NO:6.

Nucleotide sequence and the signal peptide sequence of coding Candida utilis uriKoxidase as herein defined may reside in carrier, described in this carrier, nucleotide sequence is operably connected to regulating and controlling sequence, make described regulating and controlling sequence can by be applicable to host cell (as intestinal bacteria) express as described in nucleotide sequence, namely described carrier is expression vector.Carrier of the present invention can be transformed in above-mentioned applicable host cell, has Candida utilis uriKoxidase recombinant protein as herein defined to express.Usually according to it, host cell be introduced into is selected carrier (as plasmid, clay, virus or phage vector, genomic inserts).The present invention can contain performance identical functions and the expression vector of other known in the art or known form.Once be transformed in selected host cell, carrier can not rely on the genome of host cell and copies and play function, or can be integrated into genome voluntarily.

Described carrier can comprise one or more selected marker, as provided the gene of antibiotics resistance, as provided the gene of amicillin resistance, kalamycin resistance, chlorampenicol resistant or tetracyclin resistance.Carrier can use in vitro, such as, for the preparation of RNA or for transfection or transformed host cell.

Expression vector can comprise the nucleotide sequence that this carrier can be made to copy in described host cell further.Described carrier can be pET 1-46 serial carrier (NOVAGEN company), pGEX serial carrier, pUC serial carrier, pACYC serial carrier, pUB serial carrier, pE serial carrier, pAMB serial carrier and pIJ serial carrier.Be preferably the pET1-46 serial carrier of NOVAGEN company, most preferably be pET42a (+).

The detection of above-mentioned nucleotide sequence has been come by nucleotide sequence sequenator, and whether analyze obtained gene by various molecular biology software is goal gene simultaneously.Conventional analysis software has GENTYX, Vector NTI, GenDOC, DNAman, also can carry out off-line and/or on-line search and retrieval analysis in order to comparison nucleotide sequence and protein sequence by means of BLAST and FASTA.

While mould is built, by by cDNA sequence shown in SEQ ID No.3 and 5 and the SEQ ID No.4 inferred by its clone and six amino acid sequence and database, (such as Kabat database is (see Johnson et al. respectively, 2000, Nucleic Acids Res.28:214-218.) and GenBank etc.) in sequence compare.Such as Smith-Waterman algorithm (Gusfield, 1997, in " Algorithms on Strings; Trees; and Sequences ", Cambridge University Press, or BLAST (Karlin et al. Cambridge), 1990, Proc.Natl.Acad.Sci.USA 87:2264-2268) etc. can search out and be at least about 50% with above-mentioned overall nucleotide sequence homology, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% or at least about 95% codon optimized nucleotide sequence; Or can find and be at least about 85% with above-mentioned overall amino acid sequence identity, at least about 90%, at least about 95%, at least about 98% or at least about 99% or the aminoacid sequence of identical (100%).

Term as used herein " enzyme " and term " aminoacid sequence " and/or term " polypeptide " and/or term " albumen " synonym.In some instances, term " aminoacid sequence " and term " peptide " and/or " enzyme " synonym, can alternative each otherly use.

Term as used herein " Candida utilis uriKoxidase recombinant protein " and term " the Candida utilis uriKoxidase recombinant protein of warm expression " and term " uriKoxidase recombinant protein " and/or " restructuring uriKoxidase " synonym, can alternative each otherly use.

Term " cell " herein comprises any cell of product that nucleotide sequence of the present invention or coding have the enzyme of special properties as defined herein or the nucleotide sequence of albumen and/or obtained by it, itself and term " biology " and/or term " bacterium " and/or term " microorganism " and/or term " bacterial micro-organism " synonym, replace use each other.

The term " engineering cell " relevant with the present invention or " host cell of conversion " comprise and anyly comprise the encode albumen of special properties that has as defined herein or the nucleotide sequence of enzyme and enzyme or albumen and/or the cell of product that obtained by it, and/or wherein promotor can allow the to encode enzyme of the special properties had as defined herein or the nucleotides sequence of albumen is listed in described biology and expresses.Preferred nucleotide sequence is introduced in biological genome.Term " genetically modified organism " is not encompassed in and is in self natural surroundings and is subject to the natural nucleus glycoside coding sequences that it is in the natural promoter control in self natural surroundings together simultaneously.Therefore, genetically modified organism of the present invention comprises the biology comprising following any one or combination: coding has enzyme or the nucleotide sequence of albumen, construct defined herein, carrier defined herein, plasmid defined herein, fixed cell or its product of special properties as defined herein herein.Such as, described genetically modified organism can also comprise coding to be had the enzyme of special properties as defined herein or albumen and is subject to the nucleotide sequence that promotor controls, and wherein said promotor is not connected with Candida utilis uriKoxidase recombinant protein encoding gene originally.

In yet further embodiment of the invention, provide a kind of engineering cell, wherein said engineering cell is the bacterial cell by obtaining after transforming above-mentioned expression vector and entering host cell.

Described host microorganism can be protokaryon or eukaryote.The nucleotide sequence of Candida utilis uriKoxidase recombinant protein of the present invention can available from, include but not limited to the bacterial micro-organism with subordinate: Escherichia (Escherichia), Mycobacterium (Mycobacterium), streptococcus (Streptococcus), lactobacillus (Lactobacillus), Desulfitobacterium (Desulfitobacterium), bacillus (Bacillus), campylobacter (Campylobacter), Vibrio (Vibrionaceae), XyZella (Xylella), sulfolobus solfataricus belongs to (Sulfolobus), Aeromonas (Aeromonas), streptomyces (Streptomyces), Saccharomycodes (Saccharomyces), lactococcus (Lactococcus), , Aspergillus (Aspergillus), Schizosaccharomyces (Schizosaccharomyces), listeria (Listeria), Neisseria (Neisseria), Autoinducer belongs to (Mesorhizobium), Lei Er Bordetella (Ralstonia), xanthomonas (Xanthomonas) and mycocandida (Candida), be preferably Escherichia (Escherichia). be preferably Escherichia (Escherichia).

Aptly, the nucleotide sequence of Candida utilis uriKoxidase recombinant protein of the present invention or coding Candida utilis uriKoxidase recombinant protein can available from, preferably available from following bacterial micro-organism: intestinal bacteria (E.coli), genus bacillus (Bacillus sp), campylobacter jejuni (Campylobacter jejuni), vibrios (Vibrionaceae), xyllela fastidiosa (Xylella fastidiosa), solfatara sulfolobus (Sulfolobus solfataricus), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Aeromonas hydrophila (Aeromonas hydrophila), aeromonas salmonicida (Aeromonas salmonicida), streptomyces coelicolor (Streptomyces coelicolor), streptomyces rimosus (Streptomyces rimosus), mycobacterium (Mycobacterium), micrococcus scarlatinae (Streptococcus pyogenes), Lactococcus lactis (Lactococcus lactis), Ralstonia solanacearum (Ralstonia solanacearum), xanthomonas campestris (Xanthomonas campestris), Xanthomonas axonopodis (Xanthomonas axonopodis), Candida parapsilosis (Candida parapsilosis), thermophilus streptococcus (Streptococcus thermophilus), lactobacterium helveticus (Lactobacillus helveticus), dehalogenation desulfiting bacterium (Desulfitobacterium dehalogenans), terreus (Aspergillus terreus), schizosaccharomyces pombe (Schizosaccharomyces pombe), listera innocua (Listeria innocua), listerisa monocytogenes in mjme (Listeria monocytogenes), Neisseria meningitidis (Neisseria meningitidis), Root or stem of Littleleaf Indianmulberry Autoinducer (Mesorhizobium loti).Be preferably Bacillus coli cells, be more preferably DH5 α and Rossetta Bacillus coli cells, most preferably be Rossetta Bacillus coli cells.

In one embodiment, according to Candida utilis uriKoxidase recombinant protein of the present invention can available from, preferably available from e. coli bl21 JZY003 and BL21JZY004 (Classification And Nomenclature: colon bacillus, Escherichia Coli), the microbial preservation budapest treaty that described coli strain JZY003 or JZY004 is used for patented procedure by Jin Zi source, Jilin Province Bioisystech Co., Ltd according to international recognition is deposited in China General Microbiological Culture Collection Center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center, postcode: 100101) preservation day is on 09 29th, 2014, preserving number is CGMCC9745 and CGMCC9746, it obtains respectively by aforementioned N-uricase-pET42 and the R-Uricase-pET42 expression vector of conversion.

In some embodiments of the invention, prepared some competent cells to be beneficial to external carrier and to be transferred in host cell.Conventional prepare competent cell and comprise subzero treatment, such as 16 DEG C.

In the present invention, the intestinal bacteria of indication comprise the host cell such as bacillus coli DH 5 alpha and Rossetta.Expression vector used in the present invention is prokaryotic expression carrier pET42a (+), and recombinant expression vector is converted into host cell, utilizes suitable conditional filtering recon, abduction delivering uriKoxidase recombinant protein.In the present invention, the inductor of indication is isopropyl-beta D-thio galactopyranoside (Isopropyl β-D-1-Thiogalactopyranoside, IPTG).Described separation purification method comprises ammonium sulfate precipitation, ion exchange chromatography and gel permeation chromatography.

The production technique of the present invention's mainly uriKoxidase recombinant protein, is realized by following steps:

1, Candida utilis uricase gene is that known (the NCBI number of logging in is for D32043.1; Albumen number is BAA06804.1), uriKoxidase total length 303 amino-acid residues of coding, by design Auele Specific Primer, from Candida utilis genomic dna, amplification obtains the encoding sequence of uriKoxidase.

2, an object of the present invention is to carry out codon optimized, to realize this sequence at E. coli to the DNA sequence dna of coding Candida utilis uriKoxidase.With the DNA sequence dna of method amplification coding uriKoxidase from Candida utilis uricase gene group of PCR; The sub-Optimization Software that accesses to your password (http://www.jcat.de/) the DNA encoding sequence to uriKoxidase is optimized, and makes it be suitable at expression in escherichia coli.

3, gene clone method: select pET42 carrier, restructuring uriKoxidase DNA encoding sequence clone after using NdeI and HindIII restriction enzyme site the DNA sequence dna of coding Candida utilis native uricase and codon to be optimized, in pET42 carrier, obtains N-uricase-pET42 and R-Uricase-pET42 expression plasmid respectively; When construction expression plasmid, to utilize in NdeI recognition sequence ATG as initiator codon, make expressed protein sequence and wild-type protein completely the same.

4, select intestinal bacteria Rossetta bacterial strain as Host Strains, make ectogenic Candida utilis uricase gene at E. coli.

5, purifying process: the uriKoxidase that present method is expressed, not with any label, when purifying, combinationally uses ammonium sulfate precipitation, anionresin and gel permeation chromatography, obtains the restructuring uricase protein that purity reaches more than 98%.

6, uricase activity of recombinating measures: purify obtained uriKoxidase by this technique, specific activity can reach 37.89U/mg.

In a preferred embodiment of the invention, the step of fermentation culture engineering bacteria comprises under suitable conditions:

Get 50 μ l intestinal bacteria Rossetta competent cells, be put in thawed on ice, respectively N-uricase-pET42 and R-Uricase-pET42 expression plasmid 1 μ l be converted in 50 μ l competent cells, place 60min on ice, 42 DEG C of water-bath heat shock 90S, then place 3min on ice.500 μ l are added not containing antibiotic LB substratum in competent cell, 37 DEG C, 250r/min concussion cultivation 1h, then take out on solid LB culture plate that 80 μ l products coat containing kantlex, overnight incubation in 37 DEG C of incubators, next day is from picking individual colonies flat board, be inoculated in 25ml LB substratum (containing 25mg/ml kantlex), 37 DEG C, 250r/min shaking culture spends the night.Getting 50ml overnight culture next day transfers in 1L LB substratum (containing 25mg/ml kantlex), 37 DEG C, 250r/min is cultured to OD ₆₀₀to 0.6, add IPTG and make its final concentration be 0.1mmol/L, 37 DEG C are continued shaking culture 12h, collected by centrifugation thalline, and the qualification of supernatant solubility is carried out in sampling.

In a preferred embodiment of the invention, from tunning, purifies and separates uriKoxidase recombinant protein comprises the following steps:

1. the coli somatic collected after getting fermentation culture, washs thalline 3 times with the phosphate buffered saline buffer of precooling;

2. every gram of wet thallus lysate of adding 10 times of volumes is resuspended, and adding final concentration is the N,O-Diacetylmuramidase of 1mg/ml and DTT and PMSF of 1mmol/L, acts on 30min on ice;

3. carrying out ultrasonic bacteria breaking (work 6s, cooling 9s, power are 700W, 90min) on ice bath, 14000r/min, 4 DEG C of centrifugal 60min afterwards, collect supernatant liquor.

4. ammonium sulfate precipitation: supernatant is carried out ammonium sulfate precipitation, first in supernatant liquor, solid ammonium sulfate powder is added, limit edged stirs, and makes ammonium sulfate saturation ratio reach 40%, 4 DEG C, the centrifugal 45min of 20000r/min, collect supernatant liquor, and then add intrinsic ammonium sulfate, make its saturation ratio reach 60%, stirring at room temperature 30min, 4 DEG C, the centrifugal 45min of 20000r/min, collecting precipitation thing.。

5. ion exchange chromatography: ammonium sulfate precipitation thing is dissolved in 50mmol/L glycine buffer (pH10.0), desalination is carried out with Sephadex G25, by on the sample after desalination on Resource Q anion-exchange column, carry out linear gradient elution with the 50mmol/L glycine buffer (pH10.0) containing 1M NaCl, collect the elutriant containing restructuring uriKoxidase.

6. molecular sieve purification: the component containing restructuring uriKoxidase obtained after ion-exchange merged, be concentrated into suitable volume with protein concentration pipe, loading, to Superdex 75 post, carries out molecular sieve purification, collects the elutriant containing restructuring uriKoxidase.

7. the component of collection containing target protein, detects with SDS-PAGE, merges purity and meets the requirements of component, measures protein concentration, divide and be filled in cryopreservation tube after being concentrated into certain volume by BCA method, after liquid nitrogen pre-freeze, frozen for subsequent use in-80 DEG C.

Present method has following advantage and effect: 1. present method has the advantages such as easy and simple to handle, with low cost; Present method can obtain the large and solvable recombinant protein of uriKoxidase of high purity 98% of output; The recombinant protein that present method obtains has good In vitro biological activity.

Embodiment

The cloning and expression of embodiment 1 Candida utilis uriKoxidase

1, the extraction of Candida utilis genomic dna: after freeze-drying Candida utilis (2.0120) bacterial strain by specification is required recovery, inoculate and cultivate (yeast extract 5g, peptone 10g into YPD, glucose 10g, add water to 500ml, adjust pH6.0) in, in 30 DEG C of shake-flask culture, through the visible Candida utilis normal growth of continuous two culture.Collected by centrifugation thalline, extracts test kit with pastoris genomic dna and extracts genomic dna.

2, the amplification of goal gene: according to the gene order of Candida utilis uriKoxidase in GenBank, design and synthesis pair of primers (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthesis), introduces Nde I, Hind III restriction enzyme site in primer.

P1：5’-ggA ATT CCA TAT gTC AAC AAC gCT CTC ATC-3’

P2：5’-CCC AAg CTT ACA ACT Tgg TCT TCT CC-3’

With Candida utilis genomic dna be template, P1 (Fig. 1) as shown in SEQ ID No.1 and the P2 shown in SEQ ID No.2 (Fig. 2) is the DNA sequence dna of primer PCR amplification coding uriKoxidase.PCR condition is: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations; 10min is extended after 72 DEG C.Amplified production is carried out agarose gel electrophoresis, amplification obtain 912bp as shown in SEQ ID No.4 the aminoacid sequence of uriKoxidase SEQ ID No.3 shown in coding nucleotide sequence (Fig. 3 and Fig. 4).

3, uricase gene transformation: based on the gene order of wild-type Candida utilis uriKoxidase, be optimized by codon optimized software (http://www.jcat.de/) the DNA encoding sequence to uriKoxidase, sequence (Fig. 5) as shown in SEQ ID NO.5, synthesizes complete sequence by the raw work in Shanghai.The aminoacid sequence of the uriKoxidase of being deduced by it is as shown in SEQ ID NO.6 (Fig. 6).

3, the structure of recombinant plasmid and qualification: the gene of wild-type Candida utilis uriKoxidase and the sequence after optimizing are used Nde I, Hind III double digestion respectively with prokaryotic expression plasmid pET42a (+) (Fig. 7), agarose gel electrophoresis is separated, purifying recovery.Connect through T4DNA ligase enzyme and spend the night, Transformed E .coli DH5 α competent cell, coats on the LB culture plate containing kantlex, 37 DEG C of overnight incubation.Extract recombinant plasmid after the amplification of picking mono-clonal and carry out Nde I, Hind III double digestion and PCR qualification, result display is containing 912bp goal gene fragment (Fig. 8), obtain the recombinant plasmid R-Uricase-pET42 after the gene plasmid N-Uricase-pET42 of wild-type Candida utilis uriKoxidase and sequence optimisation, confirm through order-checking, two kinds of expression plasmids build entirely true.

4, the abduction delivering of goal gene: with recombinant plasmid N-Uricase-pET42 and R-Uricase-pET42 transformation of E. coli Rossetta bacterial strain respectively, extraction plasmid, enzyme cut qualification.Picking mono-clonal is inoculated in the LB substratum containing kantlex (50mg/L), 37 DEG C of shaking culture are spent the night, in 1% ratio inoculation fresh LB, 37 DEG C of shaking culture are to logarithmic phase, add IPTG to final concentration 0.1mmol/L abduction delivering, before induction, after induction, 1.5h, 3h, 5h, 7h get 300 μ l bacterium liquid respectively, get thalline and carry out SDS-PAGE after centrifugal, testing goal protein expression situation, determines best induction time.After mass propgation, collect thalline, ultrasonication, get ultrasonic cleer and peaceful precipitation respectively and carry out SDS-PAGE qualification, determine whether target protein is solubility expression.Thalline detects through SDS-PAGE, has an obvious protein expression band at about 34kD place, conforms to 303 that infer from its gene order amino acid whose theoretical relative molecular mass 34kD, proves that restructuring uricase gene engineering bacteria successfully constructs (Fig. 9).Meanwhile, along with the prolongation of induction time, target protein expression amount increases, and after induction, 8h reaches peak value.Collect the thalline of a large amount of inducing culture, after carrying out ultrasonic bacteria breaking, full bacterium, ultrasonic cleer and peaceful precipitation are carried out SDS-PAGE, in full bacterium and bacteria break supernatant, a molecular mass is had to be about the high expression level protein band of 34kD, illustration purpose albumen successful expression in intestinal bacteria, expression amount accounts for more than 50% of bacterium total soluble protein, and exists with solvable form.

Embodiment 2 is recombinated the purifying of uriKoxidase and qualification

The purifying of target protein: by bacterium liquid 5000r/min, 4 DEG C of centrifugal 20min after induction, collect thalline, then PBS (pH7.4) is used to wash twice, bacterial sediment uses PBS resuspended again, ultrasonication thalline (whole process is all carried out in ice bath), 12000r/min, 4 DEG C of centrifugal 45min, collect supernatant, supernatant is carried out ammonium sulfate precipitation, mainly in 40% ~ 60% ammonium sulfate saturation ratio component, contain restructuring uricase protein, collect the precipitation of this component, be dissolved in 50mmol/L glycine buffer (pH10.0), desalination is carried out with Sephadex G25, then Resource Q anionresin column purification (Figure 10) is used, collect the elutriant containing restructuring uriKoxidase, molecular sieve purification (Figure 11) is carried out with Superdex 75 post, collect the elutriant containing restructuring uriKoxidase, carry out SDS-PAGE analysis, merge the component containing uricase protein, be concentrated into suitable volume, protein content is measured by BCA method,-80 DEG C of preservations after packing.

SDS-PAGE in whole purge process detects as shown in Figure 6, and in purge process, the Activity determination of each step is as shown in table 1.As can be seen from Table 1, sequence is after optimizing, and the gross activity of the uriKoxidase of expression improves 4.82%, and after a series of purifying, after optimizing, the gross activity of the target protein that sequence obtains improves 11.4%.

1. the Mass Spectrometric Identification of expressing protein and database retrieval: by the protein sample of purifying through Mass Spectrometric Identification (Figure 13), through ncbi database analysis, confirming really is Candida utilis uriKoxidase.

Embodiment 3 is recombinated the determination of activity of uriKoxidase

Activation analysis: adopt the method that (1992) such as Legoux are reported.1 μm of ol uric acid enzyme amount be oxidized to needed for wallantoin is 1 international unit (IU) of enzymic activity by per minute.In the reaction system of 5ml, add the uriKoxidase of 3ml Triethanolamine buffer (pH8.9), 1.5ml 0.2 μm of ol/L uric acid stock solution, 1 μ g (10 μ l) purifying respectively, 30 DEG C of reaction 5min, then the KOH termination reaction of 0.5ml 20% is added, measure uric acid concentration, determined wavelength is 292nm.

Calculate the activity of enzyme according to typical curve, the restructuring uricase activity of expressing from the gene of wild-type Candida utilis uriKoxidase and the sequence after optimizing does not have difference, and specific activity is respectively 36.1U/mg and 37.9U/mg.

The above results is summarized as table 1.

The recombinate activity of each purification step of uriKoxidase and protein content of table 1 detects (1L engineering bacterium fermentation liquid)

The present invention does not limit to the particular implementation described in this application, as the unitary declaration of independent aspect of the present invention.Various amendment and change is carried out when it will be understood by those skilled in the art that the spirit and scope that can not depart from the application.According to above description, except enumerating herein, the functionally equivalent purposes in the scope of the present disclosure is obvious for those skilled in the art of this area.Such change and amendment are intended to fall within the scope of the appended claims.The disclosure only by claims and with such claim the four corner that is equal to of scope limit.Should be appreciated that the disclosure is not limited to specific method, reagent, composition and biosystem, certainly, described method, reagent, composition and biosystem can change.It can also be appreciated that term used herein only for describing specific embodiment, it is restrictive for not being used for.

All patents that are referred herein or that quote, patent application, earlier application and publication are incorporated to herein by reference and in full, comprise all accompanying drawings and form, they are not contradicted with the clearly instruction of this specification sheets.Other embodiment is proposed in right.

Claims

1. a Candida utilis uriKoxidase recombinant protein, is characterized in that, described uriKoxidase recombinant protein comprises arbitrary following aminoacid sequence or has the aminoacid sequence of more than 85% identity with it:

A) aminoacid sequence shown in SEQ ID NO.4; Or

B) aminoacid sequence shown in SEQ ID NO.6.

2. the nucleic acid molecule be separated, it is characterized in that, the nucleic acid molecule of described separation comprises arbitrary following nucleotide sequence or has the nucleotide sequence of more than 50% identity with it:

A) nucleotide sequence shown in SEQ ID NO.1; Or

B) with the antisense base sequences shown in SEQ ID NO.2; Or

D) nucleotide sequence shown in SEQ ID NO.5 of the aminoacid sequence of coding SEQ ID NO:6.

3. a Candida utilis uriKoxidase recombinant protein, is characterized in that, described Candida utilis uriKoxidase recombinant protein is by expressing arbitrary nucleotide sequence shown below or having the nucleotide sequence of more than 50% identity with it and obtain:

4. an expression vector, is characterized in that, described expression vector contains arbitrary nucleotide sequence shown below or has the nucleotide sequence of more than 50% identity with it:

A) nucleotide sequence shown in SEQ ID NO.1; Or

B) with the antisense base sequences shown in SEQ ID NO.2; Or

C) nucleotide sequence shown in SEQ ID NO.3; Or

D) nucleotide sequence shown in SEQ ID NO.5.

5. expression vector as claimed in claim 4, it is characterized in that, described expression vector is pET42a (+).

6. an engineering cell, is characterized in that, described engineering cell is the bacterial cell obtained after entering host cell by the expression vector transformed described in claim 4 or 5.

7. engineering cell as claimed in claim 5, it is characterized in that, described engineering cell is bacillus coli DH 5 alpha or Rossetta cell, is preferably Rossetta cell.

8. prepare a method for Candida utilis uriKoxidase recombinant protein, it is characterized in that, described method comprises:

A) arbitrary nucleotide sequence according to claim 3 is cloned into the step of expression vector pET42a (+);

B) described expression vector pET42a (+) is transferred to the step of host cell E. coli Rossetta competent cell;

C) host cell E. coli Rossetta cell is carried out culturing step, culture condition comprises: cultivate and be divided into cultivation stage and induction period, at cultivation stage, first picking individual colonies is inoculated in the LB substratum of 25ml containing 25mg/ml kantlex, 37 DEG C of shaking culture are spent the night, get 50ml overnight culture next day to transfer in the 1L LB substratum containing 25mg/ml kantlex, 37 DEG C are cultured to OD ₆₀₀after 0.6, add IPTG, making it final concentration is 0.1mmol/L, and 37 DEG C are continued shaking culture 12h, collected by centrifugation thalline;

D) carry out any preliminary purification step by saltouing, described in saltout as ammonium sulfate precipitation, carry out the thick leach protein of classification with 40-60% neutral sulphates ammonium;

E) by anionresin, gel filtration step, described anion-exchange chromatography uses Resource Q post; Described gel-filtration uses Superdex75 10/60Hiload post, the step of the Candida utilis uriKoxidase recombinant protein of purity more than 96% of final gained target protein.

9. Candida utilis uriKoxidase recombinant protein is preparing the purposes in the medicine for the treatment of and human body high lithemia relative disease or pathological symptom, food, healthcare products and makeup thereof.

10. method as claimed in claim 8 or purposes according to claim 10, it is characterized in that, described uriKoxidase recombinant protein comprises arbitrary following aminoacid sequence or has the aminoacid sequence of more than 85% identity with it:

A) aminoacid sequence shown in SEQ ID NO.4; Or

B) aminoacid sequence shown in SEQ ID NO.6.