CN102140444B - Low temperature alkaline phosphatase and preparation method thereof - Google Patents
Low temperature alkaline phosphatase and preparation method thereof Download PDFInfo
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- CN102140444B CN102140444B CN 201010102792 CN201010102792A CN102140444B CN 102140444 B CN102140444 B CN 102140444B CN 201010102792 CN201010102792 CN 201010102792 CN 201010102792 A CN201010102792 A CN 201010102792A CN 102140444 B CN102140444 B CN 102140444B
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Abstract
The invention belongs to the field of gene engineering, and relates to novel alkaline phosphatase and a preparation method thereof. The cDNA sequence of low temperature alkaline phosphatase shown as SEQ ID NO 1 in a sequence table is cloned to a vector for genetic engineering, and expressed to obtain the low temperature alkaline phosphatase. According to codon bias of Escherichia coli, the DNA sequence of the TAB5 low temperature alkaline phosphatase is transformed, so that the DNA sequence is highly expressed in the Escherichia coli; the alkaline phosphatase is purified by nickel affinity chromatography and ion exchange chromatography; and more than 90mg of TAB5 alkaline phosphatase with the purity of over 99 percent is obtained from each liter of Escherichia coli liquid finally. The invention can be applied to research in the field of molecular biology of the field of life science, particularly multi-step reaction for heat inactivation of alkaline phosphatase, such as sample treatment before DNA sequencing and the like.
Description
Technical field
The invention belongs to the genetically engineered field, relate to a kind of dna sequence dna.Particularly, the present invention relates to a kind of alkaline phosphatase Alkaline Phosphatase (TAB5-AP) that is present in Antarctic bacteria strain TAB5, also relate to the expression of this enzyme in escherichia expression system and the method for purifying protein.
Background technology
Alkaline phosphatase is the non-specific enzyme that extensively is present in the various organisms, has vital role in organism.Most of alkaline phosphatases all are one two homodimers that identical subunit forms, and the conserved sequence of 10 groups of β-pleated sheet structures is arranged at the middle portion of enzyme.A kind of enzyme that alkaline phosphatase generally uses as the laboratory is usually used in molecular cloning non-specifically dephosphorylation group, weakens the environment from empty carrier or recombinant vectors.The fragment of being processed by Phosphoric acid esterase lacks 5 ' phosphoryl sign, therefore can avoid self-cyclisation, reduces the impact of environment directivity when synthesizing.This enzyme can be removed fully through heat inactivation, in all damping fluids activity is arranged during in addition as restriction enzyme, so be widely used in the scientific research.In addition, alkaline phosphatase has larger importance as an important physiological well-being index at clinical medicine.Pair research of medical science detection of alkaline phosphatase index is all arranged at present both at home and abroad.
Alkaline phosphatase has played irreplaceable effect in scientific research and clinical application at present.And along with the needs of experiment progressively are modified.Good alkaline phosphatase not only can shorten experimental period, the abbreviation experimental procedure, can increase the effect of gene clone experiment simultaneously.This enzyme can be removed through heat inactivation, in all damping fluids activity is arranged during in addition as restriction enzyme.That at present most study, range of application are the widest is the alkaline phosphatase Shrimp Alkaline Phosphatase that extracts from shrimp.But the procurement cost of this enzyme is higher, and easy inactivation gradually in reaction.Another kind of enzyme Calf Intestinal Alkaline Phosphotase can not heat inactivation, therefore easily cause the failure of an experiment, affects the research progress.What at present most study, range of application were the widest is the alkaline phosphatase that extracts from shrimp: Shrimp Alkaline Phosphatase (SAP).The dna sequence dna of this alkaline phosphatase and crystalline structure are revealed, and the achievement in research of related protein function is also delivered successively.SAP is with exonuclease, for removing Nucleotide before dna sequencing or the gene type from the PCR product and primer provides simple, safe and the most efficient method.But the procurement cost of this enzyme is higher, and easy inactivation gradually in reaction.Also widely used enzyme Calf Intestinal Alkaline Phosphotase (CIAP) can not heat inactivation for another kind.Therefore needing to seek better enzyme substitutes.
Summary of the invention
The objective of the invention is in order to overcome the deficiency of existing alkaline phosphatase, choose the low temperature alkaline phosphatase TAB5-AP that has announced, proof has good inactivation characteristic, theoretical according to the preference of the e. coli codon in the prokaryotic expression system, redesign the dna sequence dna of this enzyme, thereby improved expression and the effect in purifying flow out of this enzyme in prokaryotic system.
The present invention has added the dna sequence dna of 10 Histidines before the cDNA sequence of existing Phosphoric acid esterase TAB5-AP.Histidine can specificly be combined with the nickel post, helps protein purification.According to the specific preferences of intestinal bacteria to the 3rd bit base of amino acid codeword triplet, this amino acid whose codon is carried out same sense mutation, the 3rd bit base is replaced to the base of intestinal bacteria preference, revised the original series of TAB5-AP.When the nucleotides sequence of this sequence is listed in when expressing in the prokaryotic system, because intestinal bacteria have preference to the codon of replacing, thereby can improve protein expression efficient, improve the expression output of target protein in the intestinal bacteria body.
The invention provides a kind of preparation method of low temperature alkaline phosphatase, be about to the cDNA sequence clone of the low temperature alkaline phosphatase shown in sequence table SEQ ID NO 1 to engineering carrier, express, obtain low temperature alkaline phosphatase.
The present invention is optimized the codon of the dna sequence dna of existing low temperature alkaline phosphatase TAB5-AP, optimizes improved content:
1) theoretical according to colibacillary codon preference, the 3rd bit base of amino acid codeword triplet has been carried out the synonym replacement.
2) the many codon preference that exist according to intestinal bacteria, there are a plurality of Substitutions in same nucleotide site.
Can also sequence N end and (or) C holds and can connect as required different albumen labels.Preferably, at the dna sequencing fragment of the N of this enzyme cDNA end with 10 Histidines, so that purifies and separates.The length of Histidine fragment and N end and (or) position of C end can adjust as the case may be.
The expression of low temperature alkaline phosphatase of the present invention is carried out under room temperature or low temperature, thereby has saved the energy and preparation cost.Obtain the protein solution of purifying, wherein purity of protein 99.0%.Every liter of bacterium liquid obtains more than the protein 90 mg.
Particularly, at first, the cDNA sequence of synthetic low temperature alkaline phosphatase shown in sequence table SEQ ID NO 1, the i.e. nucleotide coding sequence of low temperature alkaline phosphatase of the present invention; Then, utilize gene engineering method to spend the night at 18～25 ℃ of abduction deliverings; At last, separation and purification low temperature alkaline phosphatase.
Wherein, during the nucleotide coding sequence of synthetic low temperature alkaline phosphatase, can add one by one, also can synthesize first some small segments, and then these fragments are connected according to SEQ ID NO 1.
Wherein, gene engineering method is often referred to, and the target dna fragment is cloned into the multiple clone site of carrier, then changes the host over to and copies expression.Used carrier, host and related reagent can adopt the material of genetically engineered field routine.Carrier can use conventional pet serial carrier, and such as plasmid pET28A, host cell can adopt e. coli bl21, DH5 α etc.
Wherein, the process of separation and purification can adopt routine techniques, also can carry out in accordance with the following steps:
1) the broken bacterium of Host Strains, the centrifugation that will express low temperature alkaline phosphatase go out albumen;
2) supernatant liquor in the step (1) is used Ni-NTA affinity chromatography column purification, remove foreign protein;
3) the albumen elutriant that step (2) is obtained uses the Q ion exchange column to carry out purifying.
Wherein, removing foreign protein can realize by high-efficient liquid phase chromatogram technology.
The albumen elutriant that can remove behind the foreign protein is replaced protein solution with dialysis method.Also can use the Q ion exchange column to be further purified gained albumen elutriant.
The present invention comprises that also the protein solution to the low temperature alkaline phosphatase after the separation and purification concentrates.Described concentrating can be used the ordinary skill in the art, for example with the ultrafiltration dialysis protein solution concentrated.
The invention provides a kind of novel low temperature alkaline phosphatase, its nucleotide coding sequence is shown in SEQ ID NO1.
The present invention also provides expression vector or the cloning vector that contains just like the nucleotide coding sequence shown in the SEQ ID NO 1.
The low temperature alkaline phosphatase protein solution of the purifying that preparation method of the present invention obtains, wherein purity of protein can reach 95% preferably usually more than 92%, even more than 99.0%, every liter of bacterium liquid obtains more than the protein 90 mg.
In order to overcome the deficiency of existing alkaline phosphatase, the present invention chooses the low temperature alkaline phosphatase TAB5-AP that has announced, proof has good inactivation characteristic, theoretical according to the preference of the e. coli codon in the prokaryotic expression system, redesign the dna sequence dna of this enzyme, thereby improved expression and the effect in purifying flow out of this enzyme in prokaryotic system.Obtain the protein solution of purifying, wherein purity of protein 99.0%.Every liter of bacterium liquid obtains more than the protein 90 mg.The preparation method of low temperature alkaline phosphatase of the present invention, easy and simple to handle, efficient energy-saving is all lower to working condition and equipment requirements.The present invention can be widely used in the research of life science biology field, particularly needs the polystep reaction with the alkaline phosphatase heat inactivation, such as sample preparation before the dna sequencing etc.
The present invention provides a kind of novel method and new approaches for the production of toolenzyme, so that it can be applied in commercial production and the scientific research.The invention has the beneficial effects as follows and greatly improved the output of this low temperature alkaline phosphatase in prokaryotic expression system, to the commercialization of this enzyme before death scape decisive significance is arranged.
Embodiment
Use polymerase chain reaction polymerase chain reaction (PCR) method that the TAB5-AP sequence is increased, with restriction enzyme this fragment and vector plasmid are digested, again fragment is accessed in the carrier, make up the plasmid with the TAB5-AP fragment.Be transferred at last in the intestinal bacteria competence and just screen.
Choose in the 5ml LB nutrient solution that contains 100ug/ml ammonia benzyl mycin, to activate behind the positive monoclonal and spend the night, go to subsequently in the 1L automatic induction culture medium, cultivated 4 to 5 hours prior to 37 ℃, treat that the bacterium raised growth is placed on 20 ℃ of low temperature inductions and expressed 15 hours.Collected bacterium in centrifugal 20 minutes at 5,000g rotating speed behind the abduction delivering.
With the Buffer A of wash-out Ni-NTA (20mM Tris-HCl, pH 8.0,150mM NaCl, 10%glycerol and 10mM imidazole) resuspension thalline, add carrying out ultrasonic bacteria breaking behind the Triton-X1000.2%, got supernatant in centrifugal 20 minutes lower 4 ℃ of 12,000g rotating speed.Utilize first the roughing out of Ni-NTA affinity column.Be combined rear Buffer C (20mM Tris-HCl, pH 8.0,10%glyceroland 100mM imidazole) the uncombined foreign protein of flush away with the 100mM imidazoles with medium in loading.Using Buffer B (20mM Tris-HCl, pH 8.0,500mM midazole and 10%glycerol) wash-out target protein TAP after the Buffer A balance.
Use subsequently the Q ion exchange column segment from.Carry out gradient elution with Buffer A (20mM Tris-HCl, pH 8.0and 10% glycerol) and Buffer B (20mM Tris-HCl, pH 8.0,1M NaCl and 10%glycerol) with the NaCl concentration of 0～1M.Collection contains the elute soln of TAP albumen, finally obtain purity more than 99.0% alkaline phosphatase.
The production and productivity tabular of whole protein expression and purification is in lower Table I.
Table I TAB5 alkaline phosphatase production and productivity table
SEQUENCE LISTING
＜110〉Fudan University
＜120〉new type low temperature alkaline phosphatase and preparation method thereof
<130>1009
<160>1
<170>PatentIn version 3.1
<210>1
<211>1101
<212>DNA
＜213〉artificial sequence
<400>1
atgcaccacc accaccacca ccaccaccac cacggatccg tgttggtaaa gaacgagcct 60
cagctgaaaa ctcctaagaa tgtcattctg ctgatttcgg atggcgcggg cctttcacaa 120
atatcttcga cgttttattt taaagaaggc acgccgaact acacgcaatt taagaatatt 180
gggctgatca agacttcatc gagccgcgaa gatgtcaccg attccgcgtc gggagcgact 240
gcctttagct gcggcattaa aacgtataat gcagcaattg gtgttgcgga tgattccacc 300
gccgtgaaga gcattgtaga aattgcagcc ctgaataaca ttaaaacggg cgtggttgca 360
acgtcgagca ttactcacgc gaccccagca tcattttatg cacacgcctt gaaccgtggc 420
cttgaggaag aaatcgcgat ggacatgaca gaaagtgatt tagatttttt tgccggtggt 480
ggcctgaatt atttcaccaa acgtaaagac aagaaagacg ttctcgccat tctgaaagga 540
aatcaattta caattaatac cacgggtctg actgacttca gttcgattgc ctcgaatcgc 600
aaaatgggct tcctgttagc cgacgaggcg atgcccacta tggaaaaggg ccgcgggaat 660
ttcctgtccg cggcaacgga tctggccatt cagtttctga gcaaggataa ttcagcgttt 720
ttcatcatgt ccgagggctc tcagatagac tggggcggcc acgcaaataa cgcgagttac 780
cttatctcag agattaatga ttttgacgat gctattggga cagctcttgc atttgctaaa 840
aaagatggga acactttggt gattgtgact tcggaccatg aaacgggtgg ttttaccctt 900
gccgcaaaaa aaaataaacg tgaggacggc tccgaatata gtgattacac ggaaattggc 960
ccgacgtttt ctaccggcgg gcacagcgcc accctgattc cagtctttgc ttatggcccg 1020
ggaagcgagg aatttatcgg catttacgaa aataatgaaa tttttcacaa aatcttaaag 1080
gttaccaaat ggaaccaata a 1101
Claims (9)
1. a low temperature alkaline phosphatase is characterized in that, its nucleotide coding sequence is shown in SEQ ID NO 1.
2. a method for preparing low temperature alkaline phosphatase claimed in claim 1 is characterized in that, the cDNA sequence clone of the low temperature alkaline phosphatase shown in sequence table SEQ ID NO 1 to engineering carrier, is expressed, and obtains low temperature alkaline phosphatase.
3. preparation method according to claim 2 is characterized in that, at first, and the cDNA sequence of synthetic low temperature alkaline phosphatase shown in sequence table SEQ ID NO 1; Then, utilize gene engineering method to spend the night at 18～25 ° of C abduction deliverings; At last, separation and purification low temperature alkaline phosphatase.
4. preparation method according to claim 3 is characterized in that, the process of separation and purification is:
1) the broken bacterium of Host Strains, the centrifugation that will express low temperature alkaline phosphatase go out albumen;
2) supernatant liquor in the step (1) is used Ni-NTA affinity chromatography column purification.
5. preparation method according to claim 4 is characterized in that, gained albumen elutriant is replaced protein solution with dialysis method.
6. preparation method according to claim 4 is characterized in that, uses the Q ion exchange column to be further purified gained albumen elutriant.
7. preparation method according to claim 3 is characterized in that, this preparation method comprises that also the protein solution to the low temperature alkaline phosphatase after the separation and purification concentrates.
8. preparation method according to claim 7 is characterized in that, described concentrating is with the ultrafiltration dialysis protein solution to be concentrated.
9. an expression vector or cloning vector is characterized in that, it contains just like the nucleotide coding sequence shown in the SEQ ID NO 1.
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| CN 201010102792 CN102140444B (en) | 2010-01-28 | 2010-01-28 | Low temperature alkaline phosphatase and preparation method thereof |
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| CN102140444B true CN102140444B (en) | 2013-01-02 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103233020B (en) * | 2013-04-10 | 2014-12-10 | 中国农业大学 | A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis |
| CN103233021B (en) * | 2013-04-17 | 2014-12-03 | 中国农业大学 | Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 |
| CN103233022B (en) * | 2013-04-17 | 2014-12-03 | 中国农业大学 | Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry9C |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4720458A (en) * | 1983-10-19 | 1988-01-19 | Sullivan Cornelius W | Heat sensitive bacterial alkaline phosphatase |
| CN101220368A (en) * | 2003-02-24 | 2008-07-16 | 新英格兰生物实验室公司 | Overexpression, purification and characterization of a thermolabile phosphatase |
-
2010
- 2010-01-28 CN CN 201010102792 patent/CN102140444B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4720458A (en) * | 1983-10-19 | 1988-01-19 | Sullivan Cornelius W | Heat sensitive bacterial alkaline phosphatase |
| CN101220368A (en) * | 2003-02-24 | 2008-07-16 | 新英格兰生物实验室公司 | Overexpression, purification and characterization of a thermolabile phosphatase |
Non-Patent Citations (2)
| Title |
|---|
| Maria Rina.Alkaline phosphatase from the Antarctic strain TAB5.《Eur. J. Biochem》.2000,第267卷1230-1238. * |
| Rina,M.Antarctic bacterium TAB5, phoA gene.《GeneBank》.2005,ORIGIN. * |
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