CN102140444A - Low temperature alkaline phosphatase and preparation method thereof - Google Patents
Low temperature alkaline phosphatase and preparation method thereof Download PDFInfo
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- CN102140444A CN102140444A CN2010101027925A CN201010102792A CN102140444A CN 102140444 A CN102140444 A CN 102140444A CN 2010101027925 A CN2010101027925 A CN 2010101027925A CN 201010102792 A CN201010102792 A CN 201010102792A CN 102140444 A CN102140444 A CN 102140444A
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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 South Pole bacterial 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 is formed, and the conserved sequence of 10 groups of βZhe Dies is arranged at the middle portion of enzyme.A kind of enzyme that alkaline phosphatase generally uses as the laboratory is usually used in non-specific ground dephosphorylation group in molecular cloning, weakens the environment from empty carrier or recombinant vectors.The fragment of being handled by Phosphoric acid esterase lacks 5 ' phosphoryl sign, therefore can avoid self-cyclisation, reduces the influence of environment directivity when synthesizing.This enzyme can be removed fully through heat inactivation, activity is all arranged, so be widely used in the scientific research during in addition as restriction enzyme in all damping fluids.In addition, alkaline phosphatase has bigger importance as an important physical health indicator on clinical medicine.Pair medical science detection of alkaline phosphatase Study of indexes 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, and activity is all arranged in all damping fluids during in addition as restriction enzyme.That research at present is maximum, range of application is the widest is the alkaline phosphatase Shrimp Alkaline Phosphatase that extracts from shrimp.But the procurement cost of this enzyme is higher, and inactivation gradually in reaction easily.Another kind of enzyme Calf Intestinal Alkaline Phosphotase can not heat inactivation, so cause the failure of an experiment easily, influences the research progress.What research at present was maximum, range of application is 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 and primer provides the simplest, the safest and high-efficiency method from the PCR product.But the procurement cost of this enzyme is higher, and inactivation gradually in reaction easily.The also widely used enzyme Calf of another kind Intestinal Alkaline Phosphotase (CIAP) can not heat inactivation.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 alkali Phosphoric acid esterase 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 combine 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 that intestinal bacteria are had a preference for, 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 proteic expression efficiency, improve target protein in the intravital expression output of intestinal bacteria.
The invention provides a kind of preparation method of low-temperature alkali Phosphoric acid esterase, the cDNA sequence clone that is about to the low-temperature alkali Phosphoric acid esterase shown in sequence table SEQ ID NO 1 is expressed to engineering carrier, obtains the low-temperature alkali Phosphoric acid esterase.
The present invention optimizes the codon of the dna sequence dna of existing low-temperature alkali Phosphoric acid esterase TAB5-AP, optimizes improved content:
1), the 3rd bit base of amino acid codeword triplet has been carried out the synonym replacement according to colibacillary codon preference theory.
2) the many codon preference that exist according to intestinal bacteria, same nucleotide site exist a plurality of bases to replace.
Can also sequence N end and (or) C holds and can connect different albumen labels as required.Preferably, hold the dna sequencing fragment that has 10 Histidines at the N of this enzyme cDNA, so that purifies and separates.The segmental length of Histidine and N end and (or) position of C end can adjust as the case may be.
Being expressed under room temperature or the low temperature of low-temperature alkali Phosphoric acid esterase of the present invention carried out, thereby 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 alkali Phosphoric acid esterase shown in sequence table SEQ ID NO 1, the nucleotide coding sequence of low-temperature alkali Phosphoric acid esterase promptly 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 alkali Phosphoric acid esterase.
Wherein, during the nucleotide coding sequence of synthetic low-temperature alkali Phosphoric acid esterase, can add one by one, also can synthesize some small segments earlier, and then these fragments are connected according to SEQ ID NO 1.
Wherein, gene engineering method is often referred to, and with the multiple clone site of target dna fragment cloning to carrier, changes the host then over to and duplicates expression.Used carrier, host and related reagent can adopt the material of genetically engineered field routine.Carrier can use conventional pet serial carrier, and 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 according to following steps:
1) the broken bacterium of host bacterium, the centrifugation that will express the low-temperature alkali Phosphoric acid esterase goes 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 alkali Phosphoric acid esterase 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 alkali Phosphoric acid esterase, 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 alkali 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 alkali Phosphoric acid esterase 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 alkali Phosphoric acid esterase 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, as sample preparation before the dna sequencing etc.
The present invention makes it can be applied in commercial production and the scientific research for the production of toolenzyme provides a kind of novel method and new approaches.The invention has the beneficial effects as follows and improved the output of this low-temperature alkali Phosphoric acid esterase in prokaryotic expression system greatly, 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, fragment is inserted in the carrier, structure has the segmental plasmid of TAB5-AP again.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.5, the 000g rotating speed was collected bacterium in centrifugal 20 minutes 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%, 12, following 4 ℃ of 000g rotating speed was got supernatant in centrifugal 20 minutes.Utilize the roughing out of Ni-NTA affinity column earlier.Combine Buffer C (20mM Tris-HCl, pH 8.0,10%glyceroland 100mM imidazole) the flush away uncombined foreign protein of back with medium at last sample with the 100mM imidazoles.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 proteic elute soln of TAP, finally obtain purity more than 99.0% alkaline phosphatase.
The production and productivity tabular of whole protein expression purifying is in following 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 (10)
1. a low-temperature alkali Phosphoric acid esterase is characterized in that, its nucleotide coding sequence is shown in SEQ ID NO 1.
2. a method for preparing the described low-temperature alkali Phosphoric acid esterase of claim 1 is characterized in that, the cDNA sequence clone of low-temperature alkali Phosphoric acid esterase that will be shown in sequence table SEQ ID NO 1 is expressed to engineering carrier, obtains the low-temperature alkali Phosphoric acid esterase.
3. preparation method according to claim 2 is characterized in that, at first, and the cDNA sequence of synthetic low-temperature alkali Phosphoric acid esterase shown in sequence table SEQ ID NO 1; Then, utilize gene engineering method to spend the night at 18～25 ℃ of abduction deliverings; At last, separation and purification low-temperature alkali Phosphoric acid esterase.
4. preparation method according to claim 3 is characterized in that, the process of separation and purification is:
1) the broken bacterium of host bacterium, the centrifugation that will express the low-temperature alkali Phosphoric acid esterase goes out albumen;
2) supernatant liquor in the step (1) is used Ni-NTA affinity chromatography column purification, remove foreign protein.
5. preparation method according to claim 4 is characterized in that, removes foreign protein by high-efficient liquid phase chromatogram technology.
6. preparation method according to claim 4 is characterized in that, gained albumen elutriant is replaced protein solution with dialysis method.
7. preparation method according to claim 4 is characterized in that, uses the Q ion exchange column to be further purified gained albumen elutriant.
8. preparation method according to claim 3 is characterized in that, this preparation method comprises that also the protein solution to the low-temperature alkali Phosphoric acid esterase after the separation and purification concentrates.
9. preparation method according to claim 8 is characterized in that, described concentrating is with the ultrafiltration dialysis protein solution to be concentrated.
10. expression vector or cloning vector is characterized in that it contains just like the nucleotide coding sequence shown in the SEQID NO 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233021A (en) * | 2013-04-17 | 2013-08-07 | 中国农业大学 | Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 |
| CN103233020A (en) * | 2013-04-10 | 2013-08-07 | 中国农业大学 | A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis |
| CN103233022A (en) * | 2013-04-17 | 2013-08-07 | 中国农业大学 | Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry9C |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4720458A (en) * | 1983-10-19 | 1988-01-19 | Sullivan Cornelius W | Heat sensitive bacterial alkaline phosphatase |
| WO2004076628A2 (en) * | 2003-02-24 | 2004-09-10 | New England Biolabs, Inc. | Overexpression, purification and characterization of a thermolabile phosphatase |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233020A (en) * | 2013-04-10 | 2013-08-07 | 中国农业大学 | A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis |
| 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 |
| CN103233021A (en) * | 2013-04-17 | 2013-08-07 | 中国农业大学 | Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 |
| CN103233022A (en) * | 2013-04-17 | 2013-08-07 | 中国农业大学 | Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry9C |
| 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 |
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