CN102816747B - High-temperature-resistance iron superoxide dismutase and crystallization method thereof - Google Patents
High-temperature-resistance iron superoxide dismutase and crystallization method thereof Download PDFInfo
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- CN102816747B CN102816747B CN201210284972.9A CN201210284972A CN102816747B CN 102816747 B CN102816747 B CN 102816747B CN 201210284972 A CN201210284972 A CN 201210284972A CN 102816747 B CN102816747 B CN 102816747B
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Abstract
The invention relates to a high-temperature-resistance iron superoxide dismutase and a crystallization method thereof. According to the invention, with a gene recombination technology, high-temperature-resistance FeSOD gene is constructed onto a vector pET28a; a recombinant vector pET-28a-FeSOD is introduced into E.coli BL21 (DE3) and is used in induced expression; recombinant target protein is purified with affinity chromatography and ion-exchange chromatography, such that the target protein with a purity higher than 95% is obtained; crystallization conditions of the target protein are screened by using a crystallization kit, such that protein crystals are obtained. The crystallization method provided by the invention is advantaged in simple process and high resolution of obtained crystals.
Description
Technical field
The present invention relates to DNA recombinant technology and protein technical field, particularly relate to a kind of high temperature resistant iron superoxide dismutase and crystallization method thereof.
Background technology
Superoxide-dismutase (Superoxide dismutase, be called for short SOD) as a kind of, be extensively present in biological cell, the metalloenzyme with defence oxidative injury, relevant with body aging, tumour generation, autoimmune disorder and radio-protective etc., and all there is important using value in fields such as medicine, healthcare products, foodstuff additive and makeup.
SOD is extensively present in organic sphere, and the raw material of the SOD of initial production is also animal blood, but the technique of purifying in this source is loaded down with trivial details, operational difficulty, and the Product Activity obtaining is low, and purity is also low.Utilize genetic engineering technique, the SOD of many better heat stability has successfully carried out great expression in normal temperature host, thereby has significantly reduced production cost, has expanded production application.
The protein integrity of its biologic activity that is hard to keep in vitro, however the general activity of the protein existing with crystalline structure is all more stable.The data of Collection and analysis X-ray diffraction protein crystal, just can parse the higher structure of this protein.Method of the present invention is the crystallization first of this kind of high temperature resistant iron superoxide dismutase, both at home and abroad all without report.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of high temperature resistant iron superoxide dismutase and crystallization method thereof.
For achieving the above object, the invention provides a kind of high temperature resistant iron superoxide dismutase, its aminoacid sequence is as shown in SEQ ID NO:2.
For achieving the above object, the invention provides a kind of crystallization method of above-mentioned high temperature resistant iron superoxide dismutase, described method obtains restructuring target protein by building recombinant vectors pET-28a-FeSOD and importing abduction delivering in e. coli bl21 (DE3), this restructuring target protein of the method purifying of recycling affinity chromatography and ion exchange chromatography, then by the crystallization condition of crystallization test kit screening restructuring target protein, can obtain the albumin crystal of high temperature resistant iron superoxide dismutase.
Aforesaid method comprises the following steps:
(a) by design primer, stearothermophilus ground bacillus (Geobacillus) genomic dna of take is template, by pcr amplification method, obtains high temperature resistant FeSOD gene, and its base sequence is as shown in SEQ ID NO:1;
(b) the high temperature resistant FeSOD gene of step (a) gained is connected on pET28a carrier, builds recombinant expression vector pET-28a-FeSOD, and import in e. coli bl21 (DE3), abduction delivering obtains a large amount of restructuring target proteins;
(c) restructuring target protein supernatant step (b) being obtained filters by 0.45 μ m cellulose membrane, then utilize Ni post affinity chromatography and ion exchange chromatography purification of Recombinant target protein, obtain purity and reach more than 95% restructuring target protein (have high thermotolerance, heat resisting temperature can reach 90 ℃);
(d) just the restructuring target protein after step (c) purifying concentrates, with BCA method protein quantification test kit, measure restructuring target protein concentration, when concentration reaches 5-8mg/ml, with crystallization of protein test kit, screen crystallization condition, obtain albumin crystal;
(e) albumin crystal that just step (d) forms, utilizes microscope and X ray crystalline diffraction instrument to identify.
Preferably, the concrete operations of wherein said step (b) are: by the high temperature resistant FeSOD gene of step (a) gained after Nde I and EcoR I double digestion, be connected on the pET28a of same processing carrier, build recombinant expression vector pET-28a-FeSOD, recombinant vectors pET-28a-FeSOD is imported to e. coli bl21 (DE3), obtain gene recombination engineering bacteria E.coli/pET-28a-FeSOD, by recombination engineering bacteria in 37 ℃, under 220rpm, being cultured to OD600 is 0.6 o'clock, add inductor IPTG to final concentration be 1mM, continue to cultivate 3-5 hour, collect bacterium liquid, obtain a large amount of restructuring target proteins.
Preferably, the crystallization of protein test kit of wherein said step (d) is purchased from Hampton Research company, and crystallization condition is Index
tMhR2-14461, Index
tMhR2-144 63, Index
tMhR2-144 87, Index
tMhR2-14488, Index
tMhR2-14494, PEG/lon2 Screen
tMhR2-0985, PEG/lon2 Screen
tMhR2-0987, PEG/lon2 Screen
tMhR2-09813, PEG/lon2 Screen
tMhR2-09819, Crystal Screen2
tMhR2-11248.
The present invention has following beneficial effect: crystallization method flow process provided by the present invention is simple, and the crystal resolving power obtaining is high, for studying three-dimensional structure and the function of this kind of protein, provides important condition.
Accompanying drawing explanation
Fig. 1 is the abduction delivering figure of high temperature resistant iron superoxide dismutase; M: Low molecular weight proteins standard; 1: only containing the recombinant bacterium of pET28a carrier, induce; 2: do not induce recombinant bacterium; 3; Recombinant bacterium after induction;
Fig. 2 is the electrophoretic analysis figure after resistance to high iron superoxide dismutase purifying; M: Low molecular weight proteins standard; 1: supernatant solution after the intestinal bacteria ultrasonication after abduction delivering; 2: the target protein after purifying;
Fig. 3 A is that the one-level of high temperature resistant iron superoxide dismutase is identified mass spectrum;
Fig. 3 B is that the secondary of high temperature resistant iron superoxide dismutase is identified mass spectrum;
Fig. 4 is the crystal pattern of high temperature resistant iron superoxide dismutase under visible ray;
Fig. 5 is the crystal pattern of high temperature resistant iron superoxide dismutase under UV-irradiation;
Fig. 6 is the X-ray diffractogram of high temperature resistant iron superoxide dismutase crystal.
Embodiment
Be noted that following illustrating is all exemplary, be intended to the invention provides further invention.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of conventionally understanding with the technical field of the invention personnel, and in this specification sheets, the experimental technique of unreceipted actual conditions is experimental technique routinely; In this specification sheets, test materials used is commercially available purchase product if no special instructions, and the composition of all ingredients and substratum and compound method can be referring to the operations in normal experiment handbook.
Below in conjunction with embodiment, elaborate particular content of the present invention.
Embodiment 1: the acquisition of high temperature resistant FeSOD gene
In order to remove the intron in gene, according to the sequence of the upper P.vivax SalI of NCBI (gene accession number: HM992934.1) two pairs of primers of design.
Upstream primer: 5 '-TTACATATGCGTGGGGCAAGCACGGA-3 '
Downstream primer: 5 '-GCGAATTCTTAAAACGGCTGCCAACG-3 '
Stearothermophilus ground bacillus (Geobacillus) genomic dna (Chinese Academy of Agricultural Sciences gives) of take is template, carries out pcr amplification, and specific procedure and reaction system are as follows:
Response procedures:
Reaction system:
Nde I for PCR product (Fermentas company) and two kinds of enzymes of EcoR I (Fermentas company) are carried out to double digestion, reclaim enzyme and cut after product.
Embodiment 2: the structure of recombinant expression vector pET-28a-FeSOD
By PCR product and pET-28a carrier while double digestion (Nde I, EcoRI), the fragment that enzyme is cut back to close connects with the efficient ligase enzyme of T4 DNA (Fermentas company), connect product and transform e. coli tg1 competent cell (purchased from Invitrogen company), picking list bacterium colony shakes after bacterium is cultivated and extracts recombinant expression vector pET-28a-FeSOD, utilize PCR and double digestion to identify, its base sequence is as shown in SEQ ID NO:1.
Embodiment 3: the abduction delivering of gene recombination engineering bacteria E.coli/pET-28a-FeSOD and the purifying of product
Embodiment 4: crystallization of protein
Restructuring target protein after embodiment 3 purifying is concentrated, with BCA method protein quantification test kit (purchased from BioTake company), measure restructuring target protein concentration, when concentration reaches 5-8mg/ml, with crystallization test kit (purchased from Hampton Research company), screen crystallization condition.1008 protein crystallization conditions altogether.Concrete steps are as follows:
1) aperture on 16 hole crystal growing plates is numbered to the condition of the corresponding protein reagent box screening of each numbering aperture;
2) to the crystallization damping fluid that adds successively different condition in the different test kits of 200 μ L in each hole on 16 hole crystal growing plates;
3) on the above-mentioned little bore edges that adds crystallization damping fluid, evenly fill a circle confining liquid (Vaseline);
4) with the special-purpose pipettor of crystallization, draw the protein sample of 1 μ L, then drop on silication cover glass, on each cover glass, put the protein sample of 2-3 different concns;
5) to the protein sample on above-mentioned cover glass, splash into the crystal damping fluid of 1 μ L, mixed, and carry out mark;
6) by above-mentioned, there is the cover glass turning vertical of protein sample and crystallization damping fluid to be placed on the corresponding aperture of 16 crystal pores culture plates;
7) with the lancet head of sterilizing, cover glass is pressed on gently to the edge of aperture, guarantees to seal between cover glass and aperture;
8) after etc. adding all protein examples and crystallization damping fluid, the plate of 16 orifice plates is covered, then albumin crystal culture plate is put in the crystal culturing room of 16 ℃ and cultivates;
9) whether examine under a microscope crystal every day and form, in the time of the 15 day, find that there is lenticular material and form, crystallization condition is Index
tMhR2-14461, Index
tMhR2-144 63, Index
tMhR2-144 87, Index
tMhR2-14488, Index
tMhR2-14494, PEG/lon2 Screen
tMhR2-0985, PEG/lon2 Screen
tMhR2-0987, PEG/lon2 Screen
tMhR2-09813, PEG/lon2 Screen
tMhR2-09819, Crystal Screen2
tMhR2-11248.
Embodiment 5: the evaluation of protein crystal
The albumin crystal that embodiment 4 is formed is observed under visible ray, see regular lenticular material (Fig. 4), then under the irradiation of UV-light, observe, protein crystal whiting light (Fig. 5), in order further to prove that formed crystal is crystallization of protein, this albumin crystal is pulled out with adsorbing bar, be put in RigakuX ray crystalline diffraction instrument and carry out diffraction.Result proves FeSOD albumin crystal, and X-ray diffraction resolving power is 2.5 (Fig. 6).
The above, be only the preferred embodiments of the present invention, should be understood that; for the those of ordinary skill in this technology; not departing under the prerequisite of core technology feature of the present invention, can also make some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.
SEQUENCE LISTING
<110> Tianjin Yaoyu Biotechnology Co., Ltd.
<120> high temperature resistant iron superoxide dismutase and crystallization method thereof
<130> 121255-I-CP-TJYU
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1236
<212> DNA
<213> FeSOD protein gene
<400> 1
atgcgtgggg caagcacgga tggcgcttcg tcgctgctcg agtttattgc cgagcatgac 60
ggcgagtgga cggaagaggc cgtccgcgag cttcagcgcc ttgccgatga cgtgtatgtc 120
ggtgcgcttc gccagtatgt catggaagcg gctgcgtggg gaagacaagt tgaacaggcg 180
ctgtctgcac gcaggatggc tgaggatgtt gggctttcgt ctttgttggc gtacatcgat 240
ggacacggcg atgaatggac cgaagaagcg atttacgagc tccagcgtct tgtcgatgac 300
gtatacactc gggccgtccg cctggctgat tcgtcagctg ccgatcggga agaaggggcg 360
acgcaagaac aagtcgaagg ggaatcggtg tcgcctgaac tggagagtga aaacaaagag 420
aacgaagacg gatggcttga caccagcggg acggcagagc gggttgagga cgcaaaagag 480
ccggctttta tggcggagct ctccgactcg ccgactgatg cagctgacgg cgagccggat 540
caagtggaca acgtgaccga tggaaagcga cgctgggtgg atgcggacga cggcggtgag 600
ccgcgtcaac aagtagcgcc cggccgtcat gtcttgccgc cgctgccgta ccgttatgat 660
gcgctcgagc cgcatatttc cgaagaaatc atgcgccttc accatacgaa gcatcatcaa 720
agctatgtcg acggtcttaa tcaggcggag cggatgatgg ccgaggcgcg ccggacgaac 780
aattttgatc tgttgaaaca ttgggagcgg gaagcggcgt tccacggctc cgggcattat 840
ttgcatacaa tcttttggta caacatgcat ccagagggcg gcggcgagcc gcgcggggag 900
ctgcgggcgc aaatcgagcg cgattttggc agttttaccg cctttcgccg tcatttcacc 960
gaagcggcga aaagcgccga aggggtcggc tgggcgctgc tcgtttgggc gccgcgagcg 1020
caccgtctgg aaattttgca ggcggaaaaa caccagctca tggcgcaatg ggatgtgatc 1080
ccgcttctcg tgcttgatgt gtgggagcat gcctactatt tgcaatacaa aaatgatcgg 1140
gcggcgtaca tcgaacattg gtggaacgtc gtcaactggc gcaacgttga agaacgcttt 1200
catgaggcgc agaagcttcg ttggcagccg ttttaa 1236
<210> 2
<211> 411
<212> PRT
<213> FeSOD albumen
<400> 2
Met Arg Gly Ala Ser Thr Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile
1 5 10 15
Ala Glu His Asp Gly Glu Trp Thr Glu Glu Ala Val Arg Glu Leu Gln
20 25 30
Arg Leu Ala Asp Asp Val Tyr Val Gly Ala Leu Arg Gln Tyr Val Met
35 40 45
Glu Ala Ala Ala Trp Gly Arg Gln Val Glu Gln Ala Leu Ser Ala Arg
50 55 60
Arg Met Ala Glu Asp Val Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp
65 70 75 80
Gly His Gly Asp Glu Trp Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg
85 90 95
Leu Val Asp Asp Val Tyr Thr Arg Ala Val Arg Leu Ala Asp Ser Ser
100 105 110
Ala Ala Asp Arg Glu Glu Gly Ala Thr Gln Glu Gln Val Glu Gly Glu
115 120 125
Ser Val Ser Pro Glu Leu Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly
130 135 140
Trp Leu Asp Thr Ser Gly Thr Ala Glu Arg Val Glu Asp Ala Lys Glu
145 150 155 160
Pro Ala Phe Met Ala Glu Leu Ser Asp Ser Pro Thr Asp Ala Ala Asp
165 170 175
Gly Glu Pro Asp Gln Val Asp Asn Val Thr Asp Gly Lys Arg Arg Trp
180 185 190
Val Asp Ala Asp Asp Gly Gly Glu Pro Arg Gln Gln Val Ala Pro Gly
195 200 205
Arg His Val Leu Pro Pro Leu Pro Tyr Arg Tyr Asp Ala Leu Glu Pro
210 215 220
His Ile Ser Glu Glu Ile Met Arg Leu His His Thr Lys His His Gln
225 230 235 240
Ser Tyr Val Asp Gly Leu Asn Gln Ala Glu Arg Met Met Ala Glu Ala
245 250 255
Arg Arg Thr Asn Asn Phe Asp Leu Leu Lys His Trp Glu Arg Glu Ala
260 265 270
Ala Phe His Gly Ser Gly His Tyr Leu His Thr Ile Phe Trp Tyr Asn
275 280 285
Met His Pro Glu Gly Gly Gly Glu Pro Arg Gly Glu Leu Arg Ala Gln
290 295 300
Ile Glu Arg Asp Phe Gly Ser Phe Thr Ala Phe Arg Arg His Phe Thr
305 310 315 320
Glu Ala Ala Lys Ser Ala Glu Gly Val Gly Trp Ala Leu Leu Val Trp
325 330 335
Ala Pro Arg Ala His Arg Leu Glu Ile Leu Gln Ala Glu Lys His Gln
340 345 350
Leu Met Ala Gln Trp Asp Val Ile Pro Leu Leu Val Leu Asp Val Trp
355 360 365
Glu His Ala Tyr Tyr Leu Gln Tyr Lys Asn Asp Arg Ala Ala Tyr Ile
370 375 380
Glu His Trp Trp Asn Val Val Asn Trp Arg Asn Val Glu Glu Arg Phe
385 390 395 400
His Glu Ala Gln Lys Leu Arg Trp Gln Pro Phe
405 410
Claims (1)
1. the crystallization method of a high temperature resistant iron superoxide dismutase, it is characterized in that, by gene recombination technology, build recombinant vectors pET-28a-FeSOD and import abduction delivering in e. coli bl21 (DE3) and obtain restructuring target protein, this restructuring target protein of the method purifying of recycling affinity chromatography and ion exchange chromatography, then by the crystallization condition of crystallization test kit screening restructuring target protein, can obtain the albumin crystal of high temperature resistant iron superoxide dismutase, wherein the aminoacid sequence of high temperature resistant iron superoxide dismutase is as shown in SEQ ID NO:2; Said method comprising the steps of:
(a) by design primer, stearothermophilus ground bacillus (Geobacillus) genomic dna of take is template, by pcr amplification method, obtains high temperature resistant FeSOD gene, and its base sequence is as shown in SEQ ID NO:1;
(b) by the high temperature resistant FeSOD gene of step (a) gained after Nde I and EcoR I double digestion, be connected on the pET28a of same processing carrier, build recombinant expression vector pET-28a-FeSOD, recombinant vectors pET-28a-FeSOD is imported to e. coli bl21 (DE3), obtain gene recombination engineering bacteria E.coli/pET-28a-FeSOD, it is 0.6 o'clock that recombination engineering bacteria is cultured to OD600 under 37 ℃, 220rpm, add inductor IPTG to final concentration be 1mM, continue to cultivate 3-5 hour, collect bacterium liquid, obtain a large amount of restructuring target proteins;
(c) restructuring target protein supernatant step (b) being obtained filters by 0.45 μ m cellulose membrane, then utilizes Ni post affinity chromatography and ion exchange chromatography purification of Recombinant target protein, obtains purity and reaches more than 95% restructuring target protein;
(d) just the restructuring target protein after step (c) purifying concentrates, with BCA method protein quantification test kit, measure restructuring target protein concentration, when concentration reaches 5-8mg/ml, with crystallization of protein test kit, screen crystallization condition, obtain albumin crystal, wherein said crystallization of protein test kit is purchased from Hampton Research company, and crystallization condition is Index
tMhR2-14461, Index
tMhR2-144 63, Index
tMhR2-144 87, Index
tMhR2-14488, Index
tMhR2-14494, PEG/lon2 Screen
tMhR2-0985, PEG/lon2 Screen
tMhR2-0987, PEG/lon2 Screen
tMhR2-09813, PEG/lon2 Screen
tMhR2-09819, Crystal Screen2
tMhR2-11248;
(e) albumin crystal that just step (d) forms, utilizes microscope and X ray crystalline diffraction instrument to identify.
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