CN100519580C - Angiogenin mutant as agonist, and its preparing method - Google Patents
Angiogenin mutant as agonist, and its preparing method Download PDFInfo
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- CN100519580C CN100519580C CNB2006100075215A CN200610007521A CN100519580C CN 100519580 C CN100519580 C CN 100519580C CN B2006100075215 A CNB2006100075215 A CN B2006100075215A CN 200610007521 A CN200610007521 A CN 200610007521A CN 100519580 C CN100519580 C CN 100519580C
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Abstract
The invention uses biology information to reconstruct Ang crystal structure, and construct the 3D structure, and construct the lacunae mutant that the N end lack 5 amino acid and the C end lack 12 amino acid, the reconstructed Ang mutant would be constructed by gene reconstructing and expression technology. The invention supplies a new method for curing tumor.
Description
Technical field
The invention belongs to structural molecular biology and biological anticancer technical field, particularly a kind of angiogenin mutant and preparation method as antagonist.
Background technology
1985, Fett JW etc. separated first from the human adenocarcinoma clone (HT-29) of serum-free culture and has identified a kind of protein factor, and it has the function of significant promotion angiogenesis, and with its called after angiogenin (Angiogenin, Ang).Find that at present many kinds of cells all can produce this protein factor, and almost in all tumour patients its expression level in various degree rising is all arranged.Generate element at the endotheliocyte medium vessels and can induce replying of second messenger, strengthen the adhesive power of these cells on the culture dish surface.Discover that angiogenin participates in morphological change and the angiogenesis in the ovary.In addition, it also has the function of some non-angiogenesis aspects, comprising: the flailing action that suppresses neutrophil leucocyte; Stimulate cholesterol esterification (this may be relevant with early atherosclerosis) by smooth muscle cell; In liver, have some uncertainty effects, may comprise common homeostasis etc.
Angiogenesis plays a crucial role in the generation of people and animal tumor and development, in view of angiogenin has the effect that intensive promotes vasculogenesis, simultaneously also because it has participated in multiple physiology and pathologic process, therefore its signal path is the hot issue that the various countries scholar pays close attention to always, and is at present still indeterminate about acceptor and the signal transduction pathway of Ang.But regardless of its acceptor situation with and signal path, the mutant of structural similitude and afunction all may be as the antagonist of wild-type Ang, the angiogenesis promoting function of blocking-up Ang.The key of this class mutant design is farthest to mask the function of the angiogenesis promoting of Ang, but it is structural stable to keep mutant molecule, can ensure itself and associated receptor or proteic the combination and aspect such as mutant molecule antigenicity stable like this, for the application of possible from now on clinicing aspect provides maximum potentiality.
Proteinic function depends on its space structure to a great extent.But, obviously can not satisfy the needs of structural molecular biology research far away because the existing limitation of various technology only relies on experimental technique and solves the protein steric structure problem.Theoretical prediction and structural simulation more and more reveal its importance and superiority.
Summary of the invention
The purpose of this invention is to provide a kind of angiogenin mutant and preparation method as antagonist, it is characterized in that: described angiogenin mutant nucleotides sequence as antagonist is classified as:
Tac?aca?cac?ttc?ctg?acc?cag?cac?tat?gat?gcc?aaa?cca?cag?ggc?cgg?gat?gac
aga?tac?tgt?gaa?agc?atc?atg?agg?aga?cgg?ggc?ctg?acc?tca?ccc?tgc?aaa?gac
atc?aac?aca?ttt?att?cat?ggc?aac?aag?cgc?agc?atc?aag?gcc?atc?tgt?gaa?aac
aag?aat?gga?aac?cct?cac?aga?gaa?aac?cta?aga?ata?agc?aag?tct?tct?ttc?cag
gtc?acc?act?tgc?aag?cta?cat?gga?ggt?tcc?ccc?tgg?cct?cca?tgc?cag?tac?cga
gcc?aca?gcg?ggg?ttc?aga?aac?gtt?gtt?gtt?gct?tgt?gaa?aat?ggc?tta?taa。
Its aminoacid sequence is as follows:
YTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGL。
The preparation method of described angiogenin mutant as antagonist:
1) Ang branch submodule is built
Utilizing the program ProMod II of SWISS MODEL and the Gromos96 field of force that the Ang crystalline structure has been carried out again mould builds.Guarantee that in computation process the main chain trend is fixing, at first utilize the steepest descent method to optimize for 200 steps, the parameter that adopts is TFP43B1, convergence criterion is: 2.5KJ/mol, and then utilize method of conjugate gradient to carry out further molecular mechanics optimization, the optimization step was generally for 300 steps, and convergence criterion is 2.5kJ/mol.Respectively with ribbon and backbone c atoms trend, secondary structure and three-dimensional structure are superimposed that theoretical model is analyzed.Secondary structure analysis to Ang shows, 6-13,23-33,50-55 amino acids form specific αLuo Xuanjiegou, 42-46,62-65,69-83,94-107,112-115 amino acids then form the βZhe Die structure, these zones are bigger to the integrally-built stability influence of Ang, and the secondary structure information that this and crystal structure determination obtain is in full accord.
2) analysis and the mould of ANG molecule N-terminal disappearance are built
In general, N-terminal is bigger for proteinic biological function influence.Sophisticated Ang albumen is single chain polypeptide, has the N-terminal that a sealing is arranged.According to the Ang molecular structure that mould is built, preceding 5 amino acid space structures comparatively stretch and do not form special secondary structure, participate in forming the α spiral since the 6th amino acid, so we have lacked 5 amino acid of N-terminal.The Ang molecular structure of building with mould is a template, the homology mould has been built 5 amino acid whose mutant molecule structures of N end disappearance, structure has been carried out comparative analysis after the mutant molecule structure of then mould being built and the optimization of wild-type, the backbone structure that found that the two is similar, space conformation does not change substantially, and the backbone c atoms root-mean-square displacement of the two is 0.023nm.
3) analysis and the mould of ANG molecule C-terminal disappearance are built
At first on the secondary structure of Ang, begin to participate in forming important secondary structure (βZhe Die) from the 13 amino acid of C-terminal, therefore our 12 amino acid that suddenlyd change, and the Ang molecular structure of building with the optimization mould is a template, in conjunction with the information biology theory, the homology mould has been built the three-dimensional structure of C-terminal deletion mutant.Mutant is except that the ribbon structure at C-terminal changes to some extent as can be seen, and backbone structure and main second structure characteristic do not have to change substantially.The backbone c atoms root-mean-square displacement (RMSD) of the molecular structure after C-terminal 12 amino acid whose mutant of disappearance and wild pattern are built is 0.028nm.
4) acquisition of deletion mutant gene and evaluation
According to the gene order of wild-type Ang, design mutant primer, promptly the N end lacks preceding 15 bases, back 36 bases of C end disappearance.With the wild type gene is template, obtains mutant gene by pcr amplification.
5) purifying of deletion mutant expression of gene and recombinant protein
With constructed expression vector pGEX-4T-2-AngM transformed into escherichia coli competent cell DH5 α, then through the expression of IPTG chemical induction in host bacterium BL21.
Chromatography column GSTrap FF is with the abundant balance of the PBS of pH7.3, with being stored in 4 ℃ thalline supernatant with sample on the speed of 0.5ml/min, washes post to baseline with PBS with the speed of 0.5ml/min afterwards.Chromatography column GSTrap FF is sealed, add zymoplasm (Thrombin) the 80 μ l prepared (zymoplasm is dissolved among 4 ℃ of PBS that are cooler than pH7.3 in advance by 1 μ g/ μ l), 4 ℃ of down effects 24 hours, the recombinant protein under having cut with the speed wash-out of 0.5ml/min with PBS afterwards.Continue to be eluted to baseline after reclaiming target protein, (be dissolved in 50mM Tris-CL, pH8.0) wash-out hangs over the 6ST on the pillar with the 15mM reduced glutathion then.Wash post with PBS again after wash-out finishes, then with 30% ethanol balance chromatography column GSTrap FF in order to usefulness again, 4 ℃ of preservations.
The invention has the beneficial effects as follows on the basis of Ang crystallographic structural analysis, utilize the information biology mould to build the three-dimensional structure of Ang, and under it instructs, made up 5 of Ang molecule N end disappearances and 12 amino acid whose deletion mutants of C end disappearance, and utilize gene recombination and expression technology to prepare the reorganization Ang mutant that makes up.By relevant cell system and the experiment of chicken embryo the activity of its short vasculogenesis is analyzed, the result shows: constructed Ang mutant is compared with wild-type has identical antigenicity, but can not promote huve cell, ECV304 cell proliferation, not promote the function of chick chorioallantoic membrane vasculogenesis simultaneously.The biological function that is configured to antagonism Ang of this mutant and relevant Ang afunction mutant thereof, and the biotherapy strategy of the tumor tissues angiogenesis by blocking-up Ang mediation is laid a good foundation.For clinical from now on be that the oncotherapy of means provides new method with the angiogenesis inhibitor.
Description of drawings
The stable Ang tomograph that Fig. 1 builds for mould and the optimization of process molecular mechanics obtains.
Fig. 2 builds optimum result for the mould of Ang N-terminal deletion mutant.
Fig. 3 is the three-dimensional structure superimposition figure of Ang and its N-terminal mutant.
Fig. 4 lacks the tomograph of 12 amino acid mutation bodies for C-terminal.
Fig. 5 is the superimposition figure of C-terminal deletion mutant and Ang space conformation.
Fig. 6 is the electrophoretogram analysis of RT-PCR method amplification Ang mutant gene.
Fig. 7 is the nucleotide sequence analysis of the Ang mutant gene that obtains figure as a result.
Fig. 8 is that the double digestion of recombinant expression plasmid pGEX-4T-2-AngM is identified collection of illustrative plates.
Fig. 9 is the expression of results figure of SDS-PAGE electrophoretic analysis Ang mutant in DH5 α.
Figure 10 is the influence of Ang mutant to the chick chorioallantoic membrane angiogenic action.
Embodiment
The invention provides a kind of angiogenin mutant and preparation method as antagonist.
1. experiment material and plant and instrument
1.1 bacterial strain and expression vector
Bacterial strain E.coliDH5 α (Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences) preserves, and pT-Ang plasmid (being Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences) is preserved, and expression vector pGEX-4T-2 is available from Amershampharmacia company.
1.2 enzyme and biochemical reagents
Restriction enzyme BamH I, EcoR I, Sal I, Nco I, Taq archaeal dna polymerase and T
4Dna ligase is available from Promega company; Nucleic acid molecular weight standard DL2000 is available from TaKaRa company; The molecular weight of albumen standard is available from through bio-engineering corporation of section; Affinity chromatographic column GSTrap FF and zymoplasm (Thrombin) are available from Amersham pharmacia company; Reduced glutathion (GSH), M450 methylcellulose gum are available from Sigma company.The DNA purification kit is available from vast biotech company; Plasmid extraction kit is a Promega company product.
1.3 test apparatus
The protein electrophoresis instrument is a bio-rad company product, and the protein purification instrument is a pharmacia company, SGI/Indigo
2(R4400) graphics workstation, Insight II (95.5) protein analysis software, Swiss pdb viewer 3.0, PII-350 computer, Internet Explorer 5.0, server: SWISS MODEL.
2. preparation method
2.1 the branch submodule is built process
The X ray diffractive crystal structure of angiogenin obtains, but owing in the crystal structure determination process, only obtained the atomic coordinate of heavy atom, and can't resolve for the atomic structure of hydrogen atom and side chain thereof.We utilize the program ProMod II of SWISS MODEL and the Gromos96 field of force that crystalline structure has been carried out again mould and build.Guarantee that in computation process the main chain trend is fixing, at first utilize the steepest descent method to optimize for 200 steps, the parameter that adopts is TFP43B1, convergence criterion is: 2.5KJ/mol, and then utilize method of conjugate gradient to carry out further molecular mechanics optimization, the optimization step was generally for 300 steps, and convergence criterion is 2.5kJ/mol.Fig. 1 has provided the stable theory model that obtains through molecular mechanics optimization, is the matching of further inquiring into theoretical model and experimental result, respectively with ribbon and backbone c atoms trend, secondary structure and three-dimensional structure are superimposed that theoretical model is analyzed.Secondary structure analysis to Ang shows, 6-13,23-33,50-55 amino acids form specific a spirane structure, 42-46,62-65,69-83,94-107,112-115 amino acids then form the βZhe Die structure, these zones are bigger to the integrally-built stability influence of Ang, and the secondary structure information that this and crystal structure determination obtain is in full accord.
2.2 the mould of ANG molecule N-terminal disappearance is built the result
In general, N-terminal is bigger for proteinic biological function influence.Sophisticated angiopoietin-like 4 protein is a single chain polypeptide, has the N-terminal that a sealing is arranged.According to the Ang molecular structure that mould is built, preceding 5 amino acid space structures comparatively stretch and do not form special secondary structure, participate in forming the α spiral since the 6th amino acid, so we have lacked 5 amino acid of N-terminal.The Ang molecular structure of building with mould is a template, the homology mould has been built N end disappearance 5 amino acid whose mutant molecule structures (Fig. 2), structure has been carried out comparative analysis after the mutant molecule structure of then mould being built and the optimization of wild-type, the backbone structure that found that the two is similar, space conformation does not change substantially, and the backbone c atoms root-mean-square displacement of the two is 0.023nm (Fig. 3).
2.3 the mould of ANG molecule C-terminal disappearance is built the result
At first on the secondary structure of Ang, begin to participate in forming important secondary structure (βZhe Die) from the 13 amino acid of C-terminal, therefore our 12 amino acid that suddenlyd change, and the Ang molecular structure of building with the optimization mould is a template, in conjunction with the information biology theory, the homology mould has been built the three-dimensional structure of C-terminal deletion mutant.Fig. 4 builds for the mould of 12 amino acid mutation bodies of C-terminal disappearance and optimizes the back molecular structure, and mutant is except that the ribbon structure at C-terminal changes to some extent as can be seen, and backbone structure and main second structure characteristic do not have to change substantially; Fig. 5 is 12 amino acid whose mutant of C-terminal disappearance and the wild pattern superimposed figure of molecular structure after building, and the backbone c atoms root-mean-square displacement (RMSD) of the two is 0.028nm.
2.4 the acquisition of deletion mutant gene and evaluation
According to the gene order of wild-type Ang, design mutant primer, promptly the N end lacks preceding 15 bases, back 36 bases of C end disappearance.With the wild type gene is template, obtains mutant gene (Fig. 6) by pcr amplification.
2.5 the purifying of deletion mutant expression of gene and recombinant protein
With constructed expression vector pGEX-4T-2-AngM (Fig. 8) transformed into escherichia coli competent cell DH5 α, then through the expression (Fig. 9) of IPTG chemical induction in host bacterium BL21.
Chromatography column GSTrap FF is with the abundant balance of PBS (pH7.3), with being stored in 4 ℃ thalline supernatant with sample on the speed of 0.5ml/min, washes post to baseline with PBS with the speed of 0.5ml/min afterwards.Chromatography column GSTrap FF is sealed, add the zymoplasm 80 μ l (zymoplasm is dissolved in 4 ℃ of precooling PBS pH7.3 by 1 μ g/ul) that prepared, 4 ℃ act on 24 hours, recombinant protein under having cut with the speed wash-out of 0.5ml/min with PBS afterwards, continue to be eluted to baseline after reclaiming target protein, hang over GST on the pillar with the 50mMTris-CL damping fluid preparation 15mM reduced glutathion wash-out of pH8.0 then.Wash post with PBS again after wash-out finishes, use 30% ethanol balance chromatography column GSTrap FF then, thereby construct reorganization Ang mutant (Fig. 7), preserve down at 4 ℃.Its nucleotides sequence is classified as: described angiogenin mutant nucleotides sequence as antagonist is classified as:
Tac?aca?cac?ttc?ctg?acc?cag?cac?tat?gat?gcc?aaa?cca?cag?ggc?cgg?gat?gac
aga?tac?tgt?gaa?agc?atc?atg?agg?aga?cgg?ggc?ctg?acc?tca?ccc?tgc?aaa?gac
atc?aac?aca?ttt?att?cat?ggc?aac?aag?cgc?agc?atc?aag?gcc?atc?tgt?gaa?aac
aag?aat?gga?aac?cct?cac?aga?gaa?aac?cta?aga?ata?agc?aag?tct?tct?ttc?cag
gtc?acc?act?tgc?aag?cta?cat?gga?ggt?tcc?ccc?tgg?cct?cca?tgc?cag?tac?cga
gcc?aca?gcg?ggg?ttc?aga?aac?gtt?gtt?gtt?gct?tgt?gaa?aat?ggc?tta?taa。
Its aminoacid sequence is as follows:
YTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGL。
2.6 reorganization Ang mutant protein is to the influence of ECV304 cell growth
The culture condition of ECV304 is: the DMEM substratum, and 10% calf serum, two anti-, 5% CO
2The ECV304 cell that cultivation conditions is good digests and counts, and inoculates 96 porocyte culture plates with the amount of 500,000/ml, every hole 100ul.Add the reorganization Ang mutant protein of preliminary purification with different concentration, and set up negative control group (DMED+PBS) and positive controls (DMEM+Ang).Measure the growing state of every porocyte with mtt assay.
The detected result of mtt assay shows that reorganization Ang mutant protein does not have significantly short proliferation function (table 1) to human umbilical vein clone ECV304.
Table 1.MTT method is measured the influence of Ang mutant to ECV304 cell proliferation
| Group | Concentration (ng/ml) | Sample number | The A570 value (X ± S) |
| Negative control group (PBS) | 0 | 12 | 0.57±0.00 |
| Positive controls (standard substance Ang) | 2 | 12 | 0.67±0.02 |
| Reorganization Ang mutant | 4 | 12 | 0.58±0.02 |
Annotate: reorganization Ang mutant group is compared p with the PBS group〉0.05.
2.7 reorganization Ang mutant protein is to the effect of chick chorioallantoic membrane vasculogenesis
Add sample after 3 days, add methyl alcohol, acetone balanced mix stationary liquid to the place of windowing, at room temperature fix 15 minutes, after treating that vessel inner blood on the chorioallantoic membrane solidifies, with the saucer is that film is cut at the center, puts in the plate that fills water and launches, and is attached on the filter paper take pictures (Figure 10), numeration is also carried out statistical study, and the result shows the effect (table 2) that reorganization Ang has significantly short chick chorioallantoic membrane vasculogenesis that obtains.
Table 2. reorganization Ang mutant is to the influence of chick chorioallantoic membrane blood vessel chorioallantoic membrane
| Group (μ g/ml) | Samples contg one progression | Embryo number/number of times two progression | Several 7 embryos of new vessel (X ± S) |
| Negative control group (PBS) | 0ng/ml | 8/2 | 7.38±2.2526.31±3.24 |
| Positive controls (wild-type Ang) | 5ng/ml | 8/2 | 36.50±3.3558.88±5.30 |
| Reorganization ANG mutant | 10ng/ml | 8/2 | 8.45±4.2228.12±3.11 |
Annotate: reorganization Ang mutant group is compared p with negative control group〉0.05, there was no significant difference.
2.8 the western blotting of reorganization Ang mutant identifies
Ang mutant protein electrophoresis result is carried out Immunological Identification after utilizing anti-people Ang polyclonal antibody to recombinant expressed and cutting purifying, the result shows, in relative molecular mass 14400 positions, specific hybridization band (Fig. 6) appears in the Ang mutant of expression and corresponding antibodies reaction.
Claims (4)
1. angiogenin mutant as antagonist, it is characterized in that: described angiogenin mutant aminoacid sequence as antagonist is as follows:
YTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKS
SFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGL。
2. angiogenin mutant as antagonist, it is characterized in that: described angiogenin mutant nucleotides sequence as antagonist is classified as:
Tac?aca?cac?ttc?ctg?acc?cag?cac?tat?gat?gcc?aaa?cca?cag?ggc?cgg?gat?gac
aga?tac?tgt?gaa?agc?atc?atg?agg?aga?cgg?ggc?ctg?acc?tca?ccc?tgc?aaa?gac
atc?aac?aca?ttt?att?cat?ggc?aac?aag?cgc?agc?atc?aag?gcc?atc?tgt?gaa?aac
aag?aat?gga?aac?cct?cac?aga?gaa?aac?cta?aga?ata?agc?aag?tct?tct?ttc?cag
gtc?acc?act?tgc?aag?cta?cat?gga?ggt?tcc?ccc?tgg?cct?cca?tgc?cag?tac?cga
gcc?aca?gcg?ggg?ttc?aga?aac?gtt?gtt?gtt?gct?tgt?gaa?aat?ggc?tta?taa。
3. the described angiogenin mutant as antagonist of claim 1 is characterized in that: described angiogenin mutant is compared 5 amino acid of N end disappearance with wild-type, 12 amino acid of C-terminal disappearance.
4. the preparation method of the described angiogenin mutant as antagonist of a claim 1 is characterized in that concrete preparation method is as follows:
(1) preparation of experiment material and plant and instrument
1.1 bacterial strain and expression vector
Bacterial strain E.coliDH5 α and pT-Ang plasmid are Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences, and preserve, and expression vector pGEX-4T-2 is available from Amersham pharmacia company;
1.2 enzyme and biochemical reagents
Restriction enzyme BamH I, EcoR I, Sal I, Nco I, Taq archaeal dna polymerase and T
4Dna ligase is available from Promega company; Nucleic acid molecular weight standard DL2000 is available from TaKaRa company; The molecular weight of albumen standard is available from through bio-engineering corporation of section; Affinity chromatographic column GSTrap FF and zymoplasm are available from Amershampharmacia company; Reduced glutathion, M450 methylcellulose gum are available from Sigma company, and the DNA purification kit is available from vast biotech company; Plasmid extraction kit is a Promega company product;
1.3 test apparatus
The protein electrophoresis instrument is a bio-rad company product, and the protein purification instrument is a pharmacia company, SGI/Indigo
2(R4400) graphics workstation, Insight II (95.5) protein analysis software, Swiss pdb viewer 3.0, PII-350 computer, Internet Explorer 5.0, server: SWISS MODEL;
(2) operating process
1) analysis and the mould of ANG molecule N-terminal disappearance are built
ANG molecule N-terminal is bigger for proteinic biological function influence, sophisticated Ang albumen is single chain polypeptide, has the N-terminal that a sealing is arranged, the Ang molecular structure of building according to mould, preceding 5 amino acid space structures comparatively stretch and do not form special secondary structure, participate in forming the α spiral since the 6th amino acid, so we have lacked 5 amino acid of N-terminal; The Ang molecular structure of building with mould is a template, the homology mould has been built 5 amino acid whose mutant molecule structures of N end disappearance, structure has been carried out comparative analysis after the mutant molecule structure of then mould being built and the optimization of wild-type, the backbone structure that found that the two is similar, space conformation does not change substantially, and the backbone c atoms root-mean-square displacement of the two is 0.023nm;
2) analysis and the mould of ANG molecule C-terminal disappearance are built
At first on the secondary structure of Ang, begin to participate in forming the secondary structure of βZhe Die from the 13 amino acid of C-terminal, therefore our 12 amino acid that suddenlyd change, and the Ang molecular structure of building with the optimization mould is a template, in conjunction with the information biology theory, the homology mould has been built the three-dimensional structure of C-terminal deletion mutant, and mutant is except that the ribbon structure at C-terminal changes to some extent as can be seen, and backbone structure and main second structure characteristic do not have to change substantially; The backbone c atoms root-mean-square displacement of the molecular structure after C-terminal 12 amino acid whose mutant of disappearance and wild pattern are built is 0.028nm;
3) acquisition of deletion mutant gene and evaluation
According to the gene order of wild-type Ang, design mutant primer, promptly the N end lacks preceding 15 bases, and back 36 bases of C end disappearance are template with the wild type gene, obtain mutant gene by pcr amplification;
4) purifying of deletion mutant expression of gene and recombinant protein
With constructed expression vector pGEX-4T-2-AngM transformed into escherichia coli competent cell DH5 α, then through the expression of IPTG chemical induction in host bacterium BL21; Chromatography column GSTrap FF is with the abundant balance of the PBS of pH7.3, to be stored in 4 ℃ thalline supernatant with sample on the speed of 0.5ml/min, wash post to baseline with PBS with the speed of 0.5ml/min afterwards, chromatography column GSTrap FF is sealed, add the zymoplasm 80 μ l that prepared, wherein zymoplasm is dissolved among 4 ℃ of PBS that are cooler than pH7.3 in advance by 1 μ g/ μ l, 4 ℃ of effects 24 hours down, and the recombinant protein under having cut with the speed wash-out of 0.5ml/min with PBS afterwards; Continue to be eluted to baseline after reclaiming target protein, prepare the 15mM reduced glutathion with the 50mM Tris-CL damping fluid of pH8.0 then, wash-out hangs over the GST on the pillar, after finishing, wash-out washes post with PBS again, use 30% ethanol balance chromatography column GSTrap FF then, thereby construct reorganization Ang mutant; Preserve down at 4 ℃.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4966849A (en) * | 1985-09-20 | 1990-10-30 | President And Fellows Of Harvard College | CDNA and genes for human angiogenin (angiogenesis factor) and methods of expression |
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| US4966849A (en) * | 1985-09-20 | 1990-10-30 | President And Fellows Of Harvard College | CDNA and genes for human angiogenin (angiogenesis factor) and methods of expression |
Non-Patent Citations (4)
| Title |
|---|
| Amino acid sequence of human tumor derived angiogenin. Daniel J.Strydom et al.Biochemistry,Vol.24 . 1985 |
| Amino acid sequence of human tumor derived angiogenin. Daniel J.Strydom et al.Biochemistry,Vol.24 . 1985 * |
| Crystallographic studies on the role of the C-terminal segmentof human angiogenin in defining enzymatic potency. Demetres D.Leonidas et al.Biochemistry,Vol.41 . 2002 |
| Crystallographic studies on the role of the C-terminal segmentof human angiogenin in defining enzymatic potency. Demetres D.Leonidas et al.Biochemistry,Vol.41 . 2002 * |
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