CN101629181B - Preparation method of recombinant Fas-associated death domain protein and application thereof - Google Patents
Preparation method of recombinant Fas-associated death domain protein and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, in particular to a preparation method of full-length Fas-associated death domain (FADD) protein, which utilizes a recombinant DNA technique to express and prepare the full-length FADD protein which has good stability, high yield, the same high-level structure as a wild FADD and activity for inducing cell apoptosis, and the preparation method has more convenient and faster separation and purification process and high yield. The full-length FADD protein can be applied to prepare a medicament for inducing the cell apoptosis and treat diseases such as tumors, rheumatoid arthritis, and the like. The invention also provides an expression method for expressing the full-length FADD protein in a prokaryotic expression system, which can effectively improve the yield of the full-length FADD protein.
Description
One, technical field:
The invention belongs to the genetically engineered biological technical field.
Two, background technology:
FADD (Fas-associated death domain protein; The Fas associated death domain protein) be two research groups such as Chinnaiyan, Boldin utilize yeast two-hybrid system in nineteen ninety-five screen simultaneously obtain one can with the albumen of death receptor Fas cytoplasmic domain specific effect; Because it comprises one and the proteinic death domain height of Fas homologous structural domain; Thereby be named as Fas associated death domain protein (Fas-associated death domain protein, FADD) [Boldin, people such as M.P.; 1995, J.Biol.Chem.270:7795-7798; Chinnaiyan, people such as A.M., 1995, Cell 81:505-512].208 amino acid of people FADD genes encoding; Molecular weight is 23kDa; Can be divided into three functional areas, comprise that the N relevant with apoptosis holds about 76 amino acid whose DED (death effect domain, Death Effector Domain) and 70 amino acid whose DD (death domain of C end; And about 20 the amino acid whose C-terminal fragments that in non-apoptosis, play a role death domain).As intracellular joint albumen; FADD can mediate multiple death receptor inductive apoptosis pathway; Comprise Fas, TNFR1, DR3, DR4, DR5 and p75NTR etc., generation that can be apoptosis-induced in various kinds of cell system is the apoptosis signaling molecule of classics.In death receptor inductive apoptosis pathway, dead signal (like Fas L) combines, causes the interior death domain of death receptor (like Fas) born of the same parents to form tripolymer with death receptor (like Fas), thereby recruits FADD protein through the death domain (DD) that combines FADD; And cause the FADD conformational change; Make its DED multimerization, and then recruit Gelucystine proteinase 8 (caspase 8) precursor and gather, form dead inducement signal complex body (death-inducing signaling complex; DISC); Through producing activated caspase 8, thereby excite a series of downstream caspase cascade reaction, bring out apoptosis.This apoptosis induction signal path by the FADD mediation can be expressed as simply: death receptor-FADD--caspase-8 approach; This also is to study a most thorough paths [Liu Daqing etc. at present; 2002, foreign medical science molecular biology fascicle, 24 (5): 260-263].
Big quantity research shows: the incidence and development of numerous disease (the especially generation of tumour) is relevant unusually with apoptosis.It is generally acknowledged: tumour cell is the cell hyperproliferation that causes because growth is out of control; See from apoptotic angle, think that then the apoptosis mechanism of tumour is suppressed, the result that tumour cell can not apoptosis normally take place and be eliminated.FADD plays a part very important [Zhao Dan, 2006, international Journal of Immunology 29:152-156 as the connection albumen of apoptotic signal approach in the incidence and development process of tumour; Tourneur, people such as L., 2003, Oncogene 22:2795-2804].The disappearance that FADD expresses and the generation of liver cancer are closely related; The expression rate of FADD in cancerous tissue significantly is lower than the other and non-cancer hepatic tissue of cancer, and its expression level is reduced [Shin, people such as E.C. with the increase of liver cancer grade malignancy; 1998, Cancer Lett.134:155-162; Sun, people such as B., 2000, Zhong Hua Gan Zang Bing Za Zhi 8:37-39].FADD protein expression intensity and HCC apoptosis are closely related, and the FADD expressing quantity is high more, its apoptosis degree also higher relatively [people such as Lu Dongdong, 2002, clinical liver and bladder disease magazine, 18:375-376].Find that in many patients with renal cell carcinoma bodies the FADD expression amount in the cancer cells obviously reduces [Xu, people such as H., 2009, Cancer Invest, the network edition: 2009Jun 25:1].FADD abnormal expression in nonsmall-cell lung cancer, also can play an important role [Shin, people such as M.S., 2002, Oncogene 21:4129-4136].
To linking and the function served as bridge of FADD in apoptosis with and with the relation of multiple disease, existing scholar begins to attempt to utilize the biological nature that FADD can mediating apoptosis to carry out treatment of diseases.Discover that FADD crosses the apoptosis that expression can promote pernicious cerebral glioma cell.Get into tumour cell through transient transfection FADD gene, 85% glioma cell generation apoptosis, and also this process does not need the participation [Kondo, people such as S., 1998, Human Gene Therapy 9:1599-1608] of upper reaches Fas.Utilization contains the retroviral vector stable transfection tumour cell of FADD gene, in vivo with the external survival that can both suppress tumour cell effectively.Other there are some researches show that the FADD expression of gene can induce the apoptosis of mucoepidermoid carcinoma cell [people such as Liu Daqing, 2002, Oral and Maxillofacial Surgery magazine, 12:317-320] effectively.Find that in addition expressing excessively of FADD can strengthen the susceptibility of tumour cell to drug-induced necrocytosis and CDCC, reaches better therapeutic purpose [Mishima, people such as K., 2003, Int.J.Cancer 105:593-600; Micheau, people such as O., 1999, J.Biol.Chem.274:7987-7992; Yin, people such as A., the Med.Oncol. network edition: 2009 May 5].
On this basis, have research group utilize people's telomere reversed transcriptive enzyme (hTERT) feature development a kind of new strategies in gene therapy: with the specifically expressing of hTERT gene promoter regulation and control FADD in tumour cell, be built into the hTERT/FADD expression vector; Be transfected in the tumour cell; Discovery is expressed in the male tumour cell at hTERT, and FADD also presents positive expression, and the generation of inducing apoptosis of tumour cell effectively; HTERT is expressed negative normal cell (cell of FADD high expression level does not promptly take place) then do not have effect [Komata; T. wait people, 2001, Int.J.Oncol.19:1015-1020; Koga, people such as S., 2001, Anticancer Res.21:1937-1943].
FADD also has application promise in clinical practice except important application prospects is arranged on other treatment of diseases in oncotherapy.Rheumatoid arthritis is a kind of non-organ specificity autoimmune disorder.Research shows that the apoptosis of Fas mediation is the one of the main reasons of rheumatoid arthritis patients synovium of joint cell degeneration; The signal transduction path of the FADD--caspase-8 [Kobayashi that in synovial cell's apoptotic process of rheumatoid arthritis patient, plays a significant role wherein; T. wait the people; 1999, Curr.Opin.Rheumatol.11:188-193].After utilizing Adenovirus Transfection to change the FADD gene synovial cell of rheumatoid arthritis over to, can cause the apoptosis that causes the synovial cell of pathology because of hyper-proliferative.In addition, in the rheumatoid arthritis model mice of immunodeficient, can remove the synovial cell of pathology behind local injection adenovirus-FADD (Ad-FADD), and this effect is confined to synovial tissue, and do not cause contiguous articular chondrocyte apoptosis.Thereby FADD crosses expression and can treat rheumatoid arthritis [Kobayashi, people such as T., 2000, Gene Therapy 7:527-533] effectively.
At present to be applied to other treatment of diseases such as tumour and rheumatoid arthritis mainly be through the FADD gene is advanced cell (being gene therapy methods) through transfection reagent or virus vector transfection to FADD, realizes the crossing expression of FADD and reach the purpose of disease treatment.As in the above-mentioned document the conventional method that adopts, gene therapy mainly mediates through virus vector at present.Because the spinoff that various shortcoming that virus vector exists and gene therapy show in clinical experiment, up to now, gene therapy still can't be used clinical being able to, and still awaits further improving.Since gene therapy in essence through therapeutic gene being changed in the body after genetic transcription and translate into protein and realize result of treatment directly gets into the purpose that just can reach treatment in the disease cell (like tumour or rheumatoid arthritis cell) with the transfection of FADD protein.See that technically advanced at present technology such as the liposome parcel pharmaceutical grade protein (for example FADD) of utilization carries out the vivo medicine-feeding treatment, the target administration of particular pathologies tissue (like tumour or rheumatoid arthritis cell) is feasible fully even.
Why do not use FADD protein to carry out the research report of disease treatment as medicine so far? This is very difficult because of total length FADD protein expression with complete biological function and purifying.So up to now, as the key joints molecule in the apoptosis pathway, FADD has had a plurality of structures to be resolved; Comprising hFADD-DED (1-83 amino acid) [Eberstadt, people such as M., 1998, Nature392:941-945], mFADD-DD (89-183 amino acid) [Jeong; E.J. wait people, 1999, J.Biol.Chem.274:16337-16342], hFADD-DD (93-192 amino acid) [Berglund; H. wait people, 2000, J.Mol.Biol.302:171-188], hFADD (1-191 amino acid) [Carrington; P.E. wait the people, 2006, Mol.Cell 22:599-610].Can see that above FADD is the fragment of FADD, the FADD of the 1-191 amino acids residue of wherein expressing with people such as Carrington P.E. in 2006 is for the longest, but it has still lacked 17 amino-acid residues of 192-208 position.Although in above-mentioned research; Scientists has been attempted the FADD protein that the whole bag of tricks just obtains above-mentioned various brachymemmas and has been used for partial function and structural research; Have no the laboratory can great expression and obtain the FADD protein of total length at present; The reason of searching to the bottom is that FADD protein is unstable; Be very easy to gathering,, also can't separation and purification obtain the FADD protein confession research of q.s even treat to need to use even therefore utilize genetic engineering technique to obtain the FADD protein expression of higher level.Therefore, obtain to have complete biological function, proteinic to efficiently express and prepare be that FADD further is applied to experimental study and moves towards clinical key and bottleneck as medicine to total length FADD.
Three, summary of the invention:
For overcoming the defective of present existing reorganization FADD technology of preparing; The object of the invention is to provide a kind of total length FADD proteinic preparation method; That mass production prepares is solubility, total length, have complete bioactive FADD protein, and being used to prepare can be through the medicine of apoptosis treatment such as diseases such as tumour or rheumatoid arthritis.
For achieving the above object; Technical scheme of the present invention is the recombinant mutant FADD (F25Y) that makes up a total length FADD; The Phe of the 25th of FADD is mutated into Tyr, and this total length FADD productive rate of gene engineering expression and separation and purification in prokaryotic expression system and eukaryotic expression system is higher than wild-type FADD far away.
Further, above-mentioned total length FADD protein can with small peptide label coexpression together, the small peptide label can be placed on the proteinic N of above-mentioned FADD end or C end.The small peptide label can be so that above-mentioned FADD protein can carry out purifying through affinity chromatography, make the separation and purification process convenient more, quick.
Further, above-mentioned recombinant full-lenght FADD protein has better stability than wild-type FADD in expression and purge process, and output is higher.
Further, a kind of (10-35 ℃) at low temperatures expressed above-mentioned recombinant full-lenght FADD method of protein.
Further, above-mentioned total length FADD protein and wild-type FADD have identical higher structure.
Further, above-mentioned total length FADD protein has recruitment and combines the BA of the caspase 8 in downstream, thereby has the function of cell death inducing.
Further; Because above-mentioned total length FADD protein has the function of cell death inducing; Can prepare medicine through methods such as technology such as liposome parcel FADD protein with above-mentioned total length FADD protein as medicine, carry out the application of above-mentioned total length FADD protein in disease treatment.
Compare with existing FADD protein technology of preparing, characteristic of the present invention is with the innovation part:
(1) realized the efficiently expressing and separation and purification of total length FADD of solubility first;
(2) through Fixedpoint mutation modified (25 Phe are mutated into Tyr) to FADD; Both increased proteic stability; Improve proteic output greatly; Guaranteed that again above-mentioned total length FADD higher structure does not change, still kept through recruiting the biological function of caspase 8 cell death inducings.
Four, description of drawings
The expression and purification of Fig. 1 .His-FADD wild-type protein and His-FADD (F25Y) full length protein.
1A.His-FADD the SDS-PAGE of wild-type protein expression and purification analyzes.
The bacterium bacterial protein of 1:IPTG abduction delivering; 2: the centrifugation part after the ultrasonication; 3: the centrifugal supernatant after the ultrasonication; 4: the metallic nickel affinity chromatography pass the peak; 5: the 50mM imidazoles elution peak of metallic nickel affinity chromatography; 6: the 250mM imidazoles elution peak of metallic nickel affinity chromatography; 7, the 8:G15 gel permeation chromatography passes the peak; 97,67,43,31 9: molecular weight standard article (be respectively from top to bottom:, 14kDa); The 10:DEAE chromatography passes the peak; The 0.4M NaCl elution peak of 11:DEAE chromatography.
1B.His-FADD (F25Y) SDS-PAGE of full length protein purifying expression and purification analyzes.
The bacterium bacterial protein of 1:IPTG abduction delivering; 2: the centrifugation part after the ultrasonication; 3: the centrifugal supernatant after the ultrasonication; 4: the metallic nickel affinity chromatography pass the peak; 5: the 50mM imidazoles elution peak of metallic nickel affinity chromatography; 6: the 250mM imidazoles elution peak of metallic nickel affinity chromatography; The 7:G15 gel permeation chromatography passes the peak; 116,66,45,35,25,18 8: molecular weight standard article (be respectively from top to bottom:, 14kDa).
The spectroscopic analysis of Fig. 2 .His-FADD wild-type protein and His-FADD (F25Y) full length protein.
2A. the circular dichroism spectrum analysis of extreme ultraviolet; 2B.280nm the fluorescence emission spectrum analysis under the exciting light;
2C.295nm the fluorescence emission spectrum analysis under the exciting light.
Fig. 3 .His-FADD (F25Y) full length protein and caspase 8 interaction performance analysiss.
3A. immunoblotting assay; 3B.SDS-PAGE analyze.
1: the 293T lysis supernatant of overexpression FLAG-caspase-8; 2: utilize His-GST to carry out the experimental result of pull-down as blank; 3: utilize His-FADD (F25Y) full length protein to carry out the experimental result of pull-down.
Fig. 4. the immuning fluorescent dyeing analysis of FADD wild-type protein of in eukaryotic cell, expressing and FADD (F25Y) full length protein.
4A.FADD wild-type protein; 4B.FADD (F25Y) full length protein.
Five, embodiment
Embodiment 1:
1.His-FADD wild-type protein construction of prokaryotic expression vector:
According to the gene order of FADD, (upstream primer is chemosynthesis upstream and downstream primer: 5 '-CATGCCATGGGCAGCAGCCATCATCATCATCATCACGGCATGGACCCATTCCTGGT G-3 '; Downstream draw for: 5 '-CGCGAAGCTTTCAGGGTGTTTCTGAGG-3 '), the plasmid pGEX2T-FADD that made up in the past with this laboratory is a template, and increasing with the method for PCR obtains the encoding sequence of total length wild-type FADD.The condition of PCR reaction is: 94 ℃ of thermally denatures 1 minute, and 60 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, and carried out 25 circulating reactions altogether.The PCR product carries out Nco I/Hind III double digestion after agarose gel electrophoresis reclaims; Plasmid pET28a (Novagen company) also carries out Nco I/Hind III double digestion; Using the T4DNA ligase enzyme that two double digestions are reclaimed fragment connects; Connect product transformed into escherichia coli Top10 bacterial strain competent cell; With digestion with restriction enzyme and PCR method screening positive clone, obtain plasmid pET28a-His-FADD, it reads the exactness of frame and encoding sequence through the checking of dna sequence dna sequencing analysis.In the pET28a-His-FADD plasmid, utilize upstream primer histidine-tagged, thereby the recombinant protein of expressing is the His-FADD wild-type protein 6 of the N of FADD end introducings.
2.His-FADD (F25Y) full length protein construction of prokaryotic expression vector
His-FADD (F25Y) full length protein rite-directed mutagenesis is realized through two-step pcr.The mutant primer 5 '-CTCGCGGCACAAGTACTTGAGCTCCATCAG-3 ' that at first uses the upstream primer in above-mentioned (1) and introduce point mutation (F25Y) is that template amplification goes out upper reaches fragment with the pET28a-His-FADD that obtains in above-mentioned (1).With the fragment that comprises sudden change of amplification newly as second take turns PCR upstream primer, in conjunction with the downstream primer in (1), be template with pET28a-His-FADD, amplify the encoding sequence of the rite-directed mutagenesis variant FADD (F25Y) of total length.The PCR product of purifying adopts NcoI/Hind III double digestion, is inserted between the corresponding site of pET28a.Recombinant expression vector is carried out dna sequencing, verify it and read the exactness of frame and encoding sequence.In pET28a-His-FADD (F25Y) plasmid that makes up, utilize upstream primer histidine-tagged, thereby the recombinant protein of expressing is His-FADD (F25Y) full length protein 6 of the N of FADD (F25Y) end introducings.
3.His-FADD the expression of wild-type protein and His-FADD (F25Y) full length protein in intestinal bacteria
Respectively plasmid pET28a-His-FADD and pET28a-His-FADD (F25Y) are transformed expressive host bacterium E.coli BL21 (DE3); Grow on the LB agar plate that contains kantlex; 37 ℃ the growth 16 hours after; The picking mono-clonal is seeded to fresh LB liquid nutrient medium (kantlex that contains 50mg/L), and 37 ℃ of shaking table shaking culture are treated nutrient solution OD
600Value reaches about 0.6, and the shaking table temperature is reduced to 25 ℃ and continued to cultivate half a hour, and the adding final concentration is the expression that the IPTG of 0.4mM induces target protein.Continue shaking culture after 5 hours, at 25 ℃ through centrifugal collection thalline.One liter of lysis buffer (50mM Na that expresses bacterial sediment with the 40mL precooling
3PO
4, 300mM NaCl, 10% glycerine, pH8.0) resuspended, ultrasonication thalline 10 minutes (pulse 2 seconds, 2 seconds at interval) was collected centrifugal supernatant in centrifugal 20 minutes through 12000rpm under the ice bath protection.
4.His-FADD the separation and purification of wild-type protein and His-FADD (F25Y) full length protein
In the ultrasonic degradation supernatant of collecting, add the 10mM imidazoles, be loaded into binding buffer liquid (50mMNa
3PO
4, 300mM NaCl, the 10mM imidazoles, pH8.0) the metallic nickel affinity column of pre-equilibration, last appearance finish the back with binding buffer liquid thorough washing to baseline, strengthen imidazole concentration washing foreign protein gradually, use the buffer solution elution target protein that contains the 250mM imidazoles at last.Eluted protein through metallic nickel affinity chromatography column purification uses Sephadex G15 gel chromatography to remove imidazoles immediately, and buffer system is replaced by 20mMTris-HCl, pH8.0.
Just can obtain purer His-FADD (F25Y) full length protein after imidazoles is taken off in process metallic nickel affinity chromatography column purification and G15 gel-filtration, and the His-FADD wild-type protein is also needed further through DEAE anion-exchange chromatography purifying.Be loaded into the Tris buffering (20mM Tris-HCl, pH8.0) the DEAE Sepharose Fast Flow of pre-equilibration increase the concentration of NaCl gradually in buffer system, carry out wash-out through changing His-FADD wild-type protein behind the liquid.Collect each elution peak and be used for electrophoretic analysis and Bradford concentration determination.
5. the circular dichroism spectrum analysis of protein higher structure
Use 20mM Tis-HCl, to 200 μ g/mL, scanning obtains the far-ultraviolet spectrum of 195～250nm to the damping fluid of pH8.0 on Jasco J-810 circular dichroism spectrometer with diluted sample.Concrete parameter is following: sample pool optical path 1mm, time of response 1s, sweep velocity 50nm/min, the wide 2nm of ripple, data collection intervals 0.1nm, each sample scanning three times.The data recorded deduction is converted into molar ellipticity [θ] (mdeg.cm by software kit again by the background that damping fluid scanning obtains
-2.mol
-1).
6. the fluorescent spectroscopy of protein higher structure
Use 20mM Tris-HCl, the damping fluid of pH8.0 with diluted sample to 50 μ g/mL, the fluorescence emission spectrum when on the SHIMADZURF-5301PC spectrophotofluorometer, scanning excitation wavelength respectively and being 280nm and 295nm.Concrete parameter is following: sample pool optical path 1mm, and scope 300～400nm, the sweep velocity middling speed excites, launches slit and is 3nm, data collection intervals 1nm, each sample scanning three times.The background that the data recorded deduction is obtained by damping fluid scanning.
7.His pull-down experimental analysis aleuroplast interacts outward
(1) combine the metallic nickel affinity column medium of recombinant protein to prepare:
Be kept at nickel post medium in 20% ethanol with TG (0.1) damping fluid (20mM Tris-HCl, pH8.0,10%Glycerol; 0.1M NaCl) thorough washing; Add excessive purified recombinant albumen His-FADD (F25Y), make it in the 1.5mL centrifuge tube fully reaction 4 hours, centrifugal 2 minutes of 300g, remove supernatant; Medium deposition suspends with isopyknic TG (0.1) after washing three times with the TG (0.1) of 10 times of medium volumes again.
(2) expression of FLAG-caspase-8 in the 293T cell:
The 293T cell inoculation after in the DMEM substratum that contains 10% foetal calf serum (Hyclone), penicillium mould and Streptomycin sulphate, cultivating 24 hours in (Invitrogen), is expressed eukaryon expression plasmid pRK5-FLAG-caspase-8 transfectional cell through the method for standard calcium phosphate transfection in the 10mm petridish to the 293T cell; Transfection is collecting cell after 36 hours; With precooling PBS washing once, with lysis buffer (50mMHEPES, 100mM NaCl; 10%Glycerol; 1%NP-40) suspension cell precipitates, placed the ice bath cracking 60 minutes again, and 15000rpm low-temperature centrifugation 15 minutes, cracking supernatant carry out His pull-down experimental analysis.
(3) association reaction:
Lysis supernatant (500 μ g protein content) is mixed with the metallic nickel affinity column medium 5 μ L that are combined with His-FADD (F25Y), with TG (0.1) (20mM Tris-HCl, pH 8.0; 10%Glycerol, 100mM NaCl) be diluted to 500 μ L reaction systems, on vertical mixing tank, in 4 ℃ of chromatography cabinets, react and spend the night; The centrifugal supernatant that goes is used 1mL TG (0.1) washing once, 1mL TG (1.0) (20mM Tris-HCl successively; PH8.0; 10%Glycerol, 1.0M NaCl) washed twice, each washing time 20 minutes.Last trapping medium deposition suspends with 50 μ L, 1 * SDS sample-loading buffer again, carries out the SDS-PAGE electrophoretic separation.
(4) Western Blotting immunoblotting:
After the sample process SDS-PAGE electrophoretic separation, use semidrying albumen in the PAGE glue to be transferred on the pvdf membrane constant current 1mA/cm
2Electricity changeed 3 hours.Skim-milk with PBST preparation 5% (W/V) encapsulates room temperature 1 hour in advance to film as confining liquid then.After spending the night in 1 hour or 4 ℃ with mouse monoclonal antibody anti-Flag antibody (Sigma) incubated at room, PBST washing 4 times, each 5 minutes.Use sheep anti-mouse igg-HRP (Santa Cruz) and film to hatch jointly afterwards 1 hour, equally with PBST washing 3 times, each 10 minutes.At last, with ECL system (Cell Signaling) HRP is developed the color, and the X-ray sheet is made public.
Embodiment 2:
1. wild-type FADD and FADD (F25Y) full length protein Construction of eukaryotic
With the wild-type FADD of structure and the prokaryotic expression carrier of FADD (F25Y) full length protein among the embodiment 1 is template; With the method amplification wild-type FADD of PCR and the encoding sequence of FADD (F25Y) full length protein; Introduce restriction enzyme site BamH I and Xho I (black matrix, italic) at 5 ' end and 3 ' end respectively; Used upstream primer is: 5 '-CGC
ATGGACCCATTCCTGGT-3 ', downstream primer is: 5 '-CCG
TCAGGGTGTTTCTGAGG-3 '.The condition of PCR reaction is: 94 ℃ of thermally denatures 30 seconds, and 65 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, and carried out 25 circulations altogether.The PCR product of purifying adopts BamH I/Xho I double digestion, the corresponding site of inserting pRK5-FLAG.Recombinant expression vector is carried out dna sequencing, analyze the exactness that it reads frame and encoding sequence.
2. wild-type FADD and FADD (F25Y) full length protein carrier for expression of eukaryon transfecting eukaryotic cells
Attached cell adopts this laboratory PEI method commonly used to carry out transfection (Dong W etc., 2006, ActaBiochim Biophys Sin.38:780-787).The 293T cell that growth conditions is good is with 2 * 10
5The density of/mL is inoculated in 24 orifice plates, and the 0.5mL/ hole places 37 ℃, and the 5%CO2 incubator was cultivated after 18-24 hour, carried out the PEI transfection.
The preparation of PEI/DNA sediment composite: the DNA (wild-type FADD or FADD (F25Y) full length protein carrier for expression of eukaryon) that in 50 μ L PBS, adds total amount 2 μ g; Add the PBS 50 μ L that contain 4 time μ gPEI, room temperature was placed 15 minutes behind the mixing, dropwise was added to mixture in the cell; Putting into incubator behind the mixing cultivates; Change fresh substratum after 6 hours, continue to cultivate collecting cell after the time of needs.
3.FADD protein immuning fluorescent dyeing analysis
(1) cell of transfection after 30 hours removes substratum, washes 3 times with precooling PBS;
(2) fixing: add the Paraformaldehyde 96 of 500 μ L4%, 4 ℃ 1 hour or spend the night, again with the cold PBS of 1mL wash 2 times each 3 minutes;
(3) punching: add the PBS that 500 μ L contain 0.5%Triton X-100, room temperature was placed 45 minutes, washed 3 times each 3 minutes again with cold PBS;
(4) the PBS solution that contains 3%BSA with fresh configuration encapsulates in advance, room temperature 2 hours;
(5) PBS solution dilution one anti-(1: 200) (Ye JQ etc., 2008, the BiotechnolAppl Biochem.50:143-146) with 3%BSA encapsulates and spends the night, and washes 3 times each 10 minutes with cold PBS 1mL;
(6) add fluorescently-labeled two of 3%BSA dilution and resist, 4 ℃ encapsulated 1 hour, washed 3 times each 10 minutes with the cold PBS of 1mL;
(7) add DAPI (1: 10000) and carry out nuclear dyeing, room temperature 2 minutes, 1mL PBS washes once;
(8) film-making: on slide glass, drip 20 μ L20% glycerine, then deckglass is tipped upside down on the slide glass, observe, take pictures with fluorescent microscope.
Owing to adopted the method for expressing at low temperature, His-FADD wild-type protein and His-FADD (F25Y) full-length proteins is able to improve greatly (Figure 1A and 1B) in the solubility of expression in escherichia coli product in embodiment 1.We attempt having used the expression temperature from 10 ℃-35 ℃, can both improve the solubility of FADD protein expression product to some extent.Contrast His-FADD wild-type protein and His-FADD (F25Y) full-length proteins be in the output (Figure 1A and 1B) of the expression level and the soluble fractions of expression in escherichia coli product, our discovery: the expression amount of His-FADD (F25Y) full-length proteins is higher than His-FADD wild-type protein (among Figure 1A among swimming lane 1 and Figure 1B swimming lane 1 compare); And; Under low temperature is expressed; His-FADD (F25Y) full-length proteins is almost all expressed with soluble form, and the His-FADD wild-type protein then has considerable part still to be present in the insoluble inclusion body products (among Figure 1B among swimming lane 2 and Figure 1A swimming lane 2 compare).We find that the efficient of His-FADD wild-type protein bond nickel affinity chromatography post is very low in the process of purifying; Most of His-FADD wild-type protein all flows out (swimming lane 4 among Figure 1A) during the metallic nickel affinitive layer purification in passing composition, causes the proteic recovery to reduce greatly.Target protein specificity bonded reduces the increase that also causes the non-specific combination of foreign protein, during the metallic nickel affinitive layer purification, contains more foreign protein (swimming lane 6 and 11 among Figure 1A) in the 250mM imidazoles elution peak.In further DEAE ion exchange chromatography, these foreign proteins can both combine (swimming lane 11 among Figure 1A) with the DEAE post, and the target protein major part flows out (swimming lane 10 among Figure 1A) with the form of passing, and reaches the purpose that is further purified.And after 25 phenylalanine(Phe)s are mutated into tyrosine; His-FADD (F25Y) full length protein almost all can combine with the metallic nickel affinity column; And in the 250mM imidazoles, elute; Contain foreign protein (swimming lane 6 among Figure 1B) in the 250mM imidazoles in the elution peak hardly, at last through just obtaining the target protein soluble in a large number, that purity is higher (swimming lane 7 among Figure 1B) behind the G15 gel-filtration removal imidazoles.From 1 liter of substratum, through four step separation and purification, the productive rate of His-FADD wild-type protein is 1.9mg, and the recovery is 3.2%; And for His FADD (F25Y) full length protein; As long as can obtain target product with high purity through three step separation and purification; The productive rate of His-FADD (F25Y) full length protein is 29.4mg; The recovery is 49.9%, and its productive rate is 15.5 times of His-FADD wild-type protein output, and purification step has reduced DEAE ion exchange chromatography step.
Reorganization FADD wild-type protein and FADD (F25Y) full length protein purge process and productivity ratio are
| Recombinant protein (1 liter of expression product) | Purification step | Target protein stage recovery total yield (mg) is (%) (%) |
| His-FADD | Ultrasonication crude extract nickel post affinity chromatography G15 gel permeation chromatography DEAE ion exchange chromatography | 59.7 5.6 9.4 9.4 4.6 82.1 ?7.7 1.9 41.3 ?3.2 |
| His-FADD(F25Y) | Ultrasonication crude extract nickel post affinity chromatography G15 gel permeation chromatography | 58.9 37 ?62.8 ?62.8 29.4 ?79.4 ?49.9 |
Sum up the whole purge process and the recovery of His-FADD wild-type protein and His-FADD (F25Y) full length protein, through finding that relatively both maximum difference are in this step of metallic nickel affinitive layer purification.Visible by Figure 1A; Because the His-FADD wild-type protein can not combine with the metallic nickel affinity column effectively; Cause the recovery of metallic nickel affinitive layer purification to have only 9.4%; Have only combined utilization, could express from 1L and obtain the higher target protein 1.9mg of purity the bacterium through three kinds of chromatography methods.And for His-FADD (F25Y) full length protein, the avidity of itself and metallic nickel affinity column improves greatly, and the recovery in this step is up to 62.8%.The increase of specificity bonded has reduced the non-specific binding of foreign protein simultaneously; Thereby improved the purity of target protein in the 250mM imidazoles elutriant, therefore need not to pass through again the step that is further purified of DEAE ion exchange chromatography as the His-FADD wild-type protein.Final only the need just can be expressed target recombinant albumen His-FADD (F25Y) full length protein that obtains 29.4mg the bacterium from 1L through two step chromatographies.Compare with the His-FADD wild-type protein, Phe25 is mutated into Tyr complicated originally purge process is oversimplified, and can improve proteic productive rate to a great extent, have laid a good foundation for further studying with application.
We attempted the His label is placed on the C end of FADD (F25Y) full length protein simultaneously; Perhaps use other small peptide label (like Flag) or fusion rotein (GST), these methods all do not have remarkable influence for expression, the productive rate of FADD (F25Y) full length protein.
Circular dichroism spectrum is a kind of method of measuring secondary protein structure under the solution state.(180～240nm), main chromophore is a peptide bond, and the CD spectrum of this wavelength region is comprising the information of biomacromolecule main chain conformation at the extreme ultraviolet section.The circular dichroism spectrogram of His-FADD wild-type protein and His-FADD (F25Y) full length protein extreme ultraviolet shows two negative characteristic acromion bands of a spectrum (Fig. 2 A) at 222nm and 208nm place; Characteristic with significant alpha-helix is that full α type albumen conforms to the non-total length FADD of bibliographical information.And both CD spectrum is overlapping basically, shows both not significantly differences of secondary structure, proves that the transformation that 25 Phe is mutated into Tyr can not change the secondary structure of albumen itself.
The protein fluorescence emmission spectrum that under the inducing of certain wavelength exciting light, obtains can provide luminous residues such as tryptophan in proteins and the tyrosine information of microenvironment on every side for us.The wavelength at the fluorescence emission spectrum climax of His-FADD wild-type protein and His-FADD (F25Y) full length protein about 330nm, explains that the microenvironment around luminophore Trp and the Tyr is main with hydrophobic environment basically.No matter be at 295nm (Fig. 2 B) or under the exciting light of 280nm (Fig. 2 C), the emmission spectrum high superposed of two recombinant proteins explains that the local structure around the luminophore are basic identical.
Compare through circular dichroism spectrum and two kinds of simple spectroscopic analysis of fluorescence spectrum; We can think the transformation (Phe of the 25th residue is mutated into Tyr) that His-FADD is done; Can't change Protein Folding and higher structure, therefore can use His-FADD (F25Y) to replace the total length His-FADD of wild-type further to study and use.
Phe25 is positioned at the Death Effector Domain (DED) of FADD, and according to existing knowledge, FADD interacts through the DED of DED and downstream caspase-8, the effect of performance transfer cell apoptotic signal.Although obvious variation does not take place on higher structure His-FADD (F25Y) full length protein of transforming through point mutation through the analytical proof of front, its activity that whether still has mediating apoptosis needs further checking.FADD is mainly that as the function of apoptosis protein matter it has the biological function of the proteolytic enzyme caspase 8 in recruitment and combination downstream, thereby brings into play the biological action of its cell death inducing.Therefore, whether the present invention has recruitment and combines the biological function of the proteolytic enzyme caspase 8 in downstream to carry out verification experimental verification His-FADD (F25Y) full length protein.Utilize the method for His pull-down; The recombinant protein recombine that in protokaryon, express and be purified to is to the metallic nickel affinity column; React with the FLAG-caspase-8 of overexpression in eukaryotic cell, find that His-FADD (F25Y) full-length proteins mass-energy is in external and Caspase-8 interaction (swimming lane 3 among Fig. 3 A).The His-GST that utilizes purifying proves His-FADD (F25Y) and the interactional specificity of Caspase-8 as negative control (swimming lane 2 among Fig. 3 A).Through a part of pull-down product being carried out Xylene Brilliant Cyanine G glue dyeing (Fig. 3 B), prove that His-FADD (F25Y) and His-GST protein applied sample amount are identical simultaneously.This bearing reaction recombination mutation albumen His-FADD (F25Y) full length protein of expression and purification of the present invention remain with the biological function that FADD recruited and combined Capspase-8, thereby have the function of mediating apoptosis.
In addition, we have also made up not with the FADD wild-type protein of His label and the eukaryon expression plasmid of FADD (F25Y) full length protein in embodiment 2, transfecting eukaryotic cells is expressed, the immuning fluorescent dyeing analysis that has carried out with anti-FADD monoclonal antibody.The result shows (Fig. 4): FADD wild-type protein expression level in eukaryotic cell is lower than FADD (F25Y) full length protein; And FADD (F25Y) full length protein is uniform distribution (Fig. 4 B) in eukaryotic cell, and assembling and deposited phenomenon (Fig. 4 A) still appears in eukaryotic cell in the FADD wild-type protein.Experiment also shows: FADD (F25Y) full length protein still has the biological activity of mediating apoptosis in eukaryotic cell.Therefore, no matter at prokaryotic expression system or in the eukaryotic expression expression system, whether no matter the His label arranged, the productive rate of FADD (F25Y) full length protein all is significantly higher than wild-type FADD protein.The invention provides the proteinic preparation method of a kind of preparation total length FADD, utilize present method can mass production prepare solubility, total length, have complete bioactive FADD protein.Utilize present technology such as liposome parcel FADD full length protein to carry out vivo medicine-feeding treatment, especially target administration treatment, can be through diseases such as cell death inducing treatment tumour or rheumatoid arthritiss.
Claims (1)
1. a total length FADD protein is in the application for preparing separately in the cell death inducing medicine; Wherein, A kind of total length FADD protein is meant that the 25th Phe is mutated into the total length FADD protein of Tyr, and its N end or C end can merge fusion rotein or the small peptide label with the effect of help protein purification respectively.
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| Fiona L. Scott et al.The Fas–FADD death domain complex structure unravels signalling by receptor clustering.《Nature》.2009,第457卷1019-1022，S1-S17. * |
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| CN103239736A (en) * | 2013-05-16 | 2013-08-14 | 南京大学 | Method for adjusting glycolysis and gluconeogenesis and applications of method |
| CN103239736B (en) * | 2013-05-16 | 2016-08-10 | 南京大学 | A kind of regulate glycolysis and the method for glyconeogenesis and purposes |
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