CN104164444A - Fusion protein, and preparation method and application thereof - Google Patents
Fusion protein, and preparation method and application thereof Download PDFInfo
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- CN104164444A CN104164444A CN201410377969.0A CN201410377969A CN104164444A CN 104164444 A CN104164444 A CN 104164444A CN 201410377969 A CN201410377969 A CN 201410377969A CN 104164444 A CN104164444 A CN 104164444A
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Abstract
The invention discloses a fusion protein, and a preparation method and application thereof, belonging to the field of production, analysis and application of proteins. The separation, purification and performance analysis of the calmodulin fusion protein are established, and the calmodulin fusion protein column is utilized to separate, extract and concentrate the target protein and related biomolecules in the sample, so that a new biological sample preparation and pretreatment technique capable of enhancing biological detection sensitivity is introduced.
Description
Technical field
The invention belongs to protein production, analysis and application field, is specifically related to a kind of preparation method and application thereof of fused protein.
Background technology
Calmodulin is a kind of important protein, in the information transmission relevant to intracellular calcium, brings into play important regulating effect, has the function of reconciling plurality of enzymes.On market, except needing a large amount of calmodulins as research caldesmon matter, utilize calmodulin and caldesmon to rely on the relation that calcium ion exists, also have the report that reaches combination and the object of separated calmodulin conjugated protein by the existence of adding or eliminate calcium ion recently.But utilize fusion rotein (being maltose binding protein-calmodulin fusion rotein) to reach separated herein, the method for the target material in concentrated sample is also without any report.
Have recently report to point out, heat shock protein 90 is good cancer detection marker, but does not also have a kind ofly can from biological specimen, concentrate this heat shock protein 90, is convenient to improve the report of the detection sensitivity of this protein.
Summary of the invention
The object of this invention is to provide a kind of production of simplifying protein by fused protein, purifying, analytic process and the condition of utilizing fused protein to depend on are next separated, method and the application thereof of concentrated relevant protein conjugate.
The technical solution adopted in the present invention is:
A preparation method for fused protein, comprises the following steps:
(1) the cDNA fragment of complete target gene is cloned into the expression vector of fusion rotein, during cDNA clone, before the cDNA of complete genome fragment, initiator codon (ATG) afterwards, adds the DNA sequence of Xa factor site Isoleucine-L-glutamic acid-glycine-arginine (Ile-Glu-Gly-Arg);
(2) the subclone expression vector that contains complete goal gene step (1) being obtained, make the transition or be transfected in expression host cell, make it at the required fused protein of host cell inner expression, by inoculation culture, obtain the pure clonal cell line that contains complete goal gene carrier;
(3) the pure clonal cell line that uses step (2) to contain complete goal gene carrier, quantizes to cultivate, and protein expression is processed, and acquisition quantizes, and has the host cell of object fused protein;
(4) host cell step (3) being obtained carries out cracking, obtains cell pyrolysis liquid, and protein is carried out to purifying, obtains separation, the fused protein of purifying;
(5) usage factor Xa processes fused protein, can obtain the goal gene protein of purifying.
Further, in described step (1), add the DNA sequence of Xa factor site Isoleucine-L-glutamic acid-glycine-arginine (Ile-Glu-Gly-Arg), can guarantee that expressed protein is after Xa cutting, the amino-acid sequence of the target protein matter obtaining is required, complete.
Further, protein separation and purifying are carried out by affinity column in described step (4) and (5).
Preferably, described a kind of representative fusion rotein is calmodulin fusion rotein, is to take calmodulin as the calmodulin of target protein matter and the fused protein of maltose binding protein.
By above-mentioned preparation method, the fusion rotein preparing.
An application for fused protein, the fusion rotein that utilizes separation and purification to obtain, separation, purifying and concentrated target protein matter, can be used for performance analysis and the application of protein.
Further, utilize fusion rotein, the target molecule in enriched biological sample, reaches separation, purifying, concentrated and improve the object of detection sensitivity.
Preferably, the fusion rotein that described object fused protein is maltose binding protein-calmodulin, a kind of target molecule can be used in combination and concentrated sample is heat shock protein 90.
The present invention has the following advantages:
The fused protein that this patent is introduced is produced, separation and purification and analytical procedure, and utilize fused protein separated, concentrated relevant conjugated protein or protein conjugate, reach separated, purifying and raising are analyzed, detect the object of the sensitivity of target molecule, it is the novel method that proposes first to be come by fused protein the protein conjugate in enrichment sample, its method and product, can make up the separated of specified protein and protein conjugate, many deficiencies of the aspect such as purifying and detection method, or provide more effective protein production system, its using value is very large.The present invention uses the expression of fused protein, and first the mode of production can improve solubility ratio, has simplified again the operating process of separation and purification simultaneously, has reduced cost (utilize affinity column, method is simple, and cost is low); Utilize design at two kinds of protein junction cutting parts, can easily obtain the fused protein and target protein matter (the few amino acid that meet amino acid needed order, a quite a few amino acid), be convenient to the utilizability energy of comparison fused protein and target protein matter; Utilize fused protein to come as carrier separated, concentrated relevant conjugated protein or the binding substances of protein, can be used for the separation of conjugated protein, concentrated, performance analysis, detects, and also can be used as a kind of new effective ways that improve biological detection sensitivity simultaneously.For example, calmodulin is under the condition that has calcium ion to exist, can be combined with multiple heat shock protein, and heat shock protein particularly tumour cell can secrete heat shock protein 90, so heat shock protein 90 is again the important marker of cancer detection, utilize this invention technical products, in concentrated correlated samples, the heat shock protein of (as: blood equal samples), will contribute to the early stage high-sensitivity detection of cancer.
Accompanying drawing explanation
Fig. 1 is the clone's schematic diagram that shows maltose binding protein matter and calmodulin matter fused protein;
Fig. 2 is production and the purifying schematic diagram that shows calmodulin matter;
Fig. 3 shows calmodulin matter and fused protein calcium binding experiment schematic diagram;
It is identical with calmodulin that Fig. 4 shows that calmodulin fused protein has, the function of activating phosphatase diesterase;
Fig. 5 shows the combination experiment of calmodulin fused protein and heat shock protein 90;
Fig. 6 is by the concentrated calmodulin conjugated protein of calmodulin fusion rotein post or binding substances lab diagram;
Fig. 7 utilizes calmodulin fused protein separated, the schematic diagram of purifying and concentrated caldesmon matter or binding substances.
Embodiment
Below in conjunction with drawings and embodiments, the present invention is further detailed explanation.
With reference to Fig. 1, Fig. 1 is the clone's schematic diagram that shows maltose binding protein matter and calmodulin matter fused protein.Take coli expression carrier (pMAL-c2) as experiment carrier (being purchased from New England Biolab), by PCR (Polymerase Chain Reaction, polymerase chain reaction) probe design, by the complete genome cDNA of calmodulin (Complimentary deoxyribonucleic acid, complementary DNA (cDNA)) copy, gene fragment after copying, both contained complete calmodulin cDNA, again after its setting up password, DNA (Deoxyribonucleic acid, the thymus nucleic acid) sequence that has added factor Xa.Before setting up password and after gene order, there is clone enzyme site, PCR product, after enzyme is cut, just can be cloned in carrier.After Bacillus coli cells, have calmodulin gene to insert transition, when cell strain is selected in inoculation, can obtain required clonal cell line.
With reference to Fig. 2, Fig. 2 is production and the purifying schematic diagram that shows calmodulin matter, and its middle slot 1 is the Bacillus coli cells lysate of not inducing processing; Groove 2 is that IPTG (Isopropyl β-D-1-Thiogalactopyranoside, isopropyl-β-D-thiogalactoside(IPTG)) induction calmodulin fused protein is expressed the Bacillus coli cells lysate after processing; Groove 3 is to use amylose starch affinity column separated, the calmodulin fused protein after purifying (fused protein of maltose binding protein and calmodulin); Groove 4 is that above-mentioned fused protein is carried out after factor Xa enzyme cuts, showing that the fused protein of maltose binding protein and calmodulin is cut open, and is separated into maltose binding protein matter (greatly) and two kinds of protein of calmodulin (little); Groove 5 is the protein solns after factor Xa enzyme is cut, and again makes amylose starch affinity column separating treatment, and maltose binding protein matter is adsorbed by affinity column, can obtain the calmodulin matter of separation and purification.Compare with not doing induction processing, in the Bacillus coli cells lysate after IPTG induction, the expression amount of calmodulin matter very high (groove 2).Separated through amylose starch affinity column, after purifying, obtained highly purified fused protein (groove 3).Fused protein with factor Xa cutting purifying, can obtain maltose binding protein matter and calmodulin matter (groove 4).Protein soln after factor Xa cutting, after the separation and purification of amylose starch affinity column, can obtain highly purified calmodulin matter (groove 5) again.As can be seen from Figure 2, this system is successfully produced, the separated protein with being purified into required fused protein and goal gene.
With reference to Fig. 3, Fig. 3 shows calmodulin matter and fused protein calcium binding experiment schematic diagram, its middle slot 1 is calmodulin without EGTA (ethylene glycol tetraacetic acid, ethylene glycol bis (2-amino-ethyl ether) tetraacethyl) with without CaCl
2under (calcium chloride) condition, at SDS-PAGE (SDS:Sodium dodecyl sulfate; PAGE:Polyacrylamide gel electrophoresis; SDS-PAGE) on glue, the variation on non-structure; Groove 2 is that calmodulin exists but do not have under CaCl2 condition at 5mM EGTA, on SDS-PAGE glue, and the also variation on non-structure; Groove 3 is calmodulin without EGTA but there is 5mMCaCl
2under the condition that calcium ion exists, on SDS-PAGE glue, shown the combination with calcium ion, and produced structural variation.As can be seen from Figure 3, no matter be calmodulin matter itself, or calmodulin matter fused protein, having under the condition of calcium ion, all shown and the function of calcium ion result, on SDS-PAGE glue, shown the leading movement after molecule structure change.
With reference to Fig. 4, it is identical with calmodulin that Fig. 4 shows that calmodulin fused protein has, the function of activating phosphatase diesterase (PDE).Activate the damping fluid (Buffer) of matrix with nothing and compare, the fused protein (MBP-CAM) of the maltose binding protein matter of different concns and calmodulin matter has the effect of the activating phosphatase diesterase the same with calmodulin (CAM) itself.
With reference to Fig. 5, Fig. 5 shows the combination experiment of calmodulin fused protein and heat shock protein 90.As can be seen from Figure 5, without sex change glue isotropic substance experiment, show, with the external synthetic isotropic substance S that has
35the heat shock protein 90 of mark is compared (groove 9), calmodulin fused protein and the external synthetic isotropic substance S that has
35the heat shock protein 90 of mark has formed distinctive combined belt (groove 2); The external synthetic isotropic substance S that has
35the heat shock protein 90 of mark has also formed distinctive combined belt (groove 4) with calmodulin matter, and maltose binding protein matter and the external synthetic isotropic substance S that has
35the band (groove 8) that there is no special combination between the heat shock protein 90 of mark, hence one can see that, and calmodulin fused protein has the same with calmodulin, and heat shock protein 90 binding ability.
With reference to Fig. 6, Fig. 6 is by the concentrated calmodulin conjugated protein of calmodulin fusion rotein post or binding substances lab diagram.As can be seen from Figure 6, the calmodulin fused protein post that the present invention produces has and has concentrated specific protein belt or binding substances band, shows that this fusion rotein post has function.
With reference to Fig. 7, Fig. 7 utilizes calmodulin fused protein separated, the schematic diagram of purifying and concentrated caldesmon matter or binding substances.In biological specimen, sometimes concentration or the purity of some caldesmon matter or binding substance are inadequate, or when detecting, because concentration is inadequate or purity is inadequate, and while can't detect, need separation, purifying or concentrate to improve its concentration, just can utilize fused protein post to do a pre-treatment to biological specimen, reach separated, the object of purifying or enriched target protein or binding substances.
The fused protein that this patent is introduced is produced, separation and purification and analytical procedure, and utilize fused protein separated, concentrated relevant conjugated protein or protein conjugate, reach separated, purifying and raising are analyzed, the object of the sensitivity detecting, it is the novel method that proposes first to be come by fused protein the protein conjugate in enrichment sample, its method and product, can make up the separated of specified protein and protein conjugate, many deficiencies of the aspects such as purifying and detection method, or more effective protein production system is provided, its using value is very large.The present invention uses the expression of fused protein, and first the mode of production can improve solubility ratio, has simplified again the operating process of separation and purification simultaneously, has reduced cost (utilize affinity column, method is simple, and cost is low); Utilize design at two kinds of protein junction cutting parts, can easily obtain the fused protein and target protein matter (the few amino acid that meet amino acid needed order, a quite a few amino acid), be convenient to the utilizability energy of comparison fused protein and target protein matter; Utilize fused protein to come as carrier separated, concentrated relevant conjugated protein or the binding substances of protein, can be used for the separation of conjugated protein, concentrated, performance analysis, detects, also can be used as simultaneously and improve a kind of new of biological detection sensitivity, effective ways.For example, calmodulin is under the condition that has calcium ion to exist, can be combined with multiple heat shock protein, and heat shock protein particularly tumour cell can secrete heat shock protein 90, so heat shock protein 90 is again the important marker of cancer detection, utilize this invention technical products, the heat shock protein of (as: blood equal samples) in concentrated correlated samples, will contribute to the early stage of cancer effectively to detect.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (8)
1. a preparation method for fused protein, is characterized in that, comprises the following steps:
(1) the cDNA fragment of complete target gene is cloned into the expression vector of fusion rotein, during cDNA clone, before the cDNA of complete genome fragment, initiator codon (ATG) afterwards, adds the DNA sequence of Xa factor site Isoleucine-L-glutamic acid-glycine-arginine (Ile-Glu-Gly-Arg);
(2) the subclone expression vector that contains complete goal gene step (1) being obtained, make the transition or be transfected in expression host cell, make it at the required fused protein of host cell inner expression, by inoculation culture, obtain the pure clonal cell line that contains complete goal gene carrier;
(3) the pure clonal cell line that uses step (2) to contain complete goal gene carrier, quantizes to cultivate, and protein expression is processed, and acquisition quantizes, and has the host cell of object fused protein;
(4) host cell step (3) being obtained carries out cracking, obtains cell pyrolysis liquid, and protein is carried out to purifying, obtains separation, the fused protein of purifying;
(5) usage factor Xa processes fused protein, can obtain the goal gene protein of purifying.
2. the preparation method of fused protein according to claim 1, it is characterized in that, in described step (1), add the DNA sequence of Xa factor site Isoleucine-L-glutamic acid-glycine-arginine (Ile-Glu-Gly-Arg), can guarantee that expressed protein is after Xa cutting, the amino-acid sequence of the target protein matter obtaining is required, complete.
3. the white preparation method of fusion egg matter according to claim 1, is characterized in that, protein separation and purifying are carried out by affinity column in described step (4) and (5).
4. the preparation method of fused protein according to claim 1, is characterized in that, described a kind of representative fusion rotein is calmodulin fusion rotein, is to take calmodulin as the calmodulin of target protein matter and the fused protein of maltose binding protein.
5. the preparation method described in claim 1 to 4 any one, the fusion rotein preparing.
6. an application for fused protein, is characterized in that, the fusion rotein that utilizes separation and purification to obtain, and separation, purifying and concentrated target protein matter, can be used for performance analysis and the application of protein.
7. the application of fused protein according to claim 6, is characterized in that, utilizes fusion rotein, and the target molecule in enriched biological sample reaches separation, purifying, concentrated and improve the object of detection sensitivity.
8. according to the application of the fused protein described in claim 6 or 7, it is characterized in that, described object fused protein is maltose binding protein-calmodulin fusion rotein, and a kind of target molecule can be used in combination and concentrated sample is heat shock protein 90.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109021476A (en) * | 2018-06-26 | 2018-12-18 | 东阳市特意新材料科技有限公司 | A kind of preparation method of high-strength ageing absorption resin of sodium polyacrylate |
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| CN1037396A (en) * | 1988-05-07 | 1989-11-22 | 南京农业大学 | Calcium opsonin enzyme joint immune testing medicine box |
| CN1147853A (en) * | 1994-03-10 | 1997-04-16 | 藤泽药品工业株式会社 | Method for assaying immunosuppressant |
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2014
- 2014-07-31 CN CN201410377969.0A patent/CN104164444A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1037396A (en) * | 1988-05-07 | 1989-11-22 | 南京农业大学 | Calcium opsonin enzyme joint immune testing medicine box |
| CN1147853A (en) * | 1994-03-10 | 1997-04-16 | 藤泽药品工业株式会社 | Method for assaying immunosuppressant |
Non-Patent Citations (1)
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| 王亚楠等: "核酸酶P1的原核表达、纯化及酶学特性分析", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109021476A (en) * | 2018-06-26 | 2018-12-18 | 东阳市特意新材料科技有限公司 | A kind of preparation method of high-strength ageing absorption resin of sodium polyacrylate |
| CN109021476B (en) * | 2018-06-26 | 2021-01-05 | 深圳市新拓普新材料有限公司 | Preparation method of high-strength anti-aging sodium polyacrylate water-absorbent resin |
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