CN101144093A - Recombination expression carrier and method for soluble expressing human I-type metallothionin - Google Patents
Recombination expression carrier and method for soluble expressing human I-type metallothionin Download PDFInfo
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Abstract
The present invention relates to a method for producing a human I type metal sulfur protein (Human metallothionein-I, hMT-I), in particular to the fusion expression by adopting the small molecule ubinquitin-related modifier mature peptide (Small Ubiquitin-related Modifier, SUMO) and hMT-I, and the fusion protein and the ubinquitin-related modifier protease 1 (Ubiquitin-like protease 1) are co-expressed in a prokaryotic organism. In the fermentation and expression process, the ubiquitin associative modifier gene protease 1 can hydrolyze the fusion protein which consists of the ubinquitin-related modifier mature peptide and the hMT-I, to produce the dissoluble hMT-I.
Description
Technical field
The invention belongs to the genetically engineered field.More specifically, the present invention relates to a kind of recombinant expression vector and a kind of method of expressing and producing people I shaped metal sulfoprotein with soluble form of expressing human I shaped metal sulfoprotein.
Background technology
Metallothionein(MT) (metallothionein, MT) be the low molecular mass of a class, be rich in halfcystine, can be in conjunction with heavy metal and the metal binding protein that can be produced by metal inducement.After nineteen fifty-seven was isolated metallothionein(MT) first, the researchist found that MT extensively is present in the tissue of animals and plants and eukaryotic microorganisms.
In Mammals, there are 4 kinds of MT hypotypes, be MT-I, MT-II, MT-III, MT-IV, wherein MT-I and MT-II are present in all organs, MT-III exists only in the cerebral tissue, and MT-IV then exists only in (Uchida et al., J Biol.Chem. in the squamous epithelial tissue, 1995,270:3365-3369).Typical Mammals MT has (1) lower molecular weight, generally has only 60～80 amino-acid residues; (2) high metal content; (3) halfcystine (Cys) content height can be up to 23%～33% (molar percentage); (4) has conservative Cys position and contain a large amount of Cys-X-Cys or characteristics (X is any amino acid in 20 seed amino acids) such as Cys-X-X-Cys sequence.
(structural domain of diameter 15～20U), almost spherical constitutes the tertiary structure of people MT-I, and wherein 1-29 amino acids residue forms the beta structure territory, and 32-61 amino acids residue forms αJie Gouyu by two sizableness.Two structural domains are relatively independent and be spherical, and both link to each other with 31 lysine residue by the 30th, make whole molecule be dumbbell shaped.On the separate and function of two structural domains tangible difference is arranged, wherein αJie Gouyu contains 11 Cys, can be in conjunction with 4 divalent-metal ions such as Cd
2+Or Zn
2+, perhaps in conjunction with 5～6 Cu
2+The beta structure territory contains 9 Cys, can be in conjunction with 3 divalent-metal ion (Cd
2+Or Zn
2+), perhaps 6 Cu
2+, therefore, MT molecule can in conjunction with 7～12 metal ions (Robbins et al., J Mol.Biol., 1991,221:1269-1293).
People MT-I has metabolism, heavy metal detoxification, removing free radical, the participation dna replication dna that participates in trace element in the body and transcribes, various biological function such as influence the immunizing power of protein and energy metabolism, antagonism ionizing rays, enhancing body and stress protect is (referring to Liu et al., Toxicol.Sci., 1998,46:197-203; Zeng et al., Proc.Natl.Acad.Sci.USA, 1991,88:9984-9988; Zangger et al., FASEB J, 2001,15:1303-1305; Li et al., NucleicAcids Res., 1998,26:5182-5189).In numerous functions of people I type MT, removing the detoxifcation of free radical and heavy metal knot and being has two of application prospect most.The one of the main reasons that human body aging and cancer take place is exactly the increase that interior free yl produces.Free radical character is active, has extremely strong oxygenizement, can cause DNA, RNA structural modification and biomembranous peroxidation.Under natural situation, a large amount of sulfydryls in the metallothionein(MT) exist with reduced state, and nucleophilicity that sulfydryl has make MT easily and the Electron Affinities material especially free radical interact, thereby can directly remove free radical.In addition, the strong metal binding ability that people I type MT is had make it can with toxic heavy metal ionic bond such as chromium, lead, mercury, and can excrete, thereby make human body avoid toxic action.
With regard to biologic activity, the multiple MT that finds has above-mentioned identical or similar function at present.Yet people MT-I exists scope wide, functional study get the clearest and in human body one of the most stable MT albumen, therefore, produce people I type MT and more help clinical and daily use.
The production method of Mammals or yeast MT has many reports, yet utilizes animal production and heavy metal to induce the soluble M T of acquisition, and output is extremely low and purification step is complicated (referring to ZL96100897.0; CN1803840A; CN1348010A; CN1376719A).Current, existing reported in literature utilizes protein engineering to produce the method for people MT, and use intestinal bacteria as with the proteinic preferred host cell of recombinant technology industrial production (referring to Hong et al., ProteinExpression and Purification2001,21:243-250).Although escherichia expression system has the expression amount height, be easy to advantages such as cultivation and operation and production cost be low, but, use this expression system to be difficult to obtain the MT of great amount of soluble, its reason is that the MT molecular weight is little, and contain 20 halfcystines, as easy as rolling off a log formation " inclusion body ", the i.e. active insoluble polymer of abiology.In addition, even obtain a large amount of inclusion bodys, in order to obtain the albumen of biologic activity, also must carry out denaturation renaturation to inclusion body and handle, this process is often lost a large amount of albumen.In order to solve this difficult problem, people adopt integration technology, and fusion rotein such as glutathione S-transferase (GST), Intein etc. that the MT gene is correctly folding with having assistance protein carry out amalgamation and expression.Although proteic solubility and yield increase (high energy reaches 8mg/L), but, the risk of consuming time, the expensive proteolytic enzyme expense and the protease hydrolysis target protein of existence, usually can not obtain gratifying MT output (referring to Hong et al., Protein Expression and Purification2001,21:243-250).
Small molecules ubiquitin sample modified protein (Small Ubiquitin-related Modifier, SUMO), and ubiquitin (ubiquitin Ub) have only 18% homology on primary structure, yet both tertiary structures and biological function thereof is quite similar.SUMO extensively is present in the various eukaryotic cells, multiple physiology processes such as participation adjusting apoptosis, signal transduction, rna transcription, the transportation of proteic caryoplasm and cell cycle (referring to Johnson, E.S.2004, Annu.Rev.Biochem., 73,355-382.).In vertebrates, SUMO-1 ,-2 ,-3 three members are arranged, and yeast and invertebrates only there is a related gene coded SUMO.The physiological process of SUMO modified protein such as Fig. 1.In brief, the SUMO precursor loses the partial amino-acid residue of C end under the effect of ubiquitin similar protein enzyme Ulps (ubiquitin-like proteases), expose the Gly-Gly sequence, becomes the SUMO mature peptide; Under the effect of ATP enzyme, the C-terminal Gly of this mature peptide is connected by thioester bond with the Cys residue of activating enzymes E1 (SAE1/SAE2); Then, SUMO is delivered to desmoenzyme E2 (Ubc9) through E1, and is connected with Cys93 residue on the Ubc9 avtive spot by thioester bond; Under the effect of ligase enzyme E3, the Gly of SUMO combines with the epsilon-amino of target protein Lys residue by isopeptide bond and participates in relevant physiological activity; At last, under the hydrolytic action by Ulps, SUMO separates with target protein.Whole process is a dynamically reaction rapidly.Modify differently with ubiquitin, SUMO can not cause the target protein degraded, but stability by changing target protein and Subcellular Localization etc. are regulated the function and the biological activity of target protein.
Except above-mentioned biologic activity, in recent years, SUMO is found fusion tag and the molecular chaperones that can be used as expression of recombinant proteins, has the protease inhibitor hydrolysis, significantly increases expression of recombinant proteins amount and promote target protein correctly folding, improves functions such as solubility.In addition, Ulp1 can be according to the tertiary structure of SUMO rapid hydrolysis fusion rotein, can only not cut according to the albumen primary sequence and do not resemble zymoplasm, Xa factor and enteropeptidase etc., therefore can excise SUMO like clockwork, thereby obtain the target protein of the natural N-terminal of tool and can hydrolysis target protein (Butt et al., 2005, ProteinExpression and Purification, 43,1-9; US60/482817; PCT/US03/00436).In the present invention, at first utilize SUMO significantly to increase expression amount and the solubleness of people MT-I as molecular chaperones, promote it correctly folding, the Ulp1 that expresses simultaneously in the host is according to the accurate hydrolysis fusion rotein of the tertiary structure of SUMO then, and then obtaining great amount of soluble people MT-I, this method effectively solves the risk of protease hydrolysis target protein of above-mentioned consuming time, expensive proteolytic enzyme expense, existence and the difficult problem that can not obtain gratifying MT output.
Summary of the invention
At above-mentioned research background, the invention provides and produce people I shaped metal sulfoprotein (Humanmetallothionein-I, hMT-I) method, particularly relate to and utilize small molecules ubiquitin relevant modifications factor mature peptide (Small Ubiquitin-related Modifier, SUMO) and hMT-I carry out amalgamation and expression, and this fusion rotein and ubiquitin relevant modifications factor protein enzyme 1 (Ubiquitin-like protease1) coexpression in the prokaryotic organism body.In the fermentation expression process, the fusion rotein that ubiquitin relevant modifications factor protein enzyme 1 can hydrolysis be made of molecule ubiquitin relevant modifications factor mature peptide and hMT-I is with production solubility hMT-I.
Therefore, the present invention relates to and the following:
1. the recombinant expression vector of an expressing human I shaped metal sulfoprotein, it comprises first promotor and second promotor, described first promotor is operably connected with the nucleotide sequence of the fusion rotein of coding small molecule ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein, and described second promotor is operably connected with the nucleotide sequence of coding ubiquitin relevant modifications factor protein enzyme.
2. above-mentioned 1 described recombinant expression vector, wherein said fusion rotein comprises: small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein, and two kinds of albumen is that N from fusion rotein holds C end coding successively.
3. above-mentioned 1 described recombinant expression vector, wherein said first and second promotors all are selected from the phage t7 promotor.
4. any one described recombinant expression vector among the above-mentioned 1-3, the aminoacid sequence of the people I shaped metal sulfoprotein in the wherein said fusion rotein is shown in SEQ ID No:6.
5. any one described recombinant expression vector among the above-mentioned 1-3, the aminoacid sequence of wherein said ubiquitin relevant modifications egg factor protein enzyme is shown in SEQ ID No:5.
6. any one described recombinant expression vector among the above-mentioned 1-3, the aminoacid sequence of wherein said small molecules ubiquitin relevant modifications factor mature peptide is shown in SEQ ID No:9.
7. any one described recombinant expression vector among the above-mentioned 1-3, the aminoacid sequence of wherein said fusion rotein is shown in SEQ ID No:4.
8. any one described recombinant expression vector among the above-mentioned 1-3, wherein said recombinant expression vector is derived from pET-3c.
9. method of expressing and producing people I shaped metal sulfoprotein with soluble form, this method may further comprise the steps:
1) makes up according to any one recombinant expression vector among the above-mentioned 1-8;
2) with 1) the middle recombinant expression vector transformed host cell that makes up;
3) culturing step 2 under being suitable for) with the condition of soluble form expressed fusion protein and ubiquitin relevant modifications factor protein enzyme by transformed host cells;
4) 3) host cell in give expression to the fusion rotein and the ubiquitin relevant modifications factor protein enzyme of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein respectively, and ubiquitin relevant modifications factor protein enzyme hydrolysis fusion rotein immediately;
5) remove insoluble part in the culture of described host cell, and from soluble fractions, reclaim the required active polypeptide of people I shaped metal sulfoprotein that has.
10. according to above-mentioned 9 method, wherein said being suitable for is meant 20 ℃ with the condition of soluble form expressed fusion protein and ubiquitin relevant modifications factor protein enzyme, and 0.4mmol/L IPTG induced 24 hours.
More specifically, an object of the present invention is to provide the recombinant vectors of soluble expressing human MT-I in intestinal bacteria, wherein said expression vector comprises the coding small molecule ubiquitin relevant modifications factor mature peptide that is operably connected with first promotor and the nucleotide sequence of people I shaped metal sulfoprotein fusion rotein, and described sequence downstream is connected with the nucleotide sequence of a coding total length ubiquitin relevant modifications factor protein enzyme that is operably connected with second promotor.
According to expression vector of the present invention, wherein said fusion rotein comprises: small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein, and two kinds of albumen is that N from fusion rotein holds C end coding successively.
According to expression vector of the present invention, wherein said first and second promotors all are selected from the phage t7 promotor.
According to expression vector of the present invention, the nucleotide sequence of the people I shaped metal sulfoprotein in the wherein said fusion rotein can be the sequence (SEQ ID No:1) of the wild sequence of screening in the natural origin or artificial chemosynthesis.
According to expression vector of the present invention, the nucleotide sequence of wherein said ubiquitin relevant modifications egg factor protein enzyme can be the sequence (SEQ IDNo:2) of the wild sequence of screening in the natural origin or artificial chemosynthesis.
Another object of the present invention provide a kind of in prokaryotic hosts the method with soluble form expressing human I shaped metal sulfoprotein, this method may further comprise the steps:
1) provides the nucleotide sequence (SEQ ID No:3) of the fusion rotein of coding small molecule ubiquitin relevant modifications factor mature peptide, people I shaped metal sulfoprotein;
2) provide the nucleotide sequence of coding ubiquitin relevant modifications factor protein enzyme;
3) nucleotides sequence that the middle nucleotide sequence of step (2) is connected to step (1) lists, and institute's calling sequence is connected among the suitable prokaryotic expression carrier pET-3c (Novagen co.Ltd.USA);
4) recombinant expression vector with step (3) transforms suitable e. coli host cell;
5) e. coli host cell that under being suitable for, is transformed in the culturing step (4) with the condition of soluble form expressed fusion protein and ubiquitin relevant modifications factor protein enzyme;
6) give expression to the fusion rotein (SEQ ID No:4) and the ubiquitin relevant modifications factor protein enzyme (SEQ ID No:5) of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein in the e. coli host cell respectively, and ubiquitin relevant modifications factor protein enzyme hydrolysis fusion rotein immediately;
7) remove insoluble part in the culture, and from soluble fractions, reclaim the required active polypeptide of people I shaped metal sulfoprotein (SEQ ID No:6) that has.
The method according to this invention, wherein said recombinant expression vector comprises that operability is connected to first promoter sequence that the nucleotides sequence of the fusion rotein of coding small molecule ubiquitin relevant modifications factor mature peptide (SEQ ID No:7) and people I shaped metal sulfoprotein lists, and be positioned at its downstream operability be connected to second promoter sequence that the nucleotides sequence of coding ubiquitin relevant modifications factor protein enzyme lists.
The method according to this invention, wherein said first and second promotors are phage t7 promotor (SEQ ID No:8).
The method according to this invention, wherein said being suitable for is meant 20 ℃ with the condition of soluble form expressed fusion protein and ubiquitin relevant modifications factor protein enzyme, 0.4mmol/L IPTG induced 24 hours.
The method according to this invention, wherein said small molecules ubiquitin relevant modifications factor mature peptide are meant the yeast saccharomyces cerevisiae ubiquitin relevant modifications factor (Smt3or yeast Small Ubiquitin-related Modifier) mature peptide (SEQ ID No:9).
The method according to this invention, wherein said ubiquitin relevant modifications factor protein enzyme be meant yeast saccharomyces cerevisiae ubiquitin relevant modifications factor protein enzyme 1 (ubiquitin-like protease-1, Ulp1).
The present invention relates to produce the method for people I shaped metal sulfoprotein, particularly relate to the amalgamation and expression that carries out that utilizes small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein, and this fusion rotein and ubiquitin relevant modifications factor protein enzyme 1 are at the intravital coexpression of prokaryotic organism, to produce the method for soluble human I shaped metal sulfoprotein.More particularly, the present invention relates to be used for intestinal bacteria recombinant expressed with the formal representation people I shaped metal sulfoprotein of solubility, the recombinant cell that is transformed by described recombinant expression vector, and the recombinant human I shaped metal sulfoprotein polypeptide of producing by the inventive method.The invention further relates to by the application of the people I shaped metal sulfoprotein polypeptide of method of the present invention preparation at food and cosmetic formulations.
The recombinant expression vector that the present invention is used for soluble expressing human I shaped metal sulfoprotein comprises first transcription unit of the fusion rotein that an expression is made up of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein and is used to express second transcription unit of ubiquitin relevant modifications factor protein enzyme that wherein said first transcription unit and second transcription unit are one another in series and are connected in the same recombinant vectors.
With regard to the two transcription units mode that is connected in series to each other, recombinant expression vector of the present invention at first comprises the operationally insertion site of second transcription unit of a nucleotide sequence that is operably connected to the fusion rotein that coding small molecule ubiquitin relevant modifications factor mature peptide on first promotor and people I shaped metal sulfoprotein form and.Insert the site by this, can easily in described expression vector, insert by the nucleotide sequence of coding ubiquitin relevant modifications factor protein enzyme and second startup molecular second transcription unit (SEQ ID No:10) of its upstream.
The preferred method of people I shaped metal sulfoprotein and ubiquitin relevant modifications factor protein enzyme gene of obtaining encoding is an artificial synthesis, because can avoid the influence of the sub-preference of amino acid code like this, thereby increases the expression amount of target protein.Can based on the source human body I shaped metal sulfoprotein aminoacid sequence (SEQ ID No:6) according to the described technology of people such as Itatura (Science, 1977,198:1056-1063) come synthetic gene.For example can go up sequence number based on GenBank is the synthetic people I shaped metal sulfoprotein of the described MT aminoacid sequence of NP_005937, can be based on people such as Li (Nature, 1999,398:246-251) described yeast ubiquitin relevant modifications factor protein enzyme 1 gene (SEQ IDNo:5) is synthetic.
As with the small molecules ubiquitin relevant modifications factor mature peptide gene of the nucleotide sequence amalgamation and expression of coding people I shaped metal sulfoprotein, the nucleotide sequence (SEQ ID No:7) of optimized encoding yeast saccharomyces cerevisiae small molecules ubiquitin relevant modifications factor mature peptide of the present invention.The yeast saccharomyces cerevisiae small molecules ubiquitin relevant modifications factor is a member of small molecules ubiquitin relevant modifications factor family, except having the protease inhibitor hydrolysis, significantly increasing expression of recombinant proteins amount and promote target protein correctly folding, outside the common function that most small molecules ubiquitin relevant modifications factors such as raising solubility all have, more, can be more suitable in prokaryotic hosts, expressing because it is a kind of for primary on molecular evolution.
As with the ubiquitin relevant modifications factor protein enzyme gene of the nucleotide sequence coexpression of coding small molecule ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein fusion rotein, the nucleotide sequence of optimized encoding ubiquitin relevant modifications factor protein enzyme 1 of the present invention.Contain two kinds of ubiquitin relevant modifications factor protein enzymes (Ulp1 and Ulp2) in the yeast saccharomyces cerevisiae, discover, although the fusion rotein that both equal energy hydrolysis SUMO modify, the effect of Ulp1 is more important and extensive.
Can use and anyly be suitable for therein e. coli strains with high-level efficiency coexpression desired protein (fusion rotein and the ubiquitin relevant modifications factor protein enzyme 1 formed by small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein) as the host, such bacterial strain comprises e. coli jm109, DH5, BL21, Origami (DE3) etc.The preferred host of the present invention is coli strain Origami (DE3) (Novagen co.Ltd.USA).
Can carry out synthetic, the clone of gene and operations such as structure, dna sequence analysis and the evaluation of expression vector, the conversion of host cell and the separation and purification of cultivation and expression product (referring to Sambrook et al. according to technology known in the art, Molecular Cloning:A Laboratory Manual, ColdSpring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1989).
Be purpose of the present invention, can at first utilize the nucleotide sequence of round pcr synthetic coding small molecule ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein fusion rotein directly to be connected T7 promotor downstream in the pET-3c expression vector that the present invention selects for use, obtain recombinant plasmid pET-SMT, the T7 promotor on the recombinant plasmid wherein and the gene of encoding fusion protein constitute first transcription unit.Then, utilize the nucleotide sequence of round pcr synthetic coding ubiquitin relevant modifications factor protein enzyme 1 again and be directly connected to T7 promotor downstream in the pET-3c expression vector that the present invention selects for use, obtain recombinant plasmid pET-Ulp1.Use is based on the nucleotide sequence synthetic oligonucleotide primer thing by second transcription unit of T7 promotor on the recombinant plasmid pET-Ulp1 and 1 genomic constitution of ubiquitin relevant modifications factor protein enzyme, and be template with the recombinant plasmid pET-Ulp1 that aforesaid method makes, obtain bifilar T7 promotor of synthetic and ubiquitin relevant modifications factor protein enzyme 1 gene order that two ends all have BamH I site through pcr amplification.Resulting coding T7 promotor and ubiquitin relevant modifications factor protein enzyme 1 gene order are carried out BamH I single endonuclease digestion and are connected to BamH I anticipating on the recombinant plasmid pET-SMT that makes as stated above, and then obtain connecting the recombinant vectors (pET-SMT-Ulp1) that contains coding small molecule ubiquitin relevant modifications factor mature peptide and the people I shaped metal sulfoprotein fusion rotein cDNA sequence that is under the T7 promoters driven and be in ubiquitin relevant modifications factor protein enzyme 1 gene under the T7 promoters driven with series system.Detailed process such as Fig. 2.
Can (transform or chemical process) according to a conventional method and will carry the recombinant plasmid transformed of above-mentioned first and second transcription units in suitable coli strain as electricity, and the method for knowing by those skilled in the art, for example use the LB culture medium culturing with the transformant of suitable antibiotic-screening (referring to Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989).
In the fermenting process, at first mono-clonal recombinant conversion that filters out is inoculated into and (contains 1% glucose and 100 μ g/ml penbritins) in the fresh LB substratum that contains penbritin, 30 ℃, 250rpm shaken overnight are cultivated.Then, get 100 μ l inoculums and be inoculated in the fresh LB substratum of 50ml and (contain 100 μ g/ml penbritins, but do not contain 1% glucose), during to OD600 to 0.6-1.0, add final concentration and be the IPTG of 0.4mmol/l and place 20 ℃ that 250rpm cultivated 24 hours.
After fermentation is finished, can collect thalline and separation and Culture thing supernatant by the method that those skilled in the art know, and with known three the step chromatography method (molecular sieve-ion exchange chromatography-molecular sieve) purification of soluble people I type MT (referring to ZL96100897.0), albumen behind the purifying is carried out reverse high performance liquid chromatography (HPLC) again separate, people I type MT is purified to purity is higher than 98%, silver-colored painted SDS-polyacrylamide gel electrophoresis is single band state.The structure verity of available flight mass spectrum (MOLDI-TOF), amino acid composition analysis method and amino-terminal end sequence measurement identifier I type MT, and measure reorganization MT melts combine ability with atomic absorption method and Cd/ bovine hemoglobin saturation method.
The MT of the inventive method preparation can be added pharmaceutical carrier well known to those skilled in the art or vehicle, make the formulations such as creme, emulsifying agent or aqua that are suitable for external application according to the ordinary method of cosmetic field.Also just the MT of the inventive method preparation adds carrier that the field of food technician knows or vehicle and makes according to the ordinary method of field of food and be suitable for oral solid-state or liquid-food.
Description of drawings
The physiological process of Fig. 1 .SUMO modified protein;
Fig. 2. the structure of recombinant plasmid pET-SMT-Ulp1;
Fig. 3. the synthetic step of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein antigen-4 fusion protein gene;
Fig. 4. the synthetic step of ubiquitin relevant modifications factor protein enzyme gene;
Fig. 5. enzyme is cut a recombinant human MT-I protein electrophoresis figure (left side: recombinant human MT-I albumen that obtains behind the purifying; Right: molecular weight marker);
Fig. 6. the mass spectrum of recombinant human MT-I (MOLDI-TOF) is analyzed;
Fig. 7. the proteic N-terminal of recombinant human MT-I (preceding 8 amino acids) sequencing result.
Embodiment
For the sake of clarity, below each sequence in the attached sequence table in back is made brief description:
SEQ ID NO:1: the nucleotide sequence of people I shaped metal sulfoprotein is (referring to NM_005946.2; 5 '-3 ', totally 186 bases)
SEQ ID NO:2: the nucleotide sequence of ubiquitin relevant modifications egg factor protein enzyme is (referring to NC_001148.3; 5 '-3 ', totally 666 bases)
SEQ ID NO:3: the nucleotide sequence of the fusion rotein of coding small molecule ubiquitin relevant modifications factor mature peptide, people I shaped metal sulfoprotein (5 '-3 ', totally 480 bases)
SEQ ID NO:4: the fusion rotein of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein (N end-C end, totally 159 amino acid)
SEQ ID NO:5: ubiquitin relevant modifications factor protein enzyme is (referring to NP_015305; N end-C end, totally 221 amino acid)
SEQ ID NO:6: people I shaped metal sulfoprotein is (referring to NP_005937; N end-C end, totally 61 amino acid)
SEQ ID NO:7: the nucleotide sequence of coding small molecule ubiquitin relevant modifications factor mature peptide is (referring to Schwartz D.C.et al., 2003, Trends Biochem.Sci., 28:321-328; 5 '-3 ', totally 294 bases)
SEQ ID NO:8: the nucleotide sequence of phage t7 promotor is (referring to AF525445; 5 '-3 ', totally 83 bases)
SEQ ID NO:9: yeast saccharomyces cerevisiae ubiquitin relevant modifications factor mature peptide (referring to Johnson, E.S.2004, Annu.Rev.Biochem., 73,355-382; N end-C end, totally 98 amino acid)
SEQ ID NO:10: second of the nucleotide sequence of ubiquitin relevant modifications factor protein enzyme and its upstream starts molecular second transcription unit (5 '-3 ', totally 746 bases)
SEQ ID NO:11:SUMO-F1 (5 '-3 ', totally 34 bases)
SEQ ID NO:12:SUMO-F2 (5 '-3 ', totally 59 bases)
SEQ ID NO:13:SUMO-F3 (5 '-3 ', totally 59 bases)
SEQ ID NO:14:SUMO-F4 (5 '-3 ', totally 59 bases)
SEQ ID NO:15:SUMO-R1 (5 '-3 ', totally 59 bases)
SEQ ID NO:16:SUMO-R2 (5 '-3 ', totally 59 bases)
SEQ ID NO:17:SUMO-R3 (5 '-3 ', totally 59 bases)
SEQ ID NO:18:SUMO-R4 (5 '-3 ', totally 59 bases)
SEQ ID NO:19:MT-F1 (5 '-3 ', totally 59 bases)
SEQ ID NO:20:MT-F2 (5 '-3 ', totally 59 bases)
SEQ ID NO:21:MT-R1 (5 '-3 ', totally 59 bases)
SEQ ID NO:22:MT-R2 (5 '-3 ', totally 59 bases)
SEQ ID NO:23:MT-R3 (5 '-3 ', totally 59 bases)
The nucleotide sequence of SEQ ID NO:24:S1 (5 '-3 ', totally 98 bases)
The nucleotide sequence of SEQ ID NO:25:S2 (5 '-3 ', totally 176 bases)
The nucleotide sequence of SEQ ID NO:26:S3 (5 '-3 ', totally 254 bases)
The nucleotide sequence of SEQ ID NO:27:S4 (5 '-3 ', totally 304 bases)
The nucleotide sequence of SEQ ID NO:28:M1 (5 '-3 ', totally 98 bases)
The nucleotide sequence of SEQ ID NO:29:M2 (5 '-3 ', totally 176 bases)
The nucleotide sequence of SEQ ID NO:30:M3 (5 '-3 ', totally 216 bases)
SEQ ID NO:31:T7promoter primer and T7terminator primer
T7promoter primer:TAATACGACTCACTATAGG (5 '-3 ', totally 19 bases)
T7terminator primer:CCGCTGAGCAATAACTAGC (5 '-3 ', totally 19 bases)
SEQ ID NO:32:Ulp-F1 (5 '-3 ', totally 59 bases)
SEQ ID NO:33:Ulp-F2 (5 '-3 ', totally 59 bases)
SEQ ID NO:34:Ulp-F3 (5 '-3 ', totally 59 bases)
SEQ ID NO:35:Ulp-F4 (5 '-3 ', totally 59 bases)
SEQ ID NO:36:Ulp-F5 (5 '-3 ', totally 46 bases)
SEQ ID NO:37:Ulp-F6 (5 '-3 ', totally 33 bases)
SEQ ID NO:38:Ulp-F7 (5 '-3 ', totally 59 bases)
SEQ ID NO:39:Ulp-F8 (5 '-3 ', totally 59 bases)
SEQ ID NO:40:Ulp-F9 (5 '-3 ', totally 59 bases)
SEQ ID NO:41:Ulp-R1 (5 '-3 ', totally 59 bases)
SEQ ID NO:42:Ulp-R2 (5 '-3 ', totally 59 bases)
SEQ ID NO:43:Ulp-R3 (5 '-3 ', totally 59 bases)
SEQ ID NO:44:Ulp-R4 (5 '-3 ', totally 59 bases)
SEQ ID NO:45:Ulp-R5 (5 '-3 ', totally 59 bases)
SEQ ID NO:46:Ulp-R6 (5 '-3 ', totally 59 bases)
SEQ ID NO:47:Ulp-R7 (5 '-3 ', totally 59 bases)
SEQ ID NO:48:Ulp-R8 (5 '-3 ', totally 59 bases)
SEQ ID NO:49:Ulp-R9 (5 '-3 ', totally 59 bases)
SEQ ID NO:50:UI (5 '-3 ', totally 215 bases)
SEQ ID NO:51:UII (5 '-3 ', totally 254 bases)
SEQ ID NO:52:UIII (5 '-3 ', totally 255 bases)
SEQ ID NO:53:UIV (5 '-3 ', totally 469 bases)
SEQ ID NO:54:Ulp1-T7-F (5 '-3 ', totally 53 bases)
Embodiment 1: the synthetic of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein antigen-4 fusion protein gene
Want the gene order of artificial composite coding small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein fusion rotein, must synthesize 13 oligonucleotide chains (synthetic), be respectively: SUMO-F1 (SEQ ID No:11) by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, SUMO-F2 (SEQ ID No:12), SUMO-F3 (SEQ ID No:13), SUMO-F4 (SEQ ID No:14), SUMO-R1 (SEQ ID No:15), SUMO-R2 (SEQ ID No:16), SUMO-R3 (SEQ ID No:17), SUMO-R4 (SEQ ID No:18), MT-F1 (SEQ ID No:19), MT-F2 (SEQ ID No:20), MT-R1 (SEQ ID No:21), MT-R2 (SEQID No:22) and MT-R3 (SEQ ID No:23).Synthetic detailed process such as Fig. 3.In brief, at first, the first step PCR reaction adds each 2 μ l (10 μ molL of primer SUMO-F4 and SUMO-R4
-1), dNTP (each 2.5 μ molL
-1) 10 μ l, 10x PCR pfu damping fluid 10 μ l, adding water to cumulative volume is 100 μ l.Behind 94 ℃ of sex change 5min, slowly be cooled to 55 ℃, add the pfuDNA polysaccharase again, 72 ℃ are extended 5min then, and electrophoresis detection also reclaims dna fragmentation, called after S1 (SEQ ID No:24).Carrying out the second step PCR reaction then, is template with S1, adds primer SUMO-F3 and SUMO-R3 as the upstream and downstream primer, adds dNTP (each 2.5 μ molL again
-1) 10 μ l, 10x PCR pfu damping fluid 10 μ l and pfu Taq archaeal dna polymerase 1 μ l (5U/ μ l), adding water to cumulative volume is 100 μ l, PCR reaction conditions according to standard (enters circulation behind 94 ℃ of sex change 4min, loop parameter is 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended totally 30 circulations 30 seconds) carry out the amplification of second step.Electrophoresis detection also reclaims target DNA fragment and called after S2 (SEQ ID No:25).Be template again with S2, add primer SUMO-F2 and SUMO-R2, set up reaction system and carry out pcr amplification, the target DNA fragment called after S3 of acquisition (SEQ ID No:26) according to the parameter of the second step PCR reaction as the upstream and downstream primer.Being template again with S3, is the upstream and downstream primer with SUMO-F1 and SUMO-R1, sets up reaction system and carries out pcr amplification, the target DNA fragment called after S4 (SEQID No:27) of acquisition according to the parameter of the second step PCR reaction.Set up another PCR reaction system again, add each 2 μ l (10 μ molL of primer MT-F2 and MT-R3
-1), dNTP (each 2.5 μ molL
-1) 10 μ l, 10x PCR pfu damping fluid 10 μ l, adding water to cumulative volume is 100 μ l.Behind 94 ℃ of sex change 5min, slowly be cooled to 55 ℃, add the pfuDNA polysaccharase again, 72 ℃ are extended 5min then, and electrophoresis detection also reclaims dna fragmentation, called after M1 (SEQ ID No:28).With M1 is template, adds primer MT-F1 and MT-R2 as the upstream and downstream primer, sets up reaction system and carries out pcr amplification, the target DNA fragment called after M2 of acquisition (SEQ ID No:29) according to the parameter of the second step PCR reaction.With M2 is template, adds primer MT-F1 and MT-R1 as the upstream and downstream primer, sets up reaction system and carries out pcr amplification, the target DNA fragment called after M3 (SEQ IDNo:30) of acquisition according to the parameter of the second step PCR reaction.At last, with M3 and S4 is common template, SUMO-F1 and MT-R1 are the upstream and downstream primer, set up the reaction system performing PCR amplification of going forward side by side according to the parameter of the second step PCR reaction, final acquisition length is about the PCR product of 500bp, and the product two ends have Nde I (CATATG) and BamH I (GGATCC) restriction enzyme site respectively.This PCR product with Nde I and BamH I double digestion, is connected among the expression vector pET-3c that crosses with these two kinds of enzyme same treatment then, constitutes recombinant expression plasmid pET-SMT.This plasmid is converted among the competent cell DH5.Cultivate propagation back extracting recombinant plasmid from transformant, by known method and use universal primer T7promoter primer and T7terminator primer (synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; SEQID No:31) mensuration forward and the oppositely dna sequence dna of junction fragment (providing the order-checking service by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), the result shows that the gained fragment is the nucleotide sequence of 159 amino acid whose small molecules ubiquitin relevant modifications factor mature peptides of coding and people I shaped metal sulfoprotein fusion rotein.
Embodiment 2: the structure of recombinant vectors pET-Ulp1
At first synthetic 18 oligonucleotide chains (synthetic) by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, be respectively: Ulp-F1 (SEQ ID No:32), Ulp-F2 (SEQ ID No:33), Ulp-F3 (SEQ ID No:34), Ulp-F4 (SEQ ID No:35), Ulp-F5 (SEQ ID No:36), Ulp-F6 (SEQ ID No:37), Ulp-F7 (SEQ ID No:38), Ulp-F8 (SEQID No:39), Ulp-F9 (SEQ ID No:40), Ulp-R1 (SEQ ID No:41), Ulp-R2 (SEQ ID No:42), Ulp-R3 (SEQ ID No:43), Ulp-R4 (SEQ ID No:44), Ulp-R5 (SEQ ID No:45), Ulp-R6 (SEQ ID No:46), Ulp-R7 (SEQ ID No:47), Ulp-R8 (SEQ ID No:48) and Ulp-R9 (SEQ ID No:49).According to method described in the embodiment 1 and the step among Fig. 4, utilize three big fragments of round pcr synthetic, difference called after UI (SEQ ID No:50), UII (SEQ ID No:51) and UIII (SEQID No:52).Then, be common template with UI and UII, adding Ulp-F6 and Ulp-R3 is the upstream and downstream primer, amplifies length according to parameter described in the step PCR of second among the embodiment 1 reaction and method and is about 469 big fragment UIV (SEQ ID No:53).At last, with UIV and UIII is common template, with Ulp-F6 and Ulp-R9 is the upstream and downstream primer, amplify length according to parameter described in the step PCR of second among the embodiment 1 reaction and method and be about 702bp, the PCR product of the ulp1 of Nde I (CATATG) and BamH I (GGATCC) restriction enzyme site is contained at two ends respectively.This PCR product with Nde I and BamH I double digestion, is connected among the expression vector pET-3c that crosses with these two kinds of enzyme same treatment then, constitutes recombinant expression plasmid pET-Ulp1.This plasmid is converted among the competent cell DH5.Cultivate propagation back extracting recombinant plasmid from transformant, by known method and use universal primer (T7promoter primer and T7terminator primer) to measure the forward and the reverse dna sequence dna of junction fragment, the result shows that the gained fragment is 219 proteic nucleotide sequences of amino acid whose Ulp1 of coding.
Embodiment 3: the structure of recombinant expression vector pET-SMT-Ulp1
To comprise being connected with and be in the recombinant expression vector that T7 promotor control containing down is in coding small molecule ubiquitin relevant modifications factor mature peptide and the people I shaped metal sulfoprotein fusion rotein cDNA sequence under the control of T7 promotor and is in ubiquitin relevant modifications factor protein enzyme 1 gene of T7 promotor under controlling in order to make up with series system, at first, (synthetic based on synthetic oligonucleotide primer Ulp1-T7-F:5 ' the CT GGA TCC GAA TTC GAG CTC AAG CTT CTC GAGTAA TAC GAC TCA CTA TAG GGA G3 ' of T7 promotor by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; SEQ ID NO:54).5 ' end of this primer contains a multiple clone site.PCR method according to routine, with recombinant expression vector pET-Ulp1 is template, Ulp1-T7-F and Ulp-R9 are the upstream and downstream primer, enter circulation behind 94 ℃ of sex change 4min, loop parameter is 94 ℃ of sex change 30 seconds, anneals 30 seconds for 55 ℃, 72 ℃ were extended 30 seconds, totally 30 circulations, amplification obtain 5 ' end and contain the T7 promotor of a multiple clone site and the dna fragmentation of ubiquitin relevant modifications factor protein enzyme 1 genomic constitution, and two ends all have BamH I restriction enzyme site.The dna fragmentation of T7 promotor and 1 genomic constitution of ubiquitin relevant modifications factor protein enzyme is the dna fragmentation of second transcription unit (SEQ ID NO:10) just.
This fragment is carried out single endonuclease digestion with BamH I, be connected to then among the carrier pET-SMT that crosses with BamH I same treatment, be built into recombinant expression vector pET-SMT-Ulp1.This plasmid is converted among the competent cell DH5.Cultivating propagation back extracting recombinant plasmid from transformant, all is correct by known method and direction and the amino acids coding of using universal primer (T7promoter primer and T7terminator primer) forward and backward sequencing to determine that fragment is inserted.Can utilize two multiple clone site between transcription unit to insert nucleotide sequence that length differ to adjust the distance between two units, to obtain the best efficient of transcribing.Utilize electricity to change or chemical process such as CaCl this recombinant plasmid
2Be transformed among the intestinal bacteria Origami (DE3), utilize amicillin resistance LB solid plate (10g/L peptone, 5g/l yeast extract, 10g/l sodium-chlor, 20g/l agar powder, 100 μ g/ml penbritins) to filter out recon.
The proteic separation and purification of embodiment 4:MT
After the fermentation ends, the centrifugal collection thalline of 12000rpm, the resuspended thalline of phosphate buffered saline buffer (pH7.0) of adding 0.02mol/L.Utilize ultrasonic disruption instrument smudge cells.Centrifugal 30 minutes of 18000rpm collects supernatant.With this supernatant Tris-HCl (pH7.0) equilibrated dextrane gel at first, collect the albumen elution peak of molecular weight less than 10000Kda by using 0.05mol/L.Use cation-exchange chromatography (CM-Sephadex) that the protein sample in this peak is separated once more.After washing post with Tris-HCl (pH7.0) solution of the NaCl that contains 0.1mol/l (3-5 column volume altogether), add Tris-HCl (pH7.0) the wash-out target protein that contains 0.5mol/l NaCl.Collect elution peak, and the elution peak that utilizes 18% polyacrylamide gel electrophoresis (SDS-PAGE) testing goal albumen to exist.The elution peak that will contain target protein is collected different elution peaks and is utilized polyacrylamide gel electrophoresis (SDS-PAGE) testing goal albumen location once more by carrying out desalination with ultrapure water equilibrated dextrane gel.The soln using HPLC that will contain target protein separates at last, carries out wash-out with the gradient of 0.05% trifluoroacetic acid+acetonitrile (30-60%)+water (60-30%), and the time is 60 minutes, and the MT purity of protein of collection is higher than 98%.MT albumen behind the purifying is after 18% polyacrylamide gel electrophoresis separation, and cma staining shows the single band (Fig. 5) of the about 6.2Kda of molecular weight.Mensuration (Fig. 6) by flight mass spectrum, amino acid N end order-checking (Fig. 7) and amino acid composition analysis (table 1) can confirm the verity by the MT of the present invention's preparation.According to method of the present invention, on the shake flask fermentation level, every liter of fermented liquid can obtain the recombinant human MT-I albumen of 25.2mg.
The proteic amino acid composition analysis result of table 1 recombinant human MT-I
| Amino acid | Predictor | Observed value |
| Glx (Q and E) Lys (K) Leu (L) Thr (T) Met (M) Phe (F) Gly (G) Arg (R) Asx (N and D) Ile (I) His (H) Pro (P) Tyr (Y) Val (V) Ser (S) Cys (C) Ala (A) Trp (W) | 3 8 0 3 2 0 5 0 3 1 0 2 0 0 9 20 5 0 | 3.00 8.12 0 2.89 1.87 0 4.86 0 3.50 1.20 0 2.12 0 0 9.12 20.10 4.89 0 |
| Altogether | 61 |
(Q, E) number is a standard, calculates other amino acid whose number (residue number/mol) according to the Glx that contains among every mole the reorganization hMT-I after the acid hydrolysis
Embodiment 5:Cd/ bovine hemoglobin saturation method is measured reorganization MT melts combine ability
Add the Tris-HCI damping fluid 500 μ l of 0.01mol/l pH8.6 in the protein solution of 100 μ l of above acquisition, final concentration is the CdCl of 700 μ mol/l
2The solution mixing adds mass percent and is 2% bovine hemoglobin 200 μ l mixings again, leaves standstill 10 minutes; Heating is 2-3 minute in the boiling water bath, and the centrifugal 10min of 12000rpm collects supernatant.Atomic absorption method (referring to Honda et al., 2005, Journal of Chromatography B, 820,205-210) measure wherein Cd content, the result shows that the MT that makes by invention can be in conjunction with 7 Cd ions.
Be equal to embodiment: the present invention can be presented as that other are concrete but not depart from the form of its spirit or essential characteristic, therefore think that previous embodiments is to illustrate rather than limit invention described herein in all respects.Therefore, scope of the present invention is included in claims and is equal to used modification in purpose and the scope.
Sequence table
＜110〉the former peptide bio tech ltd in Biopharmaceutial Research ﹠ Development Center of Jinan University Guangzhou
＜120〉recombinant expression vector and the method for soluble expressing human I shaped metal sulfoprotein
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Claims (10)
1. the recombinant expression vector of an expressing human I shaped metal sulfoprotein, it comprises first promotor and second promotor, described first promotor is operably connected with the nucleotide sequence of the fusion rotein of coding small molecule ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein, and described second promotor is operably connected with the nucleotide sequence of coding ubiquitin relevant modifications factor protein enzyme.
2. the described recombinant expression vector of claim 1, wherein said fusion rotein comprises: small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein, and two kinds of albumen is that N from fusion rotein holds C end coding successively.
3. the described recombinant expression vector of claim 1, wherein said first and second promotors all are selected from the phage t7 promotor.
4. any one described recombinant expression vector among the claim 1-3, the aminoacid sequence of the people I shaped metal sulfoprotein in the wherein said fusion rotein is shown in SEQ ID No:6.
5. any one described recombinant expression vector among the claim 1-3, the aminoacid sequence of wherein said ubiquitin relevant modifications egg factor protein enzyme is shown in SEQ ID No:5.
6. any one described recombinant expression vector among the claim 1-3, the aminoacid sequence of wherein said small molecules ubiquitin relevant modifications factor mature peptide is shown in SEQ ID No:9.
7. any one described recombinant expression vector among the claim 1-3, the aminoacid sequence of wherein said fusion rotein is shown in SEQ ID No:4.
8. any one described recombinant expression vector among the claim 1-3, wherein said recombinant expression vector is derived from pET-3c.
9. method of expressing and producing people I shaped metal sulfoprotein with soluble form, this method may further comprise the steps:
1) makes up according to any one recombinant expression vector among the claim 1-8;
2) with 1) the middle recombinant expression vector transformed host cell that makes up;
3) culturing step 2 under being suitable for) with the condition of soluble form expressed fusion protein and ubiquitin relevant modifications factor protein enzyme by transformed host cells;
4) 3) host cell in give expression to the fusion rotein and the ubiquitin relevant modifications factor protein enzyme of small molecules ubiquitin relevant modifications factor mature peptide and people I shaped metal sulfoprotein respectively, and ubiquitin relevant modifications factor protein enzyme hydrolysis fusion rotein immediately;
5) remove insoluble part in the culture of described host cell, and from soluble fractions, reclaim the required active polypeptide of people I shaped metal sulfoprotein that has.
10. according to the method for claim 9, wherein said being suitable for is meant 20 ℃ with the condition of soluble form expressed fusion protein and ubiquitin relevant modifications factor protein enzyme, and 0.4mmol/L IPTG induced 24 hours.
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