CN102839182A - Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system - Google Patents
Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system Download PDFInfo
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Abstract
The invention discloses a method preparing a recombinant human nerve growth factor by using an Escherichia coli expression system. Beta-subunit genes of the recombinant human nerve growth factor comprise 6 histidine purification sites, an enterokinase cleavage site and a beta-subunit gene encoding mature peptide of modified human nerve growth factor and a molecular chaperone gene. The beta-subunit genes of the recombinant human nerve growth factor have high expression quantity in the Escherichia coli; generated inclusion body protein has high-efficiency renaturation under the assist of the molecular chaperone; and the recombinant human nerve growth factor obtained by nickel column affinity chromatography after the molecular chaperone is cleavaged accurately by the enterokinase, has a purity greater than 99% and biological activity greater than 500,000 AU/mg.
Description
Technical field
The present invention relates to a kind of method of utilizing escherichia expression system to prepare RHNGF, belong to the biological medicine technology field.
Background technology
(Nerve Growth Factor is to find the earliest and most typical neurotrophic factor NGF) to NGFF, also is one of most important bioactive molecules in the neural system simultaneously.It is made up of α, β, three subunits of γ, and wherein (β-NGF) is unique subunit with BA to the β subunit, and it all has important regulation to the expression of growth, differentiation, growth, regeneration and the functional performance of maincenter and peripheral nerve unit.Present β-NGF has been developed to the medicinal application of treatment peripheral nerve injury in clinical, is widely used in nervous system disease fields such as nerve injury, hemiplegia, palsy, craniocerebral injury, cerebral palsy of children.But commercial NGF is the NGFF (mNGF) of mouse source property.The animal derived protein product of this type usually has higher immunogenicity, causes the intravital antigen-reactive of people easily, severe patient even entail dangers to life.And natural growth factor of human nerve (hNGF) is distributed in tissues such as the brain, neuroganglion, iris, heart, spleen, placenta of human body, and raw material is limited, and wherein hNGF content is low, and separation and purification is extremely difficult, can't directly extract acquisition.Utilizing genetic engineering technique to prepare RHNGF is a main path that addresses the above problem.
In each big expression system, escherichia expression system is because of its expression level is high, production cost is low, be convenient to the first-selection that advantage such as suitability for industrialized production becomes the preparation RHNGF., part eukaryotic gene expression amount in escherichia expression system is extremely low, does not even express, and reason is that the codon of eukaryote and prokaryotic organism institute preference is different.In addition, intestinal bacteria lack the processing modification system after eukaryotic protein synthesizes, and therefore, expressed proteins usually abiology is active.Investigators find that the expression amount of human in intestinal bacteria is not high, and biological activity is extremely low, mainly are because β-ngf gene and albumen particular structural thereof cause it to be difficult to effective expression in the intestinal bacteria system through analyzing.
Used more intestinal bacteria rare codon in β-ngf gene on the one hand, caused β-ngf gene not high in the expression in escherichia coli amount.
β-NGF protein structure is special on the other hand, is difficult in the intestinal bacteria that lack translation post-treatment modification system, form correct structure, causes the β-NGF protein biological activity that gives expression to extremely low, even does not have activity.The strand that natural β-NGF is made up of two 118 amino acid passes through the dimer of non covalent bond be combined into; Wherein every strand contains by 6 halfcystines and matches 3 pairs of disulfide linkage that form each other; These 3 pairs of disulfide linkage constitute the typical structural pattern of neurotrophic factor family---" cystine knot " pattern (the cystine knot); Promptly two disulfide linkage bridges (Cys58-Cys108 and Cys68-Cys110) form a ring (loop) that contains 14 residues, and Cys15 and Cys80 pass this ring and form the 3rd disulfide linkage bridge.This special construction pattern is conservative at the neurotrophic factor family camber, and is most important to the normal biological function of keeping β-NGF.β-NGF that escherichia expression system is expressed often because above-mentioned 6 halfcystines can not correct pairing, forms above-mentioned " cystine knot " structure, promptly can not form correct protein structure and cause its biological activity extremely low.
To the problems referred to above, general way is the gene that need are expressed to be carried out codon optimized, makes up the fusion rotein of Chaperones Molecular and target protein simultaneously, is folded into correct structure through Chaperones Molecular help target protein is external.
Carry out codon optimized to gene; Need to consider e. coli codon preference property; Also the factors such as joint efficiency of considered gene order and mRNA sequential structure, stability and the ribosome binding sequence of transcribing are a complicacy, the higher work of technical difficulty.
The structure of fusion rotein, the selection of Chaperones Molecular are crucial.In order to improve the folding and annealing efficiency of β-NGF, part Study person has made up the fusion rotein of Trx and disulfide linkage formation albumen and β-NGF.For example: (HE Ling-bing, WANG Yanz, WANG Ge such as HE Ling-bing; CHEN Chao, and CAO Shu-gui, Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichiu coli.CHEM. RES. CHINESEU; 2007, Vol.23 was No.2) with Trx and histidine-tagged protein and the fusion of hNGF β subunit; Utilization comes separation and purification β-NGF to the affinity chromatography of the special absorption of label protein, utilize Trx to help β-NGF simultaneously and express with the mode of solubility expression, but prokaryotic cell prokaryocyte is less; Have only when expression amount hour; Albumen could exist with the solubility mode, and when the foreign protein great expression, most albumen still can be assembled sex change; (Ding Hongmei keeps and deposits Sun Weiguo etc. for Sun Weiguo, Liu Nongle; Zhao Qiang, Xu Hua, doubly put forth energy in Shen; Shao Ningsheng, the protokaryon amalgamation and expression and the activity research thereof of RHNGF β subunit. biotechnology communication, 2009; Vol.20, the DsbA, DsbC albumen and the Trx (Trx) that No.4) disulfide linkage are formed in the protein family (Dsb) are fusion molecule, carry out amalgamation and expression with hNGF β subunit at prokaryotic expression system; Can assist the external renaturation of β-NGF though disulfide linkage forms albumen, can not discern 3 pairs of accurate positions of disulfide linkage of β-NGF, therefore also be difficult to reach ideal renaturation effect; And the end product of this research carries label protein, and is inconsistent with natural β-NGF, can't be used for drug manufacture.
Therefore, pressing for exploitation in this area is the method that purpose prepares RHNGF with low cost, high yield and high reactivity.Described preparation comprises the genetic modification that growth factor of human nerve is carried out, the amalgamation and expression of Chaperones Molecular and separation, the purifying of recombinant protein.
Summary of the invention
In view of this; One of the object of the invention is to provide a kind of RHNGF beta subunit gene; It contains 6 Histidine purifying sites, enteropeptidase cleavage site and growth factor of human nerve β subunit mature peptide fragment gene and molecular chaperone through modifying; Wherein said growth factor of human nerve β subunit mature peptide segment base is because through the improved gene of prokaryotic organism high frequency codon, its base sequence is shown in SEQ ID NO:1; Said molecular chaperone is the growth factor of human nerve β subunit leading peptide gene through modifying, and its base sequence is shown in SEQ ID NO:2.
Two of the object of the invention has been to provide the method for utilizing escherichia expression system to prepare RHNGF.
According to an aspect of the present invention, the base sequence of the growth factor of human nerve β subunit mature peptide gene of warp modification (357bp altogether) shown in SEQ ID NO:1.
Base sequence in this sequence is to have carried out the sequence that codon is transformed, and its codon is the high frequency codon of escherichia expression system, can under the prerequisite that does not change aminoacid sequence, improve the expression amount of goal gene to greatest extent.
According to a further aspect in the invention, the base sequence (309bp altogether) shown in SEQ ID NO:2 through modifying as the leading peptide gene of Chaperones Molecular.
This sequence is positioned at the growth factor of human nerve β subunit mature peptide gene order of the present invention upper reaches, and two sequences are connected by enteropeptidase cleavage site (GATGATGATGATAAA).This Chaperones Molecular can help growth factor of human nerve β subunit with the inclusion body formal representation in the correct conformation of external formation; And can after renaturation, the cutting through enteropeptidase remove; Thereby discharge with natural β-NGF deduced amino acid sequence, and have the mature peptide of higher biological activity.
The method of utilizing escherichia expression system to prepare RHNGF (rh β NGF) of the present invention comprises the steps:
(1) utilizes chemical synthesis synthetic, and it is cloned into pET28a (+) expression vector, obtain recombinant expression vector through the improved growth factor of human nerve β of prokaryotic organism high frequency codon subunit mature peptide fragment gene and molecular chaperone;
(1.1) the said gene fragment is inserted BamHI (846) and XhoI (159) site (referring to the Novagen company plasmid map) of prokaryotic expression plasmid pET28a (+) after with BamHI and XhoI restriction enzymes double zyme cutting;
(1.2) obtain recombinant expression vector pET28a (+)+[rh β NGF], through PCR and enzyme cut identify insert correctly after, its sequence exactness of sequence verification;
(2) step (1) gained recombinant expression vector is imported among the e. coli strains BL21 (λ DE3), make up the genetic engineering bacterium that contains recombinant expression vector;
When (3) culturing step (2) gained genetic engineering bacterium is to A550=0.6, add inductor IPTG, induce RHNGF rh β NGF in this genetic engineering bacterium, to express;
(4) centrifugal collection thalline, resuspended thalline ,-80 ℃ are frozen;
(5) the resuspended liquid of thalline that thaws, broken thalline, separation and purification inclusion body protein (washing separates inclusion body, 8M urea dissolving inclusion body protein);
(6) external renaturation inclusion body protein, dialysis back ultrafiltration and concentration;
(7) remove Chaperones Molecular through the enteropeptidase cutting;
(8) purifying obtains said RHNGF.
Preferably, the inductive time is 3-4 hour in the wherein said step (3).
Preferably, the concentration of inductor IPTG is 1mmol/L in the wherein said step (3).
Preferably, the resuspended liquid in the wherein said step (4) is 20mM Tris-HCl (pH8.0), and usage quantity is 20 times of volumes of thalline weight in wet base.
Preferably, the method for broken thalline is a sonioation method in the wherein said step (5), and wherein the used power of ultrasonication thalline is 400W, ultrasonic 15 seconds, 15 seconds at interval, acts on 30 minutes.
Preferably, the method for renaturation inclusion body protein is external dilution refolding method in the wherein said step (6), and in the wherein external dilution refolding liquid, arginic concentration range is 0.75-1.0mmol/L, and the pH value is 8.0-9.5.
Preferably, arginic concentration is 0.75mM in the external dilution refolding liquid that wherein said step (6) is used, and the pH value is 9.5; Redox system-reduced glutathion-Sleep-promoting factor B (GSH-GSSG) concentration is 0.5mM GSSG-5mM GSH, and the renaturation time is 3 hours; Dialyzate is 50 mM Tris-HCl damping fluids.
Preferably, the enteropeptidase in the wherein said step (7) is the recombination ox intestine kinase with 6 Histidine purifying sites.
Preferably, the enteropeptidase operative temperature in the wherein said step (7) is 21 ℃, and be 16h action time;
Preferably, the method for purifying is a nickel post affinity chromatography in the wherein said step (8).
Preferably; The filler of nickel post affinity chromatography is Ni Sepharose Fast Flow in the wherein said step (8); Use the damping fluid balance chromatography column that contains 50 mM imidazoles-0.5 M NaCl-20mMTris-HCl (pH8.0) in the chromatography process; It is the component that contains rh β NGF that the stream of collecting in the last appearance process is worn the peak, obtains purity greater than 99% through dialysis and ultrafiltration and concentration, and biological specific activity is higher than the RHNGF of 500000AU/mg.
The present invention analyzes β-ngf gene; According to the preference property of intestinal bacteria to the synonymous code selection; Eliminating rare codon, unstable sequence; Preferred on the basis of best codon, β-ngf gene is carried out design again, thereby improved the expression amount of β-NGF in intestinal bacteria to greatest extent.
The present invention is according to the constructional feature of natural β-NGF; The leading peptide of having selected β-NGF is as its Chaperones Molecular; The disulfide linkage that this peptide section can effectively be discerned among natural β-NGF forms the site, and can make it form disulfide linkage in the tram, thereby guarantees the correct folding and renaturation of β-NGF.In addition, be separation and purification that makes things convenient for target protein and the unicity that guarantees end product, the present invention has introduced 6 Histidine purifying sites at the leading peptide upper reaches, between leading peptide and mature peptide, designed the enteropeptidase cleavage site.This plan makes expression product can pass through the affinity chromatography separation and purification, and simple operation, and purification efficiency is high can discharge with natural β-NGF deduced amino acid sequence through the enteropeptidase cutting simultaneously, and has the target protein of higher biological activity.
The present invention has obtained efficiently expressing of rh β NGF through after growth factor of human nerve β subunit leading peptide and mature peptide fragment gene being carried out prokaryotic organism high frequency codon and transforming in escherichia coli expression bacterial strain BL21 (λ DE3).Molecular chaperone function by leading peptide; Rh β NGF with the inclusion body formal representation can be at external highly efficient renaturation; Rh β NGF after the renaturation can discharge rh β NGF mature peptide through the enteropeptidase cutting, can obtain purity greater than 99% through nickel post affinity chromatography, and biological specific activity is higher than the RHNGF of 500000AU/mg; This preparation method is simple and easy to do, is convenient to implement.
Description of drawings
Fig. 1 is the SDS-PAGE figure of IPTG induced concentration to the protein expression influence, and wherein 1 is standard molecular weight albumen; 2 is the empty bacterium of BL21; 3 for not adding the engineering bacteria of IPTG; 4 is IPTG concentration 0.1 mmol/L; 5 is IPTG concentration 0.5 mmol/L; 6 is IPTG concentration 1.0 mol/L; 7 is IPTG concentration 1.5 mmol/L; 8 is IPTG concentration 2.0 mmol/L; 9 is IPTG concentration 3.0 mmol/L; 10 is IPTG concentration 4.0 mmol/L; 11 is IPTG concentration 5.0 mmol/L;
Fig. 2 is the SDS-PAGE figure of induction time to the protein expression influence, and wherein 1 is standard molecular weight albumen; 2 is the empty bacterium of BL21; 3 for not adding the engineering bacteria of IPTG; 4 for inducing 0 hour; 5 for inducing 2 hours; 6 for inducing 4 hours; 7 for inducing 6 hours; 8 for inducing 8 hours; 9 for inducing 12 hours;
Fig. 3 is the SDS-PAGE figure of medium pH value to the protein expression influence, and wherein 1 is standard molecular weight albumen; 2 is the empty bacterium of BL21; 3 for not adding the engineering bacteria of IPTG; 4 is pH=5.0; 5 is pH=5.5; 6 is pH=6.0; 7 is pH=6.5; 8 is pH=7.0; 9 is pH=7.5; 10 is pH=8.0;
The inclusion body protein SDS-PAGE figure that Fig. 4 obtains for the present invention, wherein M is a standard molecular weight albumen, inclusion body protein molecular weight size is 32kDa;
The inclusion body protein Western blot figure that Fig. 5 obtains for the present invention, wherein M is a standard molecular weight albumen, inclusion body protein molecular weight size is 32kDa;
Fusion rotein after the renaturation that Fig. 6 obtains for the present invention and the target protein SDS-PAGE figure after the enteropeptidase cutting, wherein M is a standard molecular weight albumen, and 1 swimming lane is the fusion rotein after the renaturation, and the molecular weight size is 32kDa; 2 swimming lanes are the target protein after the enteropeptidase cutting, and the molecular weight size is 13.2kDa;
Fig. 7 schemes for the target protein SDS-PAGE that the present invention obtains purifying, and wherein M is a standard molecular weight albumen, and the target protein molecular weight size of purifying is 13.2kDa;
Fig. 8 schemes for the target protein Western blot that the present invention obtains purifying, and wherein M is a standard molecular weight albumen, and the target protein molecular weight size of purifying is 13.2kDa.
Embodiment
Be noted that following specifying all is exemplary, being intended to provides further explanation to the present invention.
In this manual, only if specialize, otherwise used T.T. is those skilled in the art's Essential Terms; The experimental technique of unreceipted actual conditions is by the normal experiment method in this specification sheets; Used test materials is commercially available purchase product if no special instructions in this specification sheets, and the composition of all ingredients and substratum and compound method can be referring to the operations in the normal experiment handbook.
Embodiment 1: obtain to express the genetic engineering bacterium through the growth factor of human nerve β subunit of modifying
1. with reference to prokaryotic organism preference codon table; Under the prerequisite that does not change aminoacid sequence; Base sequence to natural human nervegrowthfactor-subunit leading peptide and mature peptide is modified; And between two sequences, designed the enteropeptidase cleavage site, synthesized this sequence (base sequence is shown in SEQ ID NO:3, and the aminoacid sequence of its expression is shown in SEQ ID NO:4) through the mode of chemosynthesis
The codon that base in this sequence forms all is the high frequency codon of escherichia expression system, can under the prerequisite that does not change aminoacid sequence, improve the expression amount of goal gene to greatest extent.5 ' end GGATCC and 3 ' end CTCGAG are respectively BamHI and XhoI restriction endonuclease sites in the sequence, can make things convenient for goal gene to insert pET28a (+) prokaryotic expression carrier.Wherein GATGATGATGATAAA is the enteropeptidase cleavage site.Enteropeptidase is a kind of Tryase; It can discern N-Asp-Asp-Asp-Asp-Lys-C five amino acid residue in the peptide chain specifically; Cut point is at the carboxyl terminal of Lys; Therefore the target protein that cuts down does not have additional amino acid at aminoterminal, thereby makes the aminoacid sequence of target protein and native protein in full accord.It is strong that enteropeptidase also has identification specificity, and cutting efficiency is high, advantages such as reaction conditions gentleness.
Among the present invention; Enteropeptidase cleavage site upper reaches behaviour nervegrowthfactor-subunit leading peptide gene order; This sequence has been served as the role of Chaperones Molecular in natural β-NGF expression process; In the posttranslational modification process of β-NGF, can help mature peptide to be folded to form space conformation, and can before mature transporter peptide is to born of the same parents, be cut removal with BA.The present invention has kept its leader peptide sequences when the rh β ngf gene sequence of design prokaryotic expression, help having in external formation with the fusion rotein of inclusion body formal representation the correct conformation of BA.Cutting through enteropeptidase simultaneously can accurately separate the rh β NGF after the renaturation with leading peptide, thereby discharges the rh β NGF with high BA.
This sequence is cloned into the BamHI (846) and XhoI (159) site in pET28a (+) expression vector MCS district; Utilize the expression of the T7 promotor promotor gene of carrier; Initiator codon ATG is positioned at 294 in carrier, and there are the 6 Histidine purifying sites that a carrier carries (specifically can referring to the plasmid map of Novagen company) in its downstream.Histidine can provide coordination electronics and some metals ions such as Ni
2+Chelating, 6 Histidines can make protein adsorption in containing metal such as Ni
2+Chromatographic stuffing on, therefore can utilize metal chelate chromatography to come the purifying target protein.
2. the structure of rh β ngf gene engineering bacteria
The carrier pET28a (+) that is used to make up, host bacterium DH5 α and BL21 (λ DE3) all purchase the company in Novagen, and BamHI and XhoI enzyme are purchased in precious biotechnology (Dalian) ltd.
2.1 rh β ngf gene inserts pET28a (+) plasmid: the growth factor of human nerve beta subunit gene fragment of chemosynthesis is connected to equally on pET28a (+) expression vector that enzyme is cut through the T4 dna ligase after with BamHI, XhoI double digestion; Make up pET28a (+)+[rh β NGF] recombinant expression vector; Transformed into escherichia coli clone strain DH5 α; On the LB substratum that contains 50 μ g/ml kantlex (Kana), cultivate the screening recon for 37 ℃; Through PCR and enzyme cut identify correct after, its sequence exactness of sequence verification.
2.2 the pET28a (+) that above-mentioned evaluation is correct+[rh β NGF] recombinant expression vector extracts from clone bacterium DH5 α; Transformed into escherichia coli expression strain BL21 (λ DE3) competence; On the LB substratum that contains 50 μ g/ml kantlex, cultivate the screening recon for 37 ℃, obtain recon this moment is rh β ngf gene engineering bacteria.
The screening of the inductive condition of embodiment 2:rh β ngf gene engineering bacteria
The recombinant bacterial strain that the glycerine pipe is frozen is inoculated in fresh LB resistance substratum (containing Kana 50 μ g/ml) by 0.2%, and 37 ℃, the 220rpm shaking culture is spent the night, the activation engineering bacteria.
1, IPTG concentration is to the influence of protein expression
The activatory engineering bacteria is inoculated in respectively in the 2ml LB resistance substratum (containing Kana 50 μ g/ml); 37 ℃, the 220rpm shaking culture adds IPTG during to A550=0.6, makes its final concentration be respectively 0.1 mmol/L, 0.5 mmol/L, 1.0 mmol/L, 1.5 mmol/L, 2.0 mmol/L, 3.0 mmol/L, 4.0 mmol/L, 5.0 mmol/L; Inducing culture is the centrifuging and taking deposition after 3 hours; Use 0.5ml, the resuspended thalline of 20mmol/L Tris-HCl (pH8.0) ,-80 ℃ of multigelations three times; Centrifuging and taking deposition is carried out the SDS-PAGE electrophoresis with 0.1ml protein electrophoresis sample-loading buffer after resuspended; After the scanning of SDS-PAGE electrophoresis,, confirm that target protein accounts for the ratio of total protein through the gel imaging system analysis.
Experimental result shows (Fig. 1); IPTG concentration is under the condition of 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 3.0 mM, 4.0 mM, 5.0 mM, and the ratio that target protein accounts for total protein is respectively 31.46%, 35.23%, 40.78%, 40.82%%, 41.33%, 40.95%, 41.25%, 40.66%.Explanation is in IPTG concentration during less than 1.0 mM; Target protein accounts for the total protein ratio and rises with the rising of IPTG concentration; When IPTG concentration during greater than 1.0 mM, target protein accounts for the total protein ratio and no longer raises with IPTG concentration and significantly rise, and therefore establishing 1.0 mM is the best IPTG concentration of inductive.
2, induction time is to the influence of protein expression
The activatory engineering bacteria is inoculated in 10ml LB resistance substratum (containing Kana 50 μ g/ml), and 37 ℃, the 220rpm shaking culture adds 1.0 mM IPTG during to A550=0.6; Get 1ml bacterium liquid respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours of inducing culture, the centrifuging and taking deposition is used 0.5ml; 20mmol/resuspended the thalline of L Tris-HCl (pH8.0);-80 ℃ of multigelations three times, centrifuging and taking deposition carry out the SDS-PAGE electrophoresis with 0.1ml protein electrophoresis sample-loading buffer after resuspended, after the SDS-PAGE electrophoresis scans; Through the gel imaging system analysis, confirm that target protein accounts for the ratio of total protein.
Experimental result shows (Fig. 2), and under 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours the condition of induction time, the ratio that target protein accounts for total protein is respectively 1.25%, 36.74%, 39.87%, 38.62%, 37.55%, 33.33%.Explain that induction time is about 4 hours, it is the highest that target protein accounts for the total protein ratio, therefore establishes to be best induction time in 3.5 hours.
3, medium pH value is to the influence of protein expression
Be respectively inoculation activatory engineering bacteria in 5.0,5.5,6.0,6.5,7.0,7.5,8.0 the 2ml LB resistance substratum (containing Kana 50 μ g/ml) at pH, 37 ℃, the 220rpm shaking culture adds 1.0 mM IPTG during to A550=0.6; Inducing culture is the centrifuging and taking deposition after 3.5 hours; Use 0.5ml, the resuspended thalline of 20mmol/L Tris-HCl (pH8.0) ,-80 ℃ of multigelations three times; Centrifuging and taking deposition is carried out the SDS-PAGE electrophoresis with 0.1ml protein electrophoresis sample-loading buffer after resuspended; After the scanning of SDS-PAGE electrophoresis,, confirm that target protein accounts for the ratio of total protein through the gel imaging system analysis.
Experimental result shows (Fig. 3), and the pH value is respectively under 5.0,5.5,6.0,6.5,7.0,7.5,8.0 the condition, and the ratio that target protein accounts for total protein is respectively 12.5%, 20.36%, 25.78%, 34.33%, 39.49%, 32.61%, 31.55%.Less than 7.0 o'clock, target protein accounted for the total protein ratio and raises with pH and rise in the pH value, when pH value greater than 7.0 the time, target protein accounts for the total protein ratio and raises with pH value and reduces, so to establish pH 7.0 be the inductive optimal ph.
The abduction delivering of embodiment 3:rh β ngf gene engineering bacteria and the separation and purification of inclusion body
1. picking mono-clonal genetically engineered bacterium colony is inoculated in 50mL LB liquid nutrient medium, and 37 ℃, 220rpm shaking culture 14h;
With above-mentioned bacterium liquid kind in 3L liquid LB substratum, 37 ℃, the 220rpm shaking culture is during to A550=0.6 (about 3h); According to inductive condition groped result (like Fig. 1, Fig. 2, Fig. 3); Adding final concentration is the IPTG of 1mM, keeps 7.0,37 ℃ of medium pH values and induces 3.5 hours;
3. centrifugal (4 ℃, 8000rpm 10min), collects thalline, and is resuspended in ratio adding 20mM Tris-HCl (pH=8.0) lysis buffer of every gram thalline weight in wet base 20ml, frozen in-20 ° of C;
4. 37 ℃ of dissolving thalline suspensions carry out the ultrasonic bacterium of splitting, and power 400W ultrasonic 15 seconds, 15 seconds at interval, acts on 30 minutes.
5. centrifugal (4 ℃, 12000rpm, 10min), collecting precipitation is with the Tris-HCl washings washing that contains 1% TritonX-100 three times;
6. with the Tris-HCl washings washing that contains 2M urea once;
7. centrifugal (4 ℃, 12000rpm, 10min), and collecting precipitation (being inclusion body protein), with the lysate dissolving that contains 8M urea, in-80 ℃ of preservations, and sampling is carried out, and the SDS-PAGE electrophoresis purity is analyzed and immunoblotting is identified (Western Blot).
Experimental result shows that inclusion body protein purity is greater than 95% (Fig. 4); Be accredited as growth factor of human nerve (Fig. 5) through Western blot.
Groping of the renaturation condition of embodiment 4:h β NGF fusion rotein
The renaturation of rh β NGF fusion rotein adopts external dilution refolding method.In rh β NGF inclusion body protein solution, slowly drip renaturation buffer (flow velocity 1.66ml/min), the renaturation temperature is 10 ℃, and there are certain influence the l-arginine concentration of renaturation buffer and pH and renaturation time to annealing efficiency.
1, l-arginine concentration is to the influence of annealing efficiency
Preparation l-arginine concentration is respectively the renaturation buffer of 0.65mM, 0.75 mM, 0.85 mM, 0.95 mM, 1.05 mM, and the pH value is 9.5; Redox system is reduced glutathion-Sleep-promoting factor B (GSH-GSSG) system, and GSSG concentration is 0.5mM, and GSH concentration is 5mM; 10 ℃; Renaturation 3 hours, the centrifuging and taking supernatant is measured protein concentration with the Lowry method; Calculate Tot Prot, with this Tot Prot and the inclusion body protein total amount that adds the renaturation system than value representation renaturation yield.
Experimental result shows that the protein renaturation rate was respectively 31.36%, 40.58%, 35.12%, 34.98%, 33.71% when l-arginine concentration was 0.65mM, 0.75 mM, 0.85 mM, 0.95 mM, 1.05 mM.Therefore when explanation was 0.75 mM in l-arginine concentration, renaturation yield was the highest, established that l-arginine concentration 0.75mM is an optimum concn in the renaturation buffer.
2, renaturation buffer system pH value is to the influence of annealing efficiency
Secure ph is respectively 8.0,8.5,9.0,9.5,10.0 renaturation buffer, and l-arginine concentration is 0.75 mM; Redox system is reduced glutathion-Sleep-promoting factor B (GSH-GSSG) system, and GSSG concentration is 0.5mM, and GSH concentration is 5mM; 10 ℃; Renaturation 3 hours, the centrifuging and taking supernatant is measured protein concentration with the Lowry method; Calculate Tot Prot, with this Tot Prot and the inclusion body protein total amount that adds the renaturation system than value representation renaturation yield.
Experimental result shows that the pH value is respectively 8.0,8.5,9.0,9.5,10.0 o'clock protein renaturation rates and is respectively 33.67%, 36.88%, 38.32%, 40.53%, 31.64%.In the pH value is 9.5 o'clock, and renaturation yield is the highest, therefore establishes pH 9.5 and is best renaturation buffer pH value.
3, the renaturation time is to the influence of annealing efficiency
In l-arginine concentration is 0.75 mM, and the pH value is 9.5, and redox system is reduced glutathion-Sleep-promoting factor B (GSH-GSSG) system; GSSG concentration is 0.5mM, and GSH concentration is 5mM, carries out renaturation under 10 ℃ of conditions; Respectively at the renaturation centrifugal collection supernatant of taking a sample in 1 hour, 3 hours, 5 hours, 7 hours, 9 hours; Measure protein concentration with the Lowry method, calculate Tot Prot, with this Tot Prot and the inclusion body protein total amount that adds the renaturation system than value representation renaturation yield.
Experimental result shows that the protein renaturation rate was respectively 12.67%, 40.74%, 39.32%, 40.21%, 38.25% when the renaturation time was respectively 1 hour, 3 hours, 5 hours, 7 hours, 9 hours.The renaturation time greater than 3 hours after, with the prolongation of renaturation time, renaturation yield does not obviously improve, and therefore establishes to be the best renaturation time in 3 hours.
The renaturation of embodiment 5:h β NGF fusion rotein and enteropeptidase cutting
The renaturation of rh β NGF fusion rotein adopts external dilution refolding method, and l-arginine concentration is 0.75mM in the renaturation buffer that uses, and the pH value is 9.5; Redox system is reduced glutathion-Sleep-promoting factor B (GSH-GSSG) system, and GSSG concentration is 0.5mM, and GSH concentration is 5mM; During renaturation renaturation buffer slowly is added dropwise to (1.66ml/min) in the inclusion body solution, the renaturation temperature is 10 ℃, and the renaturation time is 3 hours;
2. collect recombinant protein solution, dialysed 24 hours with 50 mM Tris-HCl (pH 8.0) damping fluid, dialyzate of replacing in per 8 hours in 4 ℃;
3. collect dialysis back protein solution, use molecular weight cut-off to carry out ultrafiltration and concentration as the ultra-filtration membrane of 5kDa;
4. cut the fusion rotein of above-mentioned renaturation in the ratio of 1U/50 μ g with enteropeptidase, 21 ℃ of enzyme Qie Wendu, enzyme cut 16 hours time.Get enzyme simultaneously and cut the sample of front and back, adopt the SDS-PAGE electrophoresis detection.Detected result shows (Fig. 6), after the enteropeptidase cutting, can discharge the molecular weight size by the albumen of above-mentioned condition renaturation and be the target protein of 13.2kDa.
The affinity chromatography separation and purification of embodiment 6:rh β NGF and BA detect
With the damping fluid balance Ni Sepharose Fast Flow chromatography column of 50 mM imidazoles-0.5MNaCl-20mMTris-HCl (pH8.0), to collect stream in the last appearance process and wear the peak, it is the component that contains rh β NGF, warp dialysis and ultrafiltration and concentration obtain the rh β NGF of purifying.Sampling is adopted reductibility SDS-PAGE electrophoresis detection purity, and is carried out immunoblotting (Western blot) and identify; Adopt chick embryonic dorsal root ganglion culture method detection of biological active simultaneously.
Experimental result shows that the rh β NGF of preparation is single band through reductibility SDS-PAGE electrophoresis detection, and its purity reaches (Fig. 7) more than 99%; Identify that through anti human nerve growth factor monoclonal antibody Western blot it is growth factor of human nerve (Fig. 8); Mouse submaxillary gland NGFF with Nat'l Pharmaceutical & Biological Products Control Institute's distribution is reference with reference to article, adopts the chick embryonic dorsal root ganglion culture method to detect this sample biological activity, and the result shows that specific activity is higher than 500000AU/mg.
The above is merely the preferred embodiments of the present invention, should be understood that; For the those of ordinary skill in the present technique; Under the prerequisite that does not break away from core technology characteristic of the present invention, can also make some improvement and retouching, these retouchings and improvement also should belong to scope of patent protection of the present invention.
SEQUENCE LISTING
<110>Wuhan Haite Bio-pharmaceutical Co., Ltd
<120>Utilize escherichia expression system to prepare the method for RHNGF
<130>; 121229-I-CP-LYX
<160>; 4
<170>; PatentIn version 3.3
<210>; 1
<211>; 357
<212>; DNA
<213>Artificial sequence
<400>; 1
agcagcagcc atccgatttt tcatcgtggc gaatttagcg tgtgtgacag tgtcagcgtg 60
tgggttgggg ataagaccac cgccacagac atcaagggca aggaggtgat ggtgttggga 120
gaggtgaaca ttaacaacag tgtattcaaa cagtactttt ttgaaaccaa atgccgtgat 180
ccgaatccgg tggatagcgg ctgccgtggc attgatagca aacattggaa tagctattgt 240
accaccaccc atacctttgt gaaagcgctg accatggatg gcaaacaggc ggcgtggcgt 300
tttattcgta ttgataccgc gtgtgtgtgc gtgctgagcc gtaaagcggt gcgttga 357
<210>; 2
<211>; 309
<212>; DNA
<213>Artificial sequence
<400>; 2
gaaccgcata gcgaaagcaa tgtgccggcg ggccatacca ttccgcaggc gcattggacc 60
aaactgcagc atagcctgga taccgcgctg cgccgtgccc gcagcgcgcc ggcggcggcg 120
attgctgcgc gcgtggcggg ccagacccgc aacattaccg ttgatccgcg tctgtttaaa 180
aagcgtcgtc tgcgtagccc gcgtgtgctg tttagcaccc agccgccgcg tgaagcggcg 240
gatacccagg atctggattt tgaagtgggt ggtgcggcgc cgtttaaccg tacccatcgt 300
agcaaacgt 309
<210>; 3
<211>; 693
<212>; DNA
<213>Artificial sequence
<400>; 3
ggatccgaac cgcatagcga aagcaatgtg ccggcgggcc ataccattcc gcaggcgcat 60
tggaccaaac tgcagcatag cctggatacc gcgctgcgcc gtgcccgcag cgcgccggcg 120
gcggcgattg ctgcgcgcgt ggcgggccag acccgcaaca ttaccgttga tccgcgtctg 180
tttaaaaagc gtcgtctgcg tagcccgcgt gtgctgttta gcacccagcc gccgcgtgaa 240
gcggcggata cccaggatct ggattttgaa gtgggtggtg cggcgccgtt taaccgtacc 300
catcgtagca aacgtgatga tgatgataaa agcagcagcc atccgatttt tcatcgtggc 360
gaatttagcg tgtgtgacag tgtcagcgtg tgggttgggg ataagaccac cgccacagac 420
atcaagggca aggaggtgat ggtgttggga gaggtgaaca ttaacaacag tgtattcaaa 480
cagtactttt ttgaaaccaa atgccgtgat ccgaatccgg tggatagcgg ctgccgtggc 540
attgatagca aacattggaa tagctattgt accaccaccc atacctttgt gaaagcgctg 600
accatggatg gcaaacaggc ggcgtggcgt tttattcgta ttgataccgc gtgtgtgtgc 660
gtgctgagcc gtaaagcggt gcgttgactc gag 693
<210>; 4
<211>; 260
<212>; PRT
<213>Artificial sequence
<400>; 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile
35 40 45
Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu
50 55 60
Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala
65 70 75 80
Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg
85 90 95
Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu
100 105 110
Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro
115 120 125
Phe Asn Arg Thr His Arg Ser Lys Arg Asp Asp Asp Asp Lys Ser Ser
130 135 140
Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp Ser Val
145 150 155 160
Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys
165 170 175
Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys
180 185 190
Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser
195 200 205
Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr
210 215 220
Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala
225 230 235 240
Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg
245 250 255
Lys Ala Val Arg
260
Claims (9)
1. RHNGF beta subunit gene; It contains 6 Histidine purifying sites, enteropeptidase cleavage site and growth factor of human nerve β subunit mature peptide fragment gene and molecular chaperone through modifying; Wherein said growth factor of human nerve β subunit mature peptide segment base is because through the improved gene of prokaryotic organism high frequency codon, its base sequence is shown in SEQ ID NO:1; Said molecular chaperone is the growth factor of human nerve β subunit leading peptide gene through modifying, and its base sequence is shown in SEQ ID NO:2.
2. a method of utilizing escherichia expression system to prepare RHNGF is characterized in that, may further comprise the steps:
(1) utilizes the synthetic described growth factor of human nerve β of claim 1 subunit mature peptide fragment gene and the molecular chaperone of optimizing through point mutation of chemical synthesis, and it is cloned into pET28 a (+) expression vector, obtain recombinant expression vector;
(2) step (1) gained recombinant expression vector is imported among the escherichia coli expression bacterial strain BL21 (λ DE3), make up the genetic engineering bacterium that contains recombinant expression vector;
When (3) culturing step (2) gained genetic engineering bacterium is to A550=0.6, add inductor IPTG, induce RHNGF rh β NGF in this genetic engineering bacterium, to express;
(4) centrifugal collection thalline, resuspended thalline ,-80 ℃ are frozen;
(5) the resuspended liquid of thalline that thaws, broken thalline, separation and purification inclusion body protein;
(6) external renaturation inclusion body protein, dialysis back ultrafiltration and concentration;
(7) remove Chaperones Molecular through the enteropeptidase cutting;
(8) purifying obtains said RHNGF.
3. the inductive time is 3-4 hour in the method according to claim 2, wherein said step (3).
4. the concentration of inductor IPTG is 1mmol/L in the method according to claim 2, wherein said step (3).
5. method according to claim 2, used resuspended liquid is 20mM Tris-HCl (pH8.0) in the wherein said step (4), usage quantity is 20 times of volumes of thalline weight in wet base.
6. method according to claim 2, the method for broken thalline is a sonioation method in the wherein said step (5), wherein the used power of ultrasonication thalline is 400W, ultrasonic 15 seconds, 15 seconds at interval, acts on 30 minutes.
7. the method for renaturation inclusion body protein is external dilution refolding method in the method according to claim 2, wherein said step (6), and in the wherein external dilution refolding liquid, arginic concentration range is 0.75-1.0mmol/L, and the pH value is 8.0-9.5.
8. method according to claim 2, the enteropeptidase in the wherein said step (7) are the recombination ox intestine kinase with 6 Histidine purifying sites.
9. the method for purifying is a nickel post affinity chromatography in the method according to claim 2, wherein said step (8).
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| CN103834677A (en) * | 2014-03-26 | 2014-06-04 | 黄文林 | Recombinant expression method for human cryptochrome protein I (hCRY1) and application of human cryptochrome protein I (hCRY1) in preparation of radiotherapy protective agent |
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| CN105154462A (en) * | 2015-08-25 | 2015-12-16 | 大连大学 | Method for building N-glycosylation receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain) |
| CN105154461B (en) * | 2015-08-25 | 2020-05-22 | 大连大学 | Method for establishing N-glycosylation efficiency detection receptor protein model in escherichia coli by using framework protein Fn3 |
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| WO2017205996A1 (en) * | 2016-06-03 | 2017-12-07 | 黄文林 | Production technique for recombinant human cryptochrome protein i (hcry1) and combination product thereof |
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| CN110093394B (en) * | 2019-05-10 | 2023-10-03 | 重庆科润生物医药研发有限公司 | Protein inclusion body and preparation method of recombinant human beta-nerve growth factor |
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