CN102839182B - 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 recombinant human nerve growth factor, belong to the biological medicine technology field.
Background technology
Nerve growth factor (Nerve Growth Factor, NGF) is found and most typical neurotrophic factor the earliest, also is one of most important bioactive molecules in the neural system simultaneously.It is comprised of α, β, three subunits of γ, and wherein (β-NGF) is unique subunit with biologic activity to the β subunit, and it all has important regulating and controlling effect 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 for the treatment of peripheral nerve injury in clinical, is widely used in the nervous system disease fields such as nerve injury, hemiplegia, palsy, craniocerebral injury, cerebral palsy of children.But commercial NGF is the nerve growth factor (mNGF) of mouse.The animal derived protein product of this class usually has higher immunogenicity, causes easily antigen-reactive, severe patient even entail dangers to life in the human body.And natural growth factor of human nerve (hNGF) is distributed in the 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 recombinant human nerve growth factor is a main path that addresses the above problem.
In each large expression system, escherichia expression system is because its expression level is high, production cost is low, be convenient to the first-selection that the advantage such as suitability for industrialized production becomes the preparation recombinant human nerve growth factor., 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, the albumen of expression usually abiology is active.Investigators find that humanβ-NGF's Expression in Escherichia coli amount is not high, and biological activity is extremely low, mainly are because β-ngf gene and the special structure of albumen thereof cause it to be difficult to effective expression in the intestinal bacteria system by analysis.
Use 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 to form correct structure in the intestinal bacteria that lack translation post-treatment modification system, cause give expression to β-the NGF protein biological activity is extremely low, even do not have activity.The dimer that the strand that natural β-NGF is comprised of two 118 amino acid is combined into by non covalent bond, wherein every strand contains by 6 halfcystines and mutually matches 3 pairs of disulfide linkage that form, these 3 pairs of disulfide linkage consist of the typical structural pattern of neurotrophic factor family---" cystine knot " pattern (the cystine knot), namely 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 correctly match, forms above-mentioned " cystine knot " structure, namely can not form correct protein structure and cause its biological activity extremely low.
For the problems referred to above, general way is the gene that need are expressed to be carried out codon optimized, makes up simultaneously the fusion rotein of molecular chaperones and target protein, becomes correct structure by molecular chaperones help target protein in vitro folding.
Carry out codon optimized for gene, need to consider the e. coli codon Preference, the work that the factors such as joint efficiency of mRNA sequential structure, stability and the ribosome binding sequence that also should consider gene order and transcribe are complexity, technical difficulty is higher.
The structure of fusion rotein, the selection of molecular chaperones 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.Such as: (the HE Ling-bing such as HE Ling-bing, WANG Yanz, WANG Ge, 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, 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 simultaneously Trx to help β-NGF and express in the mode of solubility expression, but prokaryotic cell prokaryocyte is less, only have when expression amount hour, albumen could exist in the solubility mode, and when the foreign protein great expression, most albumen still can be assembled sex change; (the Sun Weiguo such as Sun Weiguo, Liu Nongle, Ding Hongmei, keep and deposit, Zhao Qiang, Xu Hua, doubly put forth energy in Shen, Shao Ningsheng, protokaryon amalgamation and expression and the activity research thereof of recombinant human nerve growth factor β subunit. the biotechnology communication, 2009, Vol.20, No.4) disulfide linkage is formed DsbA in the protein family (Dsb), DsbC albumen and Trx (Trx) are fusion molecule, carry out amalgamation and expression with hNGF β subunit at prokaryotic expression system, although forming albumen, disulfide linkage can assist the external renaturation of β-NGF, therefore but can not identify 3 pairs of disulfide linkage accurate locations of β-NGF, also be difficult to the renaturation effect that reaches desirable, and the end product of this research carry label protein, inconsistent with natural β-NGF, can't be used for drug manufacture.
Therefore, the method for preparing recombinant human nerve growth factor in this area in the urgent need to exploitation take low cost, high yield and high reactivity as purpose.Described preparation comprises the genetic modification that growth factor of human nerve is carried out, the amalgamation and expression of molecular chaperones and separation, the purifying of recombinant protein.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of recombinant human nerve growth factor beta subunit gene, it contains 6 Histidine purifying sites, enteropeptidase cleavage site and hNGF-β gene subunit mature peptide fragment gene and molecular chaperone through modifying, wherein said hNGF-β gene subunit mature peptide segment base is because through the gene behind the prokaryotic organism high frequency codon modify, its base sequence is shown in SEQ ID NO:1; Described molecular chaperone is the hNGF-β gene subunit leading peptide gene through modifying, and its base sequence is shown in SEQ ID NO:2.
Two of purpose of the present invention has been to provide the method for utilizing escherichia expression system to prepare recombinant human nerve growth factor.
The base sequence of according to an aspect of the present invention, modified hNGF-β gene subunit mature peptide gene is (altogether 357bp) shown in SEQ ID NO:1.
Base sequence in this sequence is the sequence of having carried out codon modify, and its codon is the high frequency codon of escherichia expression system, can under the prerequisite that does not change aminoacid sequence, improve to greatest extent the expression amount of goal gene.
The base sequence (altogether 309bp) shown in SEQ ID NO:2 of according to a further aspect in the invention, modified leading peptide gene as molecular chaperones.
This sequence is positioned at hNGF-β gene subunit mature peptide gene order of the present invention upstream, and two sequences are connected by enteropeptidase cleavage site (GATGATGATGATAAA).This molecular chaperones can help hNGF-β gene subunit with the inclusion body formal representation in the correct conformation of external formation, and can after renaturation, the cutting by enteropeptidase remove, thereby discharge with natural β-NGF aminoacid sequence on all fourly, and have the mature peptide of higher biological activity.
The method of utilizing escherichia expression system to prepare recombinant human nerve growth factor (rh β NGF) of the present invention comprises the steps:
(1) utilizes synthetic hNGF-β gene subunit mature peptide fragment gene and the molecular chaperone behind prokaryotic organism high frequency codon modify of chemical synthesis, and it is cloned into pET28a (+) expression vector, obtain recombinant expression vector;
(1.1) BamHI (846) and XhoI (159) site (referring to the Novagen company plasmid map) of prokaryotic expression plasmid pET28a (+) will be inserted behind said gene fragment usefulness BamHI and the 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 recombinant human nerve growth factor 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 isolation of occlusion bodies, 8M urea dissolving inclusion body protein);
(6) external renaturation inclusion body protein, ultrafiltration and concentration after the dialysis;
(7) remove molecular chaperones through the enteropeptidase cutting;
(8) purifying obtains described recombinant human nerve growth factor.
The time of preferably, inducing in the wherein said step (3) is 3-4 hour.
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 sonioation method in the wherein said step (5), and wherein the used power of ultrasonication thalline is 400W, and ultrasonic 15 seconds, interval 15 seconds acted on 30 minutes.
Preferably, the method for renaturation inclusion body protein is external dilution refolding method in the wherein said step (6), and wherein in the 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 the affinity chromatography method in the wherein said step (8).
Preferably, the filler of 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 loading process is worn the peak, obtain purity greater than 99% through dialysis and ultrafiltration and concentration, biological specific activity is higher than the recombinant human nerve growth factor of 500000AU/mg.
The present invention analyzes β-ngf gene, according to the Preference of intestinal bacteria to the synonymous code selection, eliminating rare codon, unstable sequence, preferred on the basis of best codon, β-ngf gene is redesigned, thereby improved to greatest extent β-NGF Expression in Escherichia coli amount.
The present invention is according to the constructional feature of natural β-NGF, selected the leading peptide of β-NGF as its molecular chaperones, the disulfide linkage that this peptide section can effectively be identified 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 the 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 in the leading peptide upstream, designed the enteropeptidase cleavage site between leading peptide and mature peptide.This design 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 aminoacid sequence on all fourly simultaneously by the enteropeptidase cutting, and have the target protein of higher biological activity.
The present invention has obtained efficiently expressing of rh β NGF by after hNGF-β gene subunit leading peptide and mature peptide fragment gene are carried out prokaryotic organism high frequency codon modify 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 affinity chromatography, biological specific activity is higher than the recombinant human nerve growth factor 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 that the IPTG induced concentration affects protein expression, 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 that induction time affects protein expression, 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 that medium pH value affects protein expression, 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;
Fig. 4 is the inclusion body protein SDS-PAGE figure that the present invention obtains, and wherein M is standard molecular weight albumen, and the inclusion body protein molecular size range is 32kDa;
Fig. 5 is the inclusion body protein Western blot figure that the present invention obtains, and wherein M is standard molecular weight albumen, and the inclusion body protein molecular size range is 32kDa;
Fig. 6 is fusion rotein after the renaturation that obtains of the present invention and the target protein SDS-PAGE figure after the enteropeptidase cutting, and wherein M is standard molecular weight albumen, and 1 swimming lane is the fusion rotein after the renaturation, and molecular size range is 32kDa; 2 swimming lanes are the target protein after the enteropeptidase cutting, and molecular size range is 13.2kDa;
Fig. 7 is the target protein SDS-PAGE figure that the present invention obtains purifying, and wherein M is standard molecular weight albumen, and the target protein molecular size range of purifying is 13.2kDa;
Fig. 8 is the target protein Western blot figure that the present invention obtains purifying, and wherein M is standard molecular weight albumen, and the target protein molecular size range of purifying is 13.2kDa.
Embodiment
Be noted that following specifying all is exemplary, be intended to the invention provides further instruction.
In this manual, unless specialize, used technical term is those skilled in the art's Essential Terms; The experimental technique of unreceipted actual conditions is experimental technique routinely 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: the genetic engineering bacterium that obtains to express modified hNGF-β gene subunit
1. with reference to prokaryotic organism preference codon table, under the prerequisite that does not change aminoacid sequence, base sequence to natural human nervegrowthfactor-β 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) by 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 to greatest extent the expression amount of goal gene.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 serine protease, it can identify 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 at aminoterminal without additional amino acid, thereby make 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, the advantages such as reaction conditions gentleness.
Among the present invention, enteropeptidase cleavage site upstream is hNGF-β gene subunit leading peptide gene order, this sequence has been served as the role of molecular chaperones in natural β-NGF expression process, in the posttranslational modification process of β-NGF, can help mature peptide to be folded to form the space conformation with biologic activity, and can before mature transporter peptide is to born of the same parents, be cut removal.The present invention has kept its leader peptide sequences when the rh β ngf gene sequence of design prokaryotic expression, be conducive to have in external formation with the fusion rotein of inclusion body formal representation the correct conformation of biologic activity.The simultaneously cutting by enteropeptidase can accurately separate the rh β NGF after the renaturation with leading peptide, thereby discharges the rh β NGF with high biologic activity.
This sequence is cloned into BamHI (846) and XhoI (159) site in pET28a (+) expression vector multiple clone site 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 metal 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
Be used for the carrier pET28a (+) of structure, Host Strains DH5 α and BL21 (λ DE3) all are purchased from Novagen company, and BamHI and XhoI enzyme are purchased from precious biotechnology (Dalian) company limited.
2.1 rh β ngf gene inserts pET28a (+) plasmid: the hNGF-β gene subunit gene fragment of chemosynthesis is connected to equally on pET28a (+) expression vector that enzyme is cut by the T4 dna ligase after with BamHI, XhoI double digestion, make up pET28a (+)+[rh β NGF] recombinant expression vector, transform escherichia coli cloning bacterial 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, transform escherichia coli expression bacterial strain BL21 (λ DE3) competence, cultivate the screening recon for 37 ℃ on the LB substratum that contains 50 μ g/ml kantlex, 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 culture base (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 on the impact of protein expression
The engineering bacteria of activation is inoculated in respectively in the 2ml LB resistance culture base (containing Kana 50 μ g/ml), 37 ℃, the 220rpm shaking culture adds IPTG during to A550=0.6, make 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 precipitation after 3 hours, use 0.5ml, the resuspended thalline of 20mmol/L Tris-HCl (pH8.0),-80 ℃ of multigelations three times, carry out the SDS-PAGE electrophoresis after the centrifuging and taking precipitation is resuspended with 0.1ml protein electrophoresis sample-loading buffer, after the scanning of SDS-PAGE electrophoresis, through the gel imaging system analysis, determine that target protein accounts for the ratio of total protein.
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 inducing.
2, induction time is on the impact of protein expression
The engineering bacteria of activation is inoculated in 10ml LB resistance culture base (containing Kana 50 μ g/ml), 37 ℃, the 220rpm shaking culture adds 1.0 mM IPTG during to A550=0.6, respectively at 0 hour of inducing culture, 2 hours, 4 hours, 6 hours, 8 hours, got 1ml bacterium liquid in 12 hours, the centrifuging and taking precipitation, use 0.5ml, 20mmol/resuspended the thalline of L Tris-HCl (pH8.0),-80 ℃ of multigelations three times, carry out the SDS-PAGE electrophoresis after the centrifuging and taking precipitation is resuspended with 0.1ml protein electrophoresis sample-loading buffer, after the scanning of SDS-PAGE electrophoresis, through the gel imaging system analysis, determine 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%.Illustrate 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 on the impact of protein expression
Be respectively 5.0 at pH, 5.5,6.0,6.5,7.0,7.5,8.0 2ml LB resistance culture base (containing Kana 50 μ g/ml) in the engineering bacteria of inoculation activation, 37 ℃, the 220rpm shaking culture adds 1.0 mM IPTG during to A550=0.6, inducing culture is the centrifuging and taking precipitation after 3.5 hours, use 0.5ml, the resuspended thalline of 20mmol/L Tris-HCl (pH8.0),-80 ℃ of multigelations three times, carry out the SDS-PAGE electrophoresis after the centrifuging and taking precipitation is resuspended with 0.1ml protein electrophoresis sample-loading buffer, after the scanning of SDS-PAGE electrophoresis, through the gel imaging system analysis, determine that target protein accounts for the ratio of total protein.
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 optimal ph of inducing.
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 (such as Fig. 1, Fig. 2, Fig. 3), adding final concentration is the IPTG of 1mM, 7.0,37 ℃ of maintain base pH values were induced 3.5 hours;
3. centrifugal (4 ℃, 8000rpm, 10min) collect thalline, and are 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, power 400W, and ultrasonic 15 seconds, interval 15 seconds acted 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), 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.Slowly drip renaturation buffer (flow velocity 1.66ml/min) in rh β NGF inclusion body protein solution, the renaturation temperature is 10 ℃, and there are certain influence the arginine concentration of renaturation buffer and pH and renaturation time to annealing efficiency.
1, arginine concentration is on the impact of annealing efficiency
Preparation 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, GSSG concentration is 0.5mM, GSH concentration is 5mM, 10 ℃, renaturation 3 hours, the centrifuging and taking supernatant liquor is measured protein concentration with the Lowry method, calculate Tot Prot, with the inclusion body protein total amount of this Tot Prot and adding 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 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 arginine concentration, renaturation yield was the highest, established that arginine concentration 0.75mM is optimum concn in the renaturation buffer.
2, renaturation buffer system pH value is on the impact of annealing efficiency
Secure ph is respectively 8.0,8.5,9.0,9.5,10.0 renaturation buffer, and arginine concentration is 0.75 mM; Redox system is reduced glutathion-Sleep-promoting factor B (GSH-GSSG) system, GSSG concentration is 0.5mM, GSH concentration is 5mM, 10 ℃, renaturation 3 hours, the centrifuging and taking supernatant liquor is measured protein concentration with the Lowry method, calculate Tot Prot, with the inclusion body protein total amount of this Tot Prot and adding 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%.Be 9.5 o'clock in the pH value, renaturation yield is the highest, therefore establishes pH 9.5 and is best renaturation buffer pH value.
3, the renaturation time is on the impact of annealing efficiency
Be 0.75 mM in arginine concentration, the pH value is 9.5, 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 the inclusion body protein total amount of this Tot Prot and adding 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 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 in 4 ℃, dialyzate of replacing in per 8 hours;
3. collecting protein solution after the dialysis, is that the ultra-filtration membrane of 5kDa carries out ultrafiltration and concentration with molecular weight cut-off;
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 simultaneously enzyme and cut the sample of front and back, adopt the SDS-PAGE electrophoresis detection.Detected result shows (Fig. 6), can discharge the target protein that molecular size range is 13.2kDa by the albumen of above-mentioned condition renaturation after the enteropeptidase cutting.
The affinity chromatography separation and purification of embodiment 6:rh β NGF and biologic activity detect
Damping fluid balance Ni Sepharose Fast Flow chromatography column with 50 mM imidazoles-0.5MNaCl-20mMTris-HCl (pH8.0), collect stream in the loading process and wear the peak, it is the component that contains rh β NGF, obtains the rh β NGF of purifying through dialysis and ultrafiltration and concentration.Sampling is adopted reductibility SDS-PAGE electrophoresis detection purity, and is carried out immunoblotting (Western blot) and identify; Adopt simultaneously chick embryonic dorsal root ganglion culture method detection of biological active.
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); Take the mouse submaxillary gland nerve growth factor reference material of Nat'l Pharmaceutical ﹠ Biological Products Control Institute distribution as reference, adopt the chick embryonic dorsal root ganglion culture method to detect this sample biological activity, the result shows that specific activity is higher than 500000AU/mg.
The above is the preferred embodiments of the present invention only, 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 feature of the present invention, can also make some improvements and modifications, 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 recombinant human nerve growth factor
<130> 121229-I-CP-LYX
<160> 4
<170> PatentIn version 3.3
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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
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attgctgcgc gcgtggcggg ccagacccgc aacattaccg ttgatccgcg tctgtttaaa 180
aagcgtcgtc tgcgtagccc gcgtgtgctg tttagcaccc agccgccgcg tgaagcggcg 240
gatacccagg atctggattt tgaagtgggt ggtgcggcgc cgtttaaccg tacccatcgt 300
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gcggcgattg ctgcgcgcgt ggcgggccag acccgcaaca ttaccgttga tccgcgtctg 180
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Claims (5)
1. a method of utilizing escherichia expression system to prepare recombinant human nerve growth factor is characterized in that, may further comprise the steps:
(1) utilizes the synthetic gene order shown in SEQ ID NO:3 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 recombinant human nerve growth factor 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) inclusion body protein of employing external dilution refolding method renaturation step (5) gained, arginine concentration is 0.75mM in the renaturation buffer that uses, the pH value is 9.5, redox system is reduced glutathion-GSH-GSSG of Sleep-promoting factor B system, GSSG concentration is 0.5mM, and GSH concentration is 5mM, during renaturation renaturation buffer slowly is added dropwise in the inclusion body solution with 1.66ml/min, the renaturation temperature is 10 ℃, and the renaturation time is 3 hours; Collect gained recombinant protein solution, in 4 ℃ with 50 mM Tris-HCl, a dialyzate was changed in pH 8.0 damping fluids dialysis 24 hours in per 8 hours; Collecting protein solution after the dialysis, is that the ultra-filtration membrane of 5kDa carries out ultrafiltration and concentration with molecular weight cut-off;
(7) in the ratio of 1U/50 μ g with the fusion rotein with recombination ox intestine kinase cutting step (6) gained in 6 Histidine purifying sites, discharge recombinant human nerve growth factor albumen; Wherein enzyme Qie Wendu is 21 ℃, and enzyme is cut 16 hours time;
(8) with 50 mM imidazoles-0.5M NaCl-20mM Tris-HCl, the damping fluid balance Ni Sepharose Fast Flow chromatography column of pH8.0, the protein solution of loading step (7) gained, collect stream in the loading process and wear the peak, obtain the recombinant human nerve growth factor of purifying through dialysis and ultrafiltration and concentration.
2. method according to claim 1, the time of inducing in the wherein said step (3) is 3-4 hour.
3. the concentration of inductor IPTG is 1mmol/L in the method according to claim 1, wherein said step (3).
4. method according to claim 1, used resuspended liquid is 20mM Tris-HCl in the wherein said step (4), pH8.0, usage quantity is 20 times of volumes of thalline weight in wet base.
5. method according to claim 1, the method for broken thalline is sonioation method in the wherein said step (5), and wherein the used power of ultrasonication thalline is 400W, and ultrasonic 15 seconds, interval 15 seconds acted on 30 minutes.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103898146A (en) * | 2012-12-31 | 2014-07-02 | 石药集团中奇制药技术（石家庄）有限公司 | Method for preparing recombined human vascular endothelial cell growth factor A |
| CN103834677B (en) * | 2014-03-26 | 2017-03-22 | 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 |
| 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 |
| CN105154462B (en) * | 2015-08-25 | 2020-06-02 | 大连大学 | Method for establishing N-glycosylation receptor protein model in escherichia coli by using framework protein Fn3 |
| CN105968182A (en) * | 2016-06-03 | 2016-09-28 | 黄文林 | Production process of recombinant human cryptochrome protein I (hCRY1) and composition thereof |
| 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 |
| CN113004388A (en) * | 2019-12-20 | 2021-06-22 | 三生国健药业（上海）股份有限公司 | Preparation method of recombinant human nerve growth factor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1334271A (en) * | 2000-07-19 | 2002-02-06 | 王革 | Renaturation process for preparing human recombinant nerve growth factor |
| CN101906423A (en) * | 2010-07-23 | 2010-12-08 | 江汉大学 | Preparation method of recombined human nerve growth factor based on insect baculovirus expression system |
-
2012
- 2012-08-07 CN CN 201210278039 patent/CN102839182B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1334271A (en) * | 2000-07-19 | 2002-02-06 | 王革 | Renaturation process for preparing human recombinant nerve growth factor |
| CN101906423A (en) * | 2010-07-23 | 2010-12-08 | 江汉大学 | Preparation method of recombined human nerve growth factor based on insect baculovirus expression system |
Non-Patent Citations (14)
| Title |
|---|
| HE Ling-bing et al..Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichia coli.《CHEM．RES．CHINESE U．》.2008,第23卷(第2期),237-240. * |
| ProNGF在大肠杆菌中的表达、纯化和复性;江汉民 等;《生物工程学报》;20080325;第24卷(第3期);509-514 * |
| 人β-NGF 蛋白的原核表达、纯化及其生物活性;朱武飞 等;《中国生物制品学杂志》;20090320;第22卷(第3期);238-241 * |
| 姜静 等.来源于E.coli和CHO表达***的重组人β-NGF性质比较.《中国生物工程杂志》.2006,第26卷(第2期),8-12. * |
| 曹志飞 等.重组牛肠激酶轻链基因在毕赤酵母中的表达与纯化.《生物技术通讯》.2006,第17卷(第1期),005-008. |
| 朱武飞 等.人β-NGF 蛋白的原核表达、纯化及其生物活性.《中国生物制品学杂志》.2009,第22卷(第3期),238-241. |
| 欧晓敏.重组人神经生长因子在原核***中不同表达策略的初步研究.《中国优秀硕士学位论文全文数据库医药卫生科技辑》.2012,E070-6. |
| 江汉民 等.ProNGF在大肠杆菌中的表达、纯化和复性.《生物工程学报》.2008,第24卷(第3期),509-514. |
| 玉米谷氨酰胺转胺酶基因的克隆及原核表达;秦兰霞 等;《食品科学》;20100201;第31卷(第3期);178 * |
| 王妍.重组人神经生长因子的克隆、表达、纯化、性质和其应用的研究.《中国博士学位论文全文数据库基础科学辑》.2006,A006-43. |
| 秦兰霞 等.玉米谷氨酰胺转胺酶基因的克隆及原核表达.《食品科学》.2010,第31卷(第3期),177-181. |
| 重组人神经生长因子在原核***中不同表达策略的初步研究;欧晓敏;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20120715;31、34-36、39、42、44、53、54、56 * |
| 重组人神经生长因子的克隆、表达、纯化、性质和其应用的研究;王妍;《中国博士学位论文全文数据库基础科学辑》;20061015;A006-43 * |
| 重组牛肠激酶轻链基因在毕赤酵母中的表达与纯化;曹志飞 等;《生物技术通讯》;20060130;第17卷(第1期);5 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148349A (en) * | 2016-06-20 | 2016-11-23 | 深圳职业技术学院 | Recombinant human nerve growth factor gene, expression vector and the method preparing recombinant human nerve growth factor |
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