CN101948844B - Protein preparation method of alpha interferon analogue - Google Patents
Protein preparation method of alpha interferon analogue Download PDFInfo
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- CN101948844B CN101948844B CN2010101022885A CN201010102288A CN101948844B CN 101948844 B CN101948844 B CN 101948844B CN 2010101022885 A CN2010101022885 A CN 2010101022885A CN 201010102288 A CN201010102288 A CN 201010102288A CN 101948844 B CN101948844 B CN 101948844B
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Abstract
The invention relates to a protein preparation method of an alpha interferon analogue, which comprises the following steps of: (1) converting a recombinant plasmid formed by cloning a gene for coding alpha interferon analogue protein to an expression vector into a host bacterium to construct an engineering bacterium, and culturing the engineering bacterium through fermentation, wherein the nucleotide sequence of the gene for coding the alpha interferon analogue protein is SEG ID NO1 or is a nucleotide sequence with the homology to SEG ID NO1 not less than 93%; (2) inducing the cultured engineering bacterium through fermentation to express target protein; and (3) purifying the target protein, wherein the purification comprises the steps of crushing the induced engineering bacterium, centrifugally crushing post thallus, diluting supernate and chromatographing. Because the chromatography during purification process adopts different multi-step chromatography sequences under different conditions, the purity of protein and the activity total yield of protein are effectively increased, experiment cost is reduced, and the loss of the target protein is reduced.
Description
Technical field
The present invention relates to the preparation method of alpha interferon analogue, relate generally to biological technical field as anti-tumor protein.
Background technology
The traditional treatment of tumour is divided into three major types, i.e. surgical operation, and radiotherapy and chemotherapy, preceding two kinds of methods are only effective to the limitation tumour, then can't implement the tumour that has spread; Chemotherapy can be treated the patient that tumour has spread, but its toxic side effect is very serious.In recent years; In the research of antitumor drug; Biotherapeutics is a dark horse, and its effect is with strong points, good effect, untoward reaction are less, and biotechnological formulation research has become the focus of people's concern; And be considered to treat tumour method the most likely, and wherein Interferon, rabbit is to use many biotherapeutics.
The BA of Interferon, rabbit can reduce three aspects, promptly antiviral, antitumor and immunomodulatory.Because Interferon, rabbit has antiviral, the antitumor and immunoregulatory biological activity of wide spectrum, has been widely used in clinical.1986 FDA (Food and Drug Adminstration) (FDA) ratify first that rhIFN α-2 α, rhIFN α-2b are clinical to be used to treat diseases such as hairy cell leukemia, chronic myelogenous leukemia, hepatitis B, hepatitis C.1990, IFN-β in 1993, IFN-y listing in succession after IFN-α series is used to recur-the remission form multiple sclerosis, the treatment of chronic granuloma etc.Along with development of biology, the production and the research and development of Interferon, rabbit pharmaceutical prod have greatly been promoted.After the Interferon, rabbit series of escherichia coli expression, differences such as Chinese hamster ovary celI, yeast are expressed the Interferon, rabbit of systems produce and are come out one after another, and are applied to clinical.Widespread use along with Interferon, rabbit; It is found that Interferon, rabbit also exists shortcomings such as low dose of curative effect is good inadequately, the escalated dose spinoff is big clinically as antitumor drug; And antitumor spectra is narrow, as the solid tumor effect is poor to hematological system tumor is effective.In order to improve efficacy of interferon therapy, to reduce side reaction, people begin Interferon, rabbit is carried out various improvement, and new forms of interferon is constantly come out.
The New-type alpha-interferon analogue of our development (group of weighing again high efficiency anti-tumor antiviral protein, English name: Novaferon), be to adopt the gene method of shuttling back and forth; 12 kinds of human alpha interferon genes are produced different, the different in size Nucleotide small segment in point of contact through enzyme method such as cut; These small segments are mixed, through many wheel pcr amplifications, the homologous fragment of differing mol will be replaced mutually again; Combination at random, thus the recruit of the intermolecular phase mutual cross of a large amount of homologous genes obtained; Employing high flux screening technology is carried out antiviral and the anti-tumour cell proliferative activity evaluation to more than 10 ten thousand clone strains, has successfully filtered out high reactivity New-type alpha-interferon albuminoid (Novaferon).This technological core point is: homologous fragment makes up at random, and carries out large-scale functional screening, wins victory with quantity.Adopt this technology that human alpha interferon is transformed, still belong to initiative so far.This high reactivity novel protein Novaferon is made up of 166 amino acid of 498 nucleotide codings, and theoretical molecular is 19314.28 dalton.Molecule includes 4 halfcystines, forms two pairs of disulfide linkage, and sugar basedization, iso-electric point are about this albumen of 6.60. and people rhIFN-α 14 homology on base and aminoacid sequence is respectively 89% (445/498) and 81% (135/166).Preliminary experiment in vitro shows, this recombinant protein N ovaferon to the growth-inhibiting effect of Daudi cell be rhIFN α-2b 200-400 doubly; In the WISH-VSV system, its antiviral specific activity is more than 10 times of rhIFN α-2b.
Though the purification process that adopts at present can reach higher purity, active total recovery is lower, in addition; Carbon/nitrogen in the substratum is than influencing the form that protein expression forms; Cross high-carbon/nitrogen ratio expressing quantity is descended, low excessively carbon/nitrogen forms insoluble form than the target protein that can make expression, and insoluble albumen exists with the form of inclusion body; Biologically active not; Therefore need through protein renaturation technology, and interferon analogue albumen contains two pairs of disulfide linkage, renaturation complex process and cost are increased.In order to be suitable for large-scale production, to improve proteic solubility expression and become urgent problem with the total recovery that increases activated protein.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, a kind of protein preparation method of alpha interferon analogue is provided, can improve the yield that biological activity protein is arranged, increase the expression amount of soluble proteins, save the technology of protein renaturation.
The present invention realizes through following technical scheme:
A kind of protein preparation method of alpha interferon analogue comprises step:
(1) the proteic gene clone of coding for alpha interferon analogue formed recombinant plasmid transformed behind the expression vector is built into engineering bacteria in the host bacterium; Again said engineering bacteria is carried out fermentation culture, the nucleotide sequence of the proteic gene of said coding for alpha interferon analogue is SEQ ID NO 1 or is not less than 93% nucleotide sequence with the homology of SEQ ID NO 1;
(2) induce the engineering bacterium expression target protein of said fermentation culture;
(3) purification of target albumen, said purification step contain the sample of target protein for fragmentation thalline, dilution supernatant, chromatography and collection after inductive engineering bacteria, centrifugal fragmentation.
Homology in the step (1) is meant the similarity degree between a plurality of sequences, also can be called homogeny in the present invention.
Said expression vector can be carrier for expression of eukaryon or prokaryotic expression carrier etc., preferred prokaryotic expression carrier pBVB, and the host strain when adopting prokaryotic expression carrier can be bacillus coli DH 5 alpha or e. coli bl21.
Before said chromatography, can the proteolytic enzyme of said engineering bacteria not handled, said chromatography can be followed successively by hydrophobic chromatography, be used for deimpurity cation-exchange chromatography, chelating chromatography and HPLC, adopts the chromatography of order like this; The chromatography process of front is the preprocessing process to all article on the chromatography of back; Sample behind the chromatography of front can directly get into the chromatography of back, has practiced thrift operation steps like this, has reduced the loss of cost and effective constituent; Wherein the HEA hydrophobic chromatography is an appearance on the high salt; Salt-free low PH wash-out, and flow velocity is higher, suits to be placed on the first step and catches thick target protein; Be used for deimpurity cation-exchange chromatography and need hang down PH, preferably salt-free upward appearance is connected more suitable with hydrophobic chromatography; Metal-chelating is little part affinity chromatography, requires the sample saliferous, is saliniferous and be used for deimpurity cationic exchange elution peak, therefore be used for deimpurity cation-exchange chromatography and be connected; Because mixed in this sample have the complete albumen and the albumen of degraded, so the last separating power that adopts is strong, the high-pressure liquid phase chromatograph that measurement sensitivity is high can improve separating effect effectively, obtains complete target protein.This chromatography order can obtain purity greater than 95%, the target protein of activated protein total recovery about 25%.Whether the sample behind said chelating chromatography and said high-pressure liquid phase chromatograph all will contain degradation fragment through the mass spectrometric detection analysis, to guarantee to collect complete segmental target protein, improves the purity of target protein.The sample of behind high-pressure liquid phase chromatograph, collecting through being used for thoroughly removing acetonitrile except that the POROS 50HS cation-exchange chromatography of acetonitrile, improves sample purity again.
Before said chromatography, can handle the proteolytic enzyme of said engineering bacteria; For adopting the not engineering bacteria of the host bacterium structure of defective of proteolytic enzyme; Usually adopt the mode of inactivated proteases to carry out described protease treatment; Engineering bacteria for the host bacterium of adopting the proteolytic enzyme defective makes up adopts the mode of arrestin enzymic activity to carry out described protease treatment usually.Carry out protease treatment to prevent the target protein degraded, adopt different protease treatment modes, improve treatment effect and purification yield for different host bacterium.
Can adopt the acidification mode to carry out described proteolytic enzyme deactivation, the said chromatography after acidification can be followed successively by reversed phase chromatography, is used for deimpurity cation-exchange chromatography, chelating chromatography and gel chromatography or can be followed successively by hydrophobic chromatography, be used for deimpurity cation-exchange chromatography, chelating chromatography and HPLC.Because the acidification cost is lower, general host bacterium is adopted this method inactivated proteases, makes test operation simple, and cost reduces.The order of chromatography can be identical with the chromatography order that does not adopt protease treatment; And the sample of behind high-pressure liquid phase chromatograph, collecting can thoroughly be removed acetonitrile through being used for except that the POROS50HS cation exchange chromatography of acetonitrile again; Improve sample purity; Such chromatography order can obtain purity all greater than 95%, the target protein of activated protein total recovery about 30%.The high-pressure liquid phase chromatograph cost is higher; Therefore also can adopt the order that is followed successively by reversed phase chromatography, is used for deimpurity cation-exchange chromatography, chelating chromatography and gel chromatography; Reversed phase chromatography can substitute hydrophobic chromatography and carry out the first step seizure crude protein; Do not need organic solvent in the gel chromatography process, can substitute performance liquid chromatography, thereby reduce experimentation cost as the final step chromatography.Such chromatography order can obtain purity all greater than 95%, the target protein of activated protein total recovery about 40%.
The process of said acidification is generally and after the dilution supernatant, adds the 6M hcl acidifying and make pH value reach 3-4; Stir 2 hours acidifying inactivated proteases at 4-8 ℃; Regulate potential of hydrogen to neutral with 1M NaOH again, filter to remove the sex change protein precipitation with filtering membrane at last.Add 6M hydrochloric acid after being employed in diluted liquid, cost is lower, can make the proteolytic enzyme complete inactivation, is fit to scale operation.
Can adopt to add the proteinase inhibitor processing mode and carry out described proteolytic enzyme deactivation, the said chromatography after proteinase inhibitor is handled can be followed successively by hydrophobic chromatography, is used for deimpurity cation-exchange chromatography, chelating chromatography, the cation-exchange chromatography that is used for concentrating sample and gel chromatography.Adopt gel chromatography can reduce experimentation cost, and the cation-exchange chromatography that is used for concentrating sample in process behind the chelating chromatography is in order to reduce appearance volume on the last chromatography, the save operation time.Then adopt proteinase inhibitor to carry out specific aim for the processing of proteolytic enzyme defective host bacterium and handle, improve the degree of functioning of protease treatment, such chromatography order can obtain purity greater than 95% pure article, the target protein of albumen total recovery about 40%.
Said proteinase inhibitor can be mixture or the commercial compound albumen enzyme inhibitors of EDTA, PMSF and TPCK, and the said proteolytic enzyme defective host strain that is used to make up said engineering bacteria can be E.coli BL21 [hsd S gal (λ cI ts857 ind1 Sam7 nin5 lac UV-5-T7)].Adopt the mixture or the compound albumen enzyme inhibitors of proteinase inhibitor, can suppress various types of protease activities in the engineering bacteria, it is high to suppress selectivity, has improved the protease treatment effect.
Said hydrophobic chromatography can be HEA HyperCel hydrophobic chromatography; Said chelating chromatography can be Chelating Sepharose FF chelating chromatography; Said HPLC can be C5 Silica performance liquid chromatography; Said gel chromatography can be the Superdex75 chromatography, and said reversed phase chromatography can be POROS 50R1 chromatography.These chromatography medias or weighting material are to select according to the self structure of target protein and characteristic, can improve the refining effect of target protein effectively, increase the yield of target protein.
Said supernatant can adopt 1MNaCl to dilute, and extension rate is 20-30, and as 20,22,25,27 or 30 etc., preferred extension rate is 25, and reasonably extension rate can increase the yield of target protein.The substratum of using in fermentation culture described in the step (1) is made up of initial substratum of fermentation and the feed supplement that stream adds usually.The initial substratum of said fermentation can be the M9 substratum; And said M9 substratum is improved M9 substratum; Said feed supplement can comprise that carbon source and nitrogenous source can be that 50% glucose solution and mass ratio are 30% yeast powder solution for mass ratio respectively as the glucose of carbon source with as the yeast powder of nitrogenous source.Adopted improved M9 substratum as the initial substratum of fermentation, and adopted certain glucose and yeast extract than row,, improved the solubility expression of target protein greatly through controlling the culture carbon/nitrogen ratio of proper ratio as feed supplement.Wherein flow the feed supplement that adds and be meant the feed supplement of adopting continuous adding mode to add.
Can be thermal induction inducing described in the step (2), said thermoinducible temperature can be 39.0-43 ℃, as: 39 ℃, 40 ℃, 41 ℃, 42 ℃ or 43 ℃; Wherein preferred 41 ℃; The said thermal induction time can be 3-4 hour, as: 3 hours, 3.5 hours or 4 hours, preferred 3.5 hours.Suitable inducing temperature and induction time can increase the expression amount of target protein.
Because the chromatography in the purge process of the present invention adopts different chromatography orders under various conditions; The total recovery (can reach the yield of 30%-50%) and the proteic purity (can reach 95%) of activated protein have been improved effectively; The chromatography process of front is the preprocessing process to all article on the chromatography of back in the order of each chromatography; Sample behind the chromatography of front can directly get into the chromatography of back, practices thrift the preceding The pretreatment of chromatography of back like this, has reduced experimentation cost; Reduce the loss of target protein, be suitable for scale operation.Adopted the improved M9 substratum of confirming through repeated screening as the initial substratum of fermentation simultaneously; And adopt certain than the glucose that is listed as and yeast extract as feed supplement; Culture carbon/nitrogen ratio through the control proper ratio; Improve the solubility expression of target protein greatly, make the content of solubility target protein account for 30% of bacterial protein, reduced proteic renaturation technology and cost.Test according to the applicant; The nucleotide sequence of the proteic gene of said coding for alpha interferon analogue in embodiment of the present invention can be selected from nucleotides sequence and classify SEQ ID NO 1 as or be not less than in 93% the nucleotide sequence any one with the homology of SEQ ID NO 1, and all can reach the effect of above expression and purifying.
Description of drawings
Fig. 1 is the construction of recombinant plasmid synoptic diagram that contains the proteic gene of coding for alpha interferon analogue among the present invention;
Fig. 2 is the proteic polyacrylamide gel electrophoresis collection of illustrative plates of d interferon analogue of the present invention;
Fig. 3 is that interferon analogue anti-tumor activity of the present invention detects collection of illustrative plates.
Embodiment
The albumen of interferon analogue is interferon-like protein again, is meant and plain function and/or the similar albumen of constitutional features that is showed of known disturbances, in this article refers to the albumen of alpha interferon analogue.
Broken bacterium is meant that means such as adopting ultrasonication carry out fragmentation to bacterial strain, makes in the cell of bacterial strain or intercellular albumen sepn is come out, and is beneficial to the electrophoresis or the purification process in later stage.
Target protein be meant testing program the albumen that will obtain, in this patent, refer to the albumen of alpha interferon analogue.
The sequence of SEQ ID NO 1 is listed in the specification sheets back with the form of sequence table in embodiment of the present invention.
Like Fig. 1, shown in 2 and 3, the invention provides a kind of protein preparation method of alpha interferon analogue, detailed process is following:
(1) structure of expression engineering bacteria
Extract total mRNA from deriving from the human peripheral blood cell, the clone obtains 12 kinds of human alpha interferon genes, then according to the nucleotide sequence of each HuIFN-α, has designed 5 ' and 3 ' end primer respectively.The 12 kinds of human alpha interferon genes that have with above-mentioned clone are template; Amplify ORFs (the Open reading frame of 12 kinds of HuIFN-α through the PCR reaction of standard; ORF); Enzyme is cut the back and is connected with the coli expression carrier pBVB that same enzyme is cut, and carries out sequential analysis at last, the plasmid with 12 kinds of human alpha interferon genes that checking makes up.
Detailed process is following: total mRNA derives from human peripheral leucocytes, and the eDNA of IFN a is through the PCR method amplification, and amplification condition is following: 10 times of amplification buffers of 2.5 μ l; 0.75 the 10mM dNTPs of μ l, 0.5 μ l 25mM sal epsom, 0.25 μ l poly synthase; 0.75 μ l mRNA; 0.75 5 of μ l ' holds primer (10 μ M, IFN a 05:5 '-tggtgctcagct (a/g) caagtc-3 '), the 3 ' primer mixture of 0.75 μ l (1.7 μ m/each; IFN a 03-1:5 '-aatcatttccatgttg (a/g) accag-3 ';
IFN?a?03-2：5′-aatcatttcccggttgtaccag-3′；
IFN?a?03-3：5′-aatcatttccatgttgaaacag-3′；
IFN?a?03-4：5′-aatcatttcaagatgagcccag-3′；
IFN?a?03-5：5′-aatgattttcatgttgaaccag-3′；
IFN?a?03-6：5′-aatcattt(c/g)(c/g)atgttgaaccag-3′；
IFN?a?03-7：5′-atgcccctgtccttttctttac；
IFN?a?03-8：5′-gagtcgtttctgtgttggatcag-3′)。
Amplified production is electrophoretic separation on 1.0% sepharose, gel-purified, and be cloned in pCRII-TOPO or the pCR-4-TOPO carrier according to manufacturer's recommendation.Utilize the ABI automatic DNA sequencer DNA to insert segmental dna sequence analysis.
All respectively at the sequence among the GeneBank relatively, the GeneBank accession number of these genes is respectively NM_024013 (IFN-α 1) to all 12 kinds of human alpha interferon genes of clone, NM_000605 (IFN-α 2); NM_010504 (IFN-α 4), NM_010505 (IFN-α 5), NM_008335 (IFN-α 6); NM_008334 (IFN-α 7), NM_008336 (IFN-α 8), NM_002171 (IFN-α 10); NM_002172 (IFN-α 14); NM_002173 (IFN-α 16), NM_021268 (IFN-α 17), NM_002175 (IFN-α 21) unanimity.
For structure (shuffling) plasmid that shuttles back and forth, according to the nucleotide sequence of each HuIFN-a, designed 5 ' and 3 ' end primer respectively, 5 ' end primer band BamHI point of contact wherein, 3 ' end primer band EcoRI point of contact.With above-mentioned clone what have Interferon, rabbit a gene is template; Amplify ORFs (the Open reading frame of 12 kinds of HuIFN-a through the PCR reaction of standard; ORF), after BamHI and EcoRI enzyme are cut, be connected with the coli expression carrier pBVB that same enzyme is cut; Carry out sequential analysis at last, the plasmid of the band human interferon a gene that checking makes up.
Plasmid with 12 kinds of aforementioned structure band human interferon a genes is a template respectively; With BVF4:5 '-agggcagcattcaaagcag-3 ' and BVR3:5 '-tcagaccgcttctgcgttctg-3 ' is primer; Carry out pcr amplification, and with the product balanced mix that increases.The method of introducing according to document (Stemmer WPC.DNAshuffling by random fragmentation and reassembly:In vitro recombination for Mecular evolution.PNAS vol:91:10747-10751,1994) is subsequently carried out DNase I enzymolysis and PCR assembling.
Product with the PCR assembling is a template, with primer BVF:5 '-gaaggctttggggtgtgtg-3 ' and BVR:5 '-aatcttctctcatccgc-3 ', carries out the secondary amplification.The secondary amplified production is connected with the coli expression carrier pBVB that same enzyme is cut with EcoR I with BamHI; Connect product transformed into escherichia coli DH5 α (supE44 Δ lacU169 hdsR17 recA1 endA1 gyrA96 thi-1 relA1) or BL21 [hsd S gal (λ cIt s857 ind1 Sam7 nin5 lac UV-5-T7)]; Be the hybridization library of human alpha interferon gene molecule, and this hybridization library is screened.
In above all pcr amplifications and PCR assembling, all use common archaeal dna polymerase (NewEngland Biolab, USA), rather than high-fidelity DNA polymerase.
The escherichia coli DH5a of fresh conversion directly coating is inoculated on the LB flat board that contains 50 μ g/ml penbritins, and under 37 ℃, spends the night.Choose single colony inoculation in 96 orifice plates, have 100 μ l to contain the penbritin LB substratum (LBA) of 50 μ g/ml in every hole, 37 ℃ shake overnight cultures.Next day, the bacterial cultures of getting the saturated growth of 10 μ l is inoculated in 96 orifice plates (copy board) successively, and 100 μ l LBA substratum are arranged in every hole; The indigenous bacteria culture plate, temporary storage is in 4 ℃ (storage plates).Copy board is placed 30 ℃, and concussion is cultured to OD600=0.4, is rapidly heated then to 42 ℃.Through 4 hours thermal induction, the microbial culture plate directly moved into-80 ℃ of refrigerators, and twice of freeze thawing.Then, again bacterial lysate liquid is diluted to required concentration, inhales appearance and add to Daudi cell culture system build-in test anti-tumour cell proliferative activity, or add to the culture system build-in test antiviral activity of Wish cell.
Each takes turns about screening 10; 000 bacterium colony, preliminary screening go out 100 anti-tumour cell proliferatives or the highest bacterium colony of antiviral activity, get into further checking property testing: according to the above-mentioned selected the highest bacterial cultures numbering of activity; From storing on the plate that the bacterium streak inoculation of preserving is dull and stereotyped in LBA; 37 ℃ of following overnight cultures are chosen single colony inoculation next day in the LBA of 1ml liquid nutrient medium, and 30 ℃, 250rpm concussion spend the night.Then, 40 μ l are organized in one of test tube at another through the microbionation of cultivating, contain the LB that 1ml has added penbritin (50 μ g/ml) in the test tube.Then,, let sample experience following steps: 42 ℃ of abduction deliverings, collect bacterial cultures, freeze-thaw circular treatment, test each bacterial lysate liquid anti-tumour cell proliferative activity and antiviral activity by the primary dcreening operation step of introducing in the preceding text.
Each is taken turns in the screening process; Through choosing about 20 anti-tumour cell proliferatives or the highest bacterium colony of antiviral activity behind the checking property testing; Extract plasmid, use BVBF:5 '-accatgaaggtgacgctc-3 ' plasmid and insertion fragment thereof to be carried out automated dna sequence analysis as primer.On the basis of sequential analysis; Reject the identical clone of indivedual dna sequence dnas; Use the flanking sequence of the MCS upstream and downstream of expression vector pBVB then; Design primer BVBF:5 '-accatgaaggtgacgctc-3 ' and BVR:5 '-aatcttctctcatccgc-3 ' carry out pcr amplification, and product is used for the structure in next round reorganization library, repeats above-mentioned screening step then.
Take turns the antiviral and the highest escherichia coli cloning of anti-tumour cell proliferative activity that obtains the screening from above-mentioned five; Extract plasmid and order-checking; Its with, the proteic polynucleotide sequence called after of encoding novel SEQID NO 1, the above-mentioned plasmid that contains polynucleotide sequence (SEQ ID NO 1) is used for next step and makes up recombinant expression plasmid and engineering bacteria.
The polynucleotide sequence that manual work is added the SEQ ID NO 1 of initiator codon ATG is cloned in the derivable pBVB carrier of temperature; Be positioned under the regulation and control of PRPL promotor, recombinant expressed matter draws title to be respectively pNOVAFERON (shown in Figure 1 be the structure schema of this plasmid).Recombinant expression plasmid is transformed into respectively among escherichia coli DH5a or the BL21, obtains expressing engineering bacteria E.coli-DH5 α-509 or E.coli-BL21-509, it is frozen to express engineering bacterial strain.
(2) fermentation expression of engineering bacteria
Frozen expression engineering bacteria is inoculated in the LB substratum that contains the 50ug/ml penbritin in 1: 100 ratio, and shake-flask culture is about 10 hours under 30 ℃, 180rpm, is primary seed solution.Primary seed solution is inoculated in 1: 100 ratio contains an amount of antibiotic LB substratum, the about 12-13 of shake-flask culture hour is secondary seed under 30 ℃, 250rpm.During OD600=2.0-3.0, thalline is in the logarithmic phase initial stage, and the growth in this period is vigorous, and the seed of this moment adapts to new growing environment more easily.The secondary seed solution inoculative proportion is that inoculation in 1: 10 goes into to ferment in the initial substratum, and the about 13-15 of shake-flask culture hour is ferment-seeded under 30 ℃, 180rpm.
Ferment-seeded also joins in the fermentor tank by 1: 10 volume, is the initial substratum of fermentation in the fermentor tank, and culture temperature is controlled at 30 ℃, and ammoniacal liquor is regulated PH to 6.98 ± 0.02, and dissolved oxygen rate maintains more than 30% through stirring velocity and air flow.The modified version M9 of the initial substratum of described fermentation for confirming through repeated screening, the prescription of said improved M9 substratum is: the 1.0L substratum comprises Na
2HPO
412.0g, K
2HPO
43g, NaCl 0.5g, Trytone 10.0g, Yeast Extract 15.0g, Glucose 3.75g, NH
4Cl 1.0g and MgSO
40.3g the glucose in the said modified version M9 substratum is to add NH behind the independent autoclaving
4Cl and MgSO
4Be that the degerming of configuration after-filtration adds.
According to each cultivation stage engineering bacteria growth feed supplement (being flow feeding) in the fermenting process.The carbon source that feed supplement is used is 50% (mass ratio) glucose solution, and nitrogenous source is 30% (mass ratio) yeast powder solution, regulates pH value with the ammoniacal liquor of 25% (volume ratio), does skimmer with the bubble enemy solution of 20% (volume ratio).Owing to cross high-carbon/nitrogen ratio expressing quantity is descended; Cross low carbon/nitrogen and form insoluble form than the target protein that can make expression; Therefore adopt glucose and yeast extract as feed supplement, the fermentor tank of 50L approximately adds 1800ml 50% glucose solution, 2300ml 30% yeast powder solution; Ammoniacal liquor with 500ml 25% is regulated pH value; Through control culture carbon/nitrogen ratio, obtain suitable engineering bacteria growth velocity and protein expression speed, make the content of target protein account for 30% of bacterial protein.
Under the condition that the technical configuration of fermenter equipment allows, can adopt auto-feeding feed supplement mode, the feed supplement amount of designing unit's time; By device program control feed supplement pump; Automatic limited flow feeding, the parameter value variation control of fermentor tank is steady, and the growth conditions of bacterium is very stable.What of feed supplement amount then are the sizes according to the fermentor tank capacity, and the volume of fermented liquid and the nectar degree in the fermenting process, final nectar degree (OD600 value) are confirmed the feed supplement amount.
When the engineering bacteria growth reaches proper density, induce 42 ℃ of inducible proteins to express through being warmed up to for 30 ℃ from culture temperature.Induce in intensification to add quick repairing material in the process so that comparatively sufficient nutrition to be provided, keep dissolved oxygen rate more than 20% through strengthening stirring velocity with air flow, abduction delivering stopped the centrifugal receipts bacterium of fermenting in 3.5 hours.
The initial volume of fermentor tank top fermentation at the 50L working volume is 45L, heats up when being cultured to OD600=7.0-8.0 and induces, and in 30 minutes, temperature is risen to 42.0 ℃, keeps 41 ℃, induces 3.5 hours.The whole OD600 that ferments is 17.0-19.0.With this understanding, zymophyte reaches that 2100g bacterium weight in wet base/more than the 45L fermented liquid, the expression amount of target protein is about 33.0%.Through to the broken bacterium rear electrophoresis analytical proof of fermentation thalline, realize that with upper type the soluble proteins of alpha interferon analogue is expressed.Can obtain the efficient target protein expression of solubility at high proportion at this moment in section and this TR.Cross low inducing temperature and the counterseal protein-active is kept and minimizing target protein expression amount, too high inducing temperature is than increasing the ratio that target protein forms insoluble form.
When the albumen of structural similitude efficiently expressed in intestinal bacteria with it, majority presented the form of inclusion body, and therefore the albumen of inclusion body form biologically active not needs through protein renaturation technology.This interferon-like protein contains two pairs of disulfide linkage, and renaturation complex process and cost are increased, and influences protein yield simultaneously because of annealing efficiency is low.Solubility expression can save the technology of protein renaturation and direct purification obtains the albumen of biologically active.
(3) purifying process of target protein
Two kinds of engineering bacterium expression target proteins all are to be expressed in born of the same parents' pericentral siphon chamber with soluble form, and behind the bacterial cell disruption, the content of target protein accounts for 30% of bacterial protein.The theoretical molecular of target protein is 19314.28Da, and iso-electric point 6.60, the ratio of hydrophobic amino acid are 33.73%.
According to the different treatment mode to engineering bacteria proteolytic enzyme, the purifying mode of target protein is respectively following four kinds of purifying process.
Target protein purifying process one: (purifying process of proteolytic enzyme of said engineering bacteria not being handled before the chromatography)
The purifying process research initial stage is the albumen supernatant that adopts E.coli-DH5 α-509 engineering bacterium expression.The pure article of target protein electrophoresis level that behind the multistep purifying, obtain; But through mass spectrometric detection; Except that the intact proteins of most of molecular weight near 19314.28Da, remain the protein degradation that molecular weight is respectively 18622.15Da and 18578.89Da in the purification of samples, these two kinds of protein degradations are than the molecular weight little 692Da of difference and the 736Da of intact proteins; Maybe for PROTEIN C terminal amino acid residue by due to the proteasome degradation, cause aminoacid deletion.
According to above-mentioned property of protein prediction; Through purification schemes is explored, confirmed following purifying process: bacteria breaking (processing ultrasonication after the homogenate with phosphate-buffered salt PH8.0 solution) → centrifuging and taking supernatant → dilute (diluting 10 times) → HEA HyperCel hydrophobic chromatography → be used for deimpurity POROS 50HS cation-exchange chromatography → Chelating Sepharose FF chelating chromatography to obtain the mixture of purpose intact proteins and protein degradation with 1M NaCl with 1: 10 ratio.The sample that uses the chelating chromatography to obtain is pure as electrophoresis, needs mass spectroscopy can find that partial amino-acid residue degrade proteins exists.Because the albumen of three kinds of molecular form is more or less the same at aspects such as molecular weight, iso-electric point, hydrophobicitys in the mixture, so C5Silica performance liquid (HPLC) chromatographic separating process on probation.C5 Silica HPLC separates this sample, and with each fraction collection sample of mass spectroscopy, protein degradation is positioned at the back 1/3 of HPLC elution peak, should not be collected in the final sample.
Frozen expression bacterial classification inoculation is contained the LB substratum of 50ug/mg penbritin in 50ml, and shake-flask culture is about 10 hours under 30 ℃, 180rpm, is primary seed solution.Primary seed solution is inoculated in 4000ml in 1: 100 ratio contains an amount of antibiotic LB substratum, divide 12 bottles shake bottle under 30 ℃, 250rpm the about 12-13 of shake-flask culture hour be secondary seed.Inoculation in OD600=2.0-3.0 hour 1: 10 goes into to ferment in the initial substratum.
The autoclaving 40L initial liquid medium that ferments in advance in the fermentor tank; Temperature is controlled at 30 ℃ after adding seed liquor; Ammoniacal liquor is regulated PH to 6.98 ± 0.02, and dissolved oxygen rate maintains more than 30% through stirring velocity and air flow, according to each cultivation stage engineering bacteria growth feed supplement of fermenting process.Ammoniacal liquor with 25% is regulated pH value, does skimmer with 20% bubble enemy solution.Adopt auto-feeding feed supplement mode, replenishing 50% glucose solution is carbon source, and 30% yeast powder solution is nitrogenous source, and the feed supplement amount is confirmed according to the nectar degree in fermentation parameter and the fermenting process.When the growth of engineering bacteria density reaches OD600=7.0-8.0, induce 41 ℃ of inducible proteins to express through being warmed up to from culture temperature.Induce in intensification and to add quick repairing material in the process so that comparatively sufficient nutrition to be provided; Keep dissolved oxygen rate more than 20% through strengthening stirring velocity and air flow; Abduction delivering stopped fermentation in 3.5 hours; Final volume is about 45L, and OD600 is that the centrifugal receipts of 17.0-19.0. bacterium obtains 2100g bacterium weight in wet base approximately, and the expression amount of target protein is about 33.0%.
Purifying process: bacteria breaking → centrifuging and taking supernatant → with 10 times → HEA of 1M NaCl dilution HyperCel hydrophobic chromatography → be used for deimpurity POROS 50HS cation-exchange chromatography → Chelating Sepharose FF chelating chromatography to obtain the mixture of purpose intact proteins and protein degradation.Because the albumen of three kinds of molecular form is more or less the same at aspects such as molecular weight, iso-electric point, hydrophobicitys in the mixture, so use C5 Silisca HPLC separation method.The intact proteins position is the mixing zone of three kinds of molecular weight proteins at preceding 2/3 one of elution peak afterwards, therefore only collects preceding 2/3 part.Collect after be used for removing the acetonitrile that the POROS 50HS cation exchange chromatography of acetonitrile is thoroughly removed elutriant, can obtain purity greater than 95% pure article.Active total recovery is about 25%, the albumen of its biological activity ratio's aminoacid deletion (protein degradation) height.Detect the molecular weight of target protein be consistent with theoretical molecular (as shown in Figure 2) through polyacrylamide gel electrophoresis.
Target protein purifying process two: (the preceding a kind of purifying process that the proteolytic enzyme of said engineering bacteria is carried out acidification of chromatography)
Can the amino acid whose material of protein degradation be enzyme, the first method that suppresses degraded be to add the enzyme spcificity suppressor factor, and advantage is that selectivity is high; Shortcoming is the suppressor factor expensive; Second method is that enzyme denaturation is lost activity, and advantage is that cost is low, and shortcoming is a poor selectivity.Consider the scale operation cost, we have selected second method, and target protein has antiacid preferably sex change ability with 6M hcl acidifying inactivated proteases in employing.In order to prevent the degraded of target protein, after broken bacterium discharges with target protein, reach PH3-4 with the 6M hcl acidifying immediately.The co-precipitation of target protein is diluted with salts solution supernatant before the acidifying, and is added immediately when preventing the sour metaprotein deposition of part in the supernatant, can avoid supernatant to produce new deposition again, thereby influence the treatment effect of proteolytic enzyme.Earlier supernatant was done 1: 10,1: 20 or 1: 30 three extent of dilution with 1M NaCl diluent; Acidifying then, the contrast target protein recovery, protein-active two association; It is 80.5% that 1: 30 times of dilution yield can reach yield, and the result shows that 1: 30 Dilution ratio is best.1: 25,1: 30 two extent of dilution relatively again, 1: 25 the recovery is 80.16%, with 1: 30 basic near and can reduce extension rate; Be more suitable in suitability for industrialized production; As be lower than 1: 20 and influence protein yield, the too high sample volume that makes is bigger than normal, increases the chromatography time and causes waste; And protein recovery is not seen increase, and therefore final definite extension rate is 1: 25.
With broken (processing ultrasonication after the homogenate with phosphate-buffered salt PH8.0 the solution) → albumen supernatant dilution → acidifying → POROS 50R1 reversed phase chromatography of E.coli-DH5 α-509 engineering bacteria that fermentation obtains → be used for deimpurity POROS 50HS cation-exchange chromatography → Chelating Sepharose FF chelating chromatography → Superdex75 gel chromatography with 1: 10 ratio; Can obtain purity greater than 95% pure article, active total recovery about 30%.Before the Superdex75 gel chromatography, can through the POROS 50HS cation-exchange chromatography that being used for concentrating sample, reduce the applied sample amount and the chromatography time of final gel chromatography.The target protein that obtains is through mass spectrometric detection, and molecular weight is 19313.07Da, is consistent with theoretical molecular.
Actication of culture, seed liquor preparation, fermenting process are with embodiment 1.
E.coli-DH5 α-509 engineering bacteria that fermentation obtains is broken; Supernatant is done dilution in 1: 25 with 1M NaCl diluent; The supernatant diluent reaches pH3-4 with the 6M hcl acidifying immediately, places 2 hours acidifying inactivated proteases at 4-8 ℃, with 0.45 μ M bag type filter solids removed by filtration impurity.Filter albumen supernatant → POROS 50R1 reversed phase chromatography → be used for deimpurity POROS 50HS cation-exchange chromatography → Chelating Sepharose FF chelating chromatography → be used for POROS 50HS cation-exchange chromatography → Superdex75 gel chromatography of concentrating sample; Can obtain purity greater than 95% pure article, active total recovery about 30%.The target protein that obtains is through mass spectrometric detection, and molecular weight is 19313.07Da, be consistent with theoretical molecular, and the target protein of biological activity ratio's aminoacid deletion is high.
Target protein purifying process three: (the preceding another kind of purifying process that the proteolytic enzyme of said engineering bacteria is carried out acidification of chromatography)
Acidification technique is with target protein purifying process two; With broken (processing ultrasonication after the homogenate with phosphate-buffered salt PH8.0 the solution) → albumen supernatant dilution → acidifying → HEA HyperCel hydrophobic chromatography of E.coli-DH5 α-509 engineering bacteria that fermentation obtains → be used for deimpurity POROS 50HS cation-exchange chromatography → Chelating Sepharose FF chelating chromatography → C5 Silica performance liquid chromatography → be used to remove POROS 50HS cation-exchange chromatography of acetonitrile with 1: 10 ratio; Can obtain purity greater than 95% pure article, active total recovery about 40%.The target protein that obtains is through mass spectrometric detection, and molecular weight is 19313.07Da, be consistent with theoretical molecular, and the target protein that the preceding two kinds of purification process of target protein biological activity ratio obtain is high.
Actication of culture, seed liquor preparation, fermenting process are with embodiment 1.
E.coli-DH5 α-509 engineering bacteria that fermentation obtains is broken; Supernatant is done dilution in 1: 25 with 1M NaCl diluent; The supernatant diluent reaches PH3-4 with the 6M hcl acidifying immediately; Stir 2 hours acidifying inactivated proteases at 4-8 ℃, regulate potential of hydrogen to weakly alkaline (PH=7.2-7.4) with 1M NaOH again, with 0.45 μ M bag type filter solids removed by filtration impurity.Filter albumen supernatant → HEA HyperCel hydrophobic chromatography → be used for deimpurity POROS 50HS cation-exchange chromatography → Chelating Sepharose FF chelating chromatography → C5 Silica performance liquid chromatography → be used for removing POROS 50HS cation-exchange chromatography of acetonitrile; Can obtain purity greater than 95% pure article, active total recovery about 40%.If the volume of the sample before the C5 Silica performance liquid chromatography is bigger, the POROS 50HS cation-exchange chromatography that also can be used for concentrating sample earlier reduces the running time with concentrating sample.The target protein that obtains is through mass spectrometric detection, and molecular weight is 19313.07Da, be consistent with theoretical molecular, and the target protein that the preceding two kinds of purification process of target protein biological activity ratio obtain is high.
Target protein purifying process four: (proteolytic enzyme to said engineering bacteria before the chromatography adds the purifying process that proteinase inhibitor is handled)
E.coli BL21 [hsd S gal (λ cI ts857 ind1 Sam7 nin5 lac UV-5-T7)] is a kind of protease-deficient engineering host bacterium, and protease content is lower, only needs the small amounts of protease suppressor factor just can suppress its degraded to target protein.The method that adopts proteolytic enzyme defective host strain to add broken bacterium under the specific proteins enzyme inhibitors condition can be avoided the degraded of proteolytic enzyme to target protein, simplifies purification step, improves protein yield.Bacteria protease divides from the mechanism of action; Generally include metalloprotease (like Carboxypeptidase A/B; Endopeptidase N etc.), Tryase (like endopeptidase C, carboxypeptidase y, Proteinase K), dredge basic proteolytic enzyme (like cathepsin C) and mixed type enzyme, can distinguish EDTA (Ethylenediaminetetraacetic acid), PMSF (Phenylmethanesulfony fluoride), TPCK suppressor factor inhibitory enzyme activities such as (Tosyl-L-phenylalanine chloromethyl ketone).We use above-mentioned three types inhibitor screening suppressor factor respectively, and the result is all satisfied inadequately, thus consider it is a small amount of simultaneously existence of enzyme of multiple type of action, use compound enzyme inhibitors after, respond well, so adopt compound albumen enzyme inhibitors.
We select the proteinase inhibitor of three kinds of different effects mechanism for use: EDTA, PMSF and TPCK.Different because of the dissolution conditions of three kinds of compounds, three kinds of compounds are prepared storage liquid respectively, and EDTA is soluble in water, and with phosphate-buffered salt PH8.0 solution preparation 100mM/L solution, PMSF and TPCK are soluble in organic solvent, are mixed with the aqueous isopropanol of 50mM/L.Use the final concentration of liquid to reach 1.0-10.0mM/L, 0.1-1.0mM/L, 0.1-1.0mM/L respectively.
The E.coli-BL21-509 engineering bacteria that fermentation obtains is processed homogenate with 1: 10 ratio with phosphate-buffered salt PH8.0 solution; Add simultaneously behind the three specific specificity proteinase inhibitor mixings and to reset and add 10 times → HEA of 1M NaCl dilution HyperCel hydrophobic chromatography → be used for deimpurity POROS 50HS cationic exchange → Chelating Sepharose FF chelating chromatography → be used for POROS 50HS cation-exchange chromatography → Superdex75 gel chromatography of concentrating sample on 0-10 ℃ of broken bacterium → centrifugal acquisition albumen; Can obtain purity greater than 95% pure article, total recovery about 40%.The target protein that obtains is consistent with theoretical molecular through mass spectrometric detection, detects through polyacrylamide gel electrophoresis also to be consistent with theoretical molecular.This method is used to treat the preparation of medicine, the detection of Virahol residual quantity in the detection of the extra increase proteinase inhibitor residual quantity of needs and the finished product in stoste detects.Simultaneously can't remove isomers.
Actication of culture, seed liquor preparation, fermenting process are with embodiment 1.
E.coli-DH5 α-509 engineering bacteria that fermentation obtains is processed homogenate with 1: 10 ratio with phosphate-buffered salt PH8.0 solution; 0-10 ℃ of broken bacterium is broken after adding three specific specificity proteinase inhibitor mixings simultaneously; This three specific specificity proteinase inhibitor is respectively EDTA, PMSF, TPCK; Its final concentration is respectively resets and add 10 times → HEA of 1M NaCl dilution HyperCel hydrophobic chromatography → be used for deimpurity POROS 50HS cationic exchange → Chelating Sepharose FF chelating chromatography → be used for POROS 50HS cation-exchange chromatography → Superdex75 gel chromatography of concentrating sample on 2.0mM/L, 0.4mM/L, the 0.4mM/L → albumen; Can obtain purity greater than 95% pure article, active total recovery about 40%.The target protein that obtains is consistent with theoretical molecular through mass spectrometric detection.
More than the test parameter of various chromatographies following:
1, hydrophobic chromatography parameter:
The balance chromatography column: balance liquid for the solution that is made into by the PB of final concentration 20mM, 0.02% Tween80 (mass volume ratio) and final concentration 1M NaCl (solution PH=7.5-8), the PB of final concentration 20mM specifically fill a prescription for TV be the Na that contains 6.23g in the solution of 1000ml
2HPO
412H
2The NaH of O and 0.41g
2PO
42H
2O.
Equilibrium velocity 200ml/min, starting time is 15min;
Last appearance: go up appearance flow velocity 200-260ml/min;
Wash-out: with balance liquid balance 15min, the elutriant (PH=6-6.5 of elutriant) of using PB and 0.02%Tween80 by 20mM to be made into again carries out wash-out, wash-out impurity and partial pigment before the wash-out; Elution flow rate is 180-200ml/min;
Collect: by the Na of final concentration 20mM
2HPO
4The collection liquid that-Hydrocerol A and 0.02% Tween80 (mass volume ratio) are made into (collecting the PH=3.5-4 of liquid) is collected main peak, the Na of final concentration 20mM
2HPO
4The concrete prescription of-Hydrocerol A is the Na that contains 2.3g in the solution of 1000ml for TV
2HPO
412H
2The Hydrocerol A of O and 1.42g.
Flow velocity 200ml/min during collection.At last with the liquid of collecting with 1 times of the diluted of PH=4.4; The solution of diluent for being made into by the Tween80 of final concentration 25mM AAB and 0.02%, the concrete prescription of final concentration 25mMAAB are to contain the anhydrous sodium acetate of 1g and the glacial acetic acid of 0.73ml in the solution of 1000ml for TV.
2, be used for deimpurity POROS 50HS cation-exchange chromatography parameter:
Balance: balance liquid is the solution that is made into (PH=4-4.5) of the Tween80 of final concentration 25mM AAB and 0.02%; Equilibrium velocity is 20ml/min, and starting time is 10min;
Last appearance: go up appearance flow velocity 20ml/min;
Wash-out and collection: wash-out is preceding with balance liquid balance 10min; Adopt gradient elution; Earlier carry out the wash-out first time with elutriant I, elution flow rate 18-20ml/min carries out wash-out and the collect main peak second time with elutriant II again; Elution flow rate is about 18-20ml/min, and last main peak transfers PH to 7.6-8.0 with 6MNaOH.The solution that elutriant I is made into for the Tween80 by final concentration 25mM AAB and 0.02%, and PH=4-4.5; Elutriant II is for by final concentration 25mM AAB, final concentration being the NaCl of 1.2M and the solution that 0.02% Tween80 is made into, and PH=4-4.5.
3, Chelating Sepharose FF chelating chromatography parameter:
Balance: balance liquid is the solution (PH=7.5-8 of solution) that PB, 0.02% Tween80 and the final concentration 0.5M NaCl of final concentration 20mM is made into; Equilibrium velocity 20ml/min, starting time is 15min;
Last appearance: go up appearance flow velocity 20ml/min;
Wash-out and collection: wash-out is preceding with balance liquid balance 10min; With elutriant I and elutriant II difference wash-out; Elutriant I is the solution (its PH=4.5-5) that final concentration 25mM AAB, 0.02% Tween80 and final concentration 0.5M NaCl are made into, and elution flow rate 20ml/min washes impurity peaks; Elutriant II is the Na of final concentration 20mM
2HPO
4The solution (its PH=3.8) that-Hydrocerol A, 0.02% Tween80 and final concentration are made into for 0.5M NaCl, elution flow rate 20ml/min washes impurity peaks once more; With collecting liquid wash-out target protein, collecting liquid is the Na of final concentration 20mM again
2HPO
4The solution (its PH=3.6) that-Hydrocerol A, 0.02% Tween80 and 0.5MNaCl are made into; Flow velocity is about 15ml/min during collection; Collect the back sample with 3 times of the diluted of PH=4.4, said diluent is the solution that the Tween80 of final concentration 25mM AAB and 0.02% is made into.
4, the POROS 50HS cation-exchange chromatography parameter that is used for concentrating sample
Balance: balance liquid is the solution (its PH=4-4.5) that the Tween80 of final concentration 25mM AAB and 0.02% is made into, equilibrium velocity 20ml/min, and starting time is 5min;
Last appearance: go up appearance flow velocity 20ml/min;
Collect: collect full peak with collection liquid, collection liquid is the solution (its PH=7.5-8) that PB, 0.02% Tween80 and the 0.5M NaCl of final concentration 20mM is made into, and flow velocity is about 20ml/min during collection.
5, C5 Silica performance liquid chromatography parameter
Balance: balance liquid is the solution (its PH=3-4) that the TFA (volume ratio of trifluoroacetic acid) of pure water and 0.02% is made into, equilibrium velocity 6ml/min, and starting time is 20min;
Last appearance: go up all article and transfer PH=3-4, last appearance flow velocity 6ml/min with 30%TFA;
Wash-out and collection: the buffered soln I (its PH=3-4) that is made into the TFA of pure water and 0.02% and cross post 20 minutes respectively with the buffered soln II (PH=3-4) that 0.07%TFA is made into 35% acetonitrile (volume ratio); The buffered soln III (PH=3-4) that is made into 35% acetonitrile and 0.07%TFA then and carry out gradient elution with the buffered soln IV (PH=3-4) that 55% acetonitrile and 0.08%TFA are made into collects main peak; Main peak is used 5 times of the diluted of PH=4.4 immediately behind the wash-out, the solution that diluent is made into for the Tween80 by the 25mM AAB of final concentration and 0.02%.
6, be used for removing the POROS 50HS cation-exchange chromatography parameter of acetonitrile:
Balance: the solution (its PH=4.4) that balance liquid is made into for the Tween80 by final concentration 25mM AAB and 0.02%, starting time 3min, equilibrium velocity 20ml/min;
Last appearance: go up appearance flow velocity 20ml/min;
Wash-out and collection: buffered soln one (its PH=4.4) wash-out that at first is made into the Tween80 of final concentration 25mM AAB and 0.02% is to reduce the residual of acetonitrile; Buffered soln two (its PH=4.4) wash-out that is made into the PB of final concentration 20mM, 0.02% Tween80 and 0.5M NaCl again; Reduce the residual of acetonitrile once more; Use final concentration to collect full peak as the collection liquid (PH=7.6) that the PB of 20mM, 0.02% Tween80 and 0.5M NaCl are made at last, protein concentration is controlled at 1.0-1.5mg/ml.
7, gel chromatography parameter
Balance: balance liquid is that final concentration is the PB of 20mM and the mixing solutions (PH=7-8) that 0.15M NaCl is made into, and equilibrium velocity is 6ml/min;
Last appearance: go up appearance flow velocity 6ml/min;
Wash-out and collection: use final concentration to carry out wash-out as the elutriant (PH=7-8) that PB and the final concentration 0.15M NaCl of 20mM is made into, elution flow rate is 6ml/min, collects the solution that elutes.
8, reversed phase chromatography POROS 50R1 parameter
Balance: balance liquid is pure water (accent PH is 3-4), and equilibrium velocity is 15ml/min, and starting time is 5min;
Last appearance: go up appearance flow velocity 15ml/min;
Wash-out and collection:, use the eluant solution of 40% acetonitrile (accent PH is 3-4) to collect target peak again with the eluant solution impurity peaks of 15% acetonitrile (accent PH is 3-4).
(4) quantitatively determined of anti-tumor activity
Cell proliferation active (antiproliferation activity) is one of major function of recombination high efficiency anti-tumor viral protein injection liquid.But different cells have different antiproliferative activities, do not have unified clone to be used to detect the antitumor proliferation activity of human interferon at present, adopt the Daudi cell to carry out the anti-tumour cell proliferative activity of human interferon in the world.Have homology because of recombinant human anti-tumor viral protein and I type disturb, thus, we select the most general clone Daudi cell at present.Carry out anti-tumour cell proliferative activity with Daudi (ATCC CCL-213) cell and measure, three kinds of detection methods are arranged.Direct cell counting, isotropic substance infiltration method and WST-1 method.Through a large amount of experiments relatively, the WST-1 method is optimum selection.
Novagenetics Inc. company with my company cooperates adopts the antitumor proliferation activity of Daudi cell detection, through a large amount of preliminary experiments, its objective is stability and the accuracy of confirming optimum cell inoculum density and this method; After having confirmed preliminary experimental technique; Further cooperating with " China Research Centre of Medicine Biological Products Standardization " in foundation and the perfecting process; Launched the methodological study of stability, circulation ratio, accuracy and the recovery; Through nearly 2 months experiment, both sides have set up the method for standard specifications jointly.Detect multiple batches of recombination high efficiency anti-tumor viral protein injection liquid stoste with this method, its anti-tumour cell proliferative specific activity is not less than 2.0 * 106U/mg.
The quantitatively determined of embodiment 5 New-type alpha-interferon analogue anti-tumor activities
(1) bed board: get the centrifugal collection of cultured cells, become every 1mL to contain 4.0 * 10 with complete inoculum preparation
3The cell suspension of individual cell is inoculated in the 96 porocyte culture plates, every hole 100 μ L.In temperature is to cultivate about 2 hours under 36.5 ℃, 5% CO2gas incubator condition.
(2) determination of activity is with reference to the preparation of article: be diluted to every 1ml with complete culture solution and contain 15000U, operation under aseptic condition.
(3) preparation of trial-product: the New-type alpha-interferon analogue is diluted with complete culture solution by protein content in advance.
(4) preparation sample gradient: every hole adds the 180*1 complete culture solution in 96 porocyte culture plates, in the capable corresponding hole of B, adds the reference article of 20*1/ hole 4.2 preparations or the trial-product of 4.3 preparations.Do 10 times of dilutions from the capable 20*1/ of getting of B hole to C is capable, down do 10 times of dilutions successively to D is capable with the capable 20*1/ of getting of reason C hole, totally 6 extent of dilution, each extent of dilution is done 2 holes, and the surplus liquid of 180*1 is stayed in every hole.
(5) application of sample: take out the Tissue Culture Plate of 4.1 preparations, with corresponding this Tissue Culture Plate, the every hole 100*1 of moving into of the sample gradient of (4) preparation.In temperature is 36.5 ℃, cultivates 6 days under the 5% CO2gas incubator condition.
(6) dyeing: cultivated the back the 7th day, and in Tissue Culture Plate, added staining fluid WST-1, every hole 20*1 is 36.5 ℃ in cultivating the temperature inside the box, 5% carbon dioxide conditions held 4 hours.
(7) colorimetric: using ELIASA is to measure wavelength with 450nm, and 690nm is that reference wavelength detects absorbancy, and the record result.
(8) experimental result
Anti-tumour cell proliferative activity is 1.7 * 107U/mL, and the anti-tumour cell proliferative specific activity is 1.5 * 107U/mg, and raw data is seen Fig. 3.
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Claims (6)
1. the protein preparation method of an alpha interferon analogue is characterized in that comprising step:
(1) the proteic gene clone of coding for alpha interferon analogue formed recombinant plasmid transformed behind the expression vector is built into engineering bacteria in the host bacterium; Again said engineering bacteria is carried out fermentation culture, the nucleotide sequence of the proteic gene of said coding for alpha interferon analogue is SEQ ID NO 1;
(2) induce engineering bacterium expression target protein after the said fermentation culture;
(3) the said target protein of purifying, purification step are the sample that broken thalline, dilution supernatant, chromatography and collection after inductive engineering bacteria, centrifugal fragmentation contains target protein,
The proteolytic enzyme of said engineering bacteria is not handled before the said chromatography, said chromatography is followed successively by hydrophobic chromatography, is used for deimpurity cation-exchange chromatography, chelating chromatography and HPLC; Before the perhaps said chromatography proteolytic enzyme of said engineering bacteria is handled; It is the not engineering bacteria of defective of proteolytic enzyme that the mode of employing inactivated proteases is handled the host bacterium; Adopt the mode of arrestin enzymic activity to handle the engineering bacteria that the host bacterium is the proteolytic enzyme defective; The mode of said inactivated proteases is an acidification; Said chromatography after acidification is followed successively by reversed phase chromatography, is used for deimpurity cation-exchange chromatography, chelating chromatography and gel chromatography or be followed successively by hydrophobic chromatography, be used for deimpurity cation-exchange chromatography, chelating chromatography and HPLC; Said arrestin enzymic activity mode is for adding proteinase inhibitor; Said chromatography after proteinase inhibitor is handled is followed successively by hydrophobic chromatography, is used for deimpurity cation-exchange chromatography, chelating chromatography, the cation-exchange chromatography that is used for concentrating sample and gel chromatography, and said proteinase inhibitor is mixture or the commercial compound albumen enzyme inhibitors of EDTA, PMSF and TPCK.
2. the protein preparation method of alpha interferon analogue as claimed in claim 1; It is characterized in that before said chromatography the proteolytic enzyme of said engineering bacteria handled and adopting the mode of inactivated proteases to handle the host bacterium is that proteolytic enzyme is not under the situation of the engineering bacteria of defective; The process of said acidification makes pH value reach 3-4 for after the dilution supernatant, adding the 6M hcl acidifying; Stir 2 hours acidifying inactivated proteases at 4-8 ℃; Regulate potential of hydrogen to neutral with 1M NaOH again, filter to remove the sex change protein precipitation with filtering membrane at last.
3. the protein preparation method of alpha interferon analogue as claimed in claim 1; Under the situation that it is characterized in that before said chromatography not the proteolytic enzyme of said engineering bacteria is handled; Said hydrophobic chromatography is a HEA HyperCel hydrophobic chromatography; Said to be used for deimpurity cation-exchange chromatography be POROS 50HS cationic exchange, and said chelating chromatography is a Chelating Sepharose FF chelating chromatography, and said HPLC is a C5 Silica performance liquid chromatography.
4. the protein preparation method of alpha interferon analogue as claimed in claim 2; It is characterized in that said hydrophobic chromatography is a HEA HyperCel hydrophobic chromatography; Said to be used for deimpurity cation-exchange chromatography be POROS 50HS cationic exchange, and said chelating chromatography is a Chelating Sepharose FF chelating chromatography, and said HPLC is a C5 Silica performance liquid chromatography; Said gel chromatography is the Superdex75 chromatography, and said reversed phase chromatography is a POROS 50R1 chromatography.
5. the protein preparation method of alpha interferon analogue as claimed in claim 1; It is characterized in that before said chromatography the proteolytic enzyme of said engineering bacteria handled and adopt the mode of arrestin enzymic activity to handle under the situation of engineering bacteria that the host bacterium is the proteolytic enzyme defective; Said hydrophobic chromatography is a HEA HyperCel hydrophobic chromatography; Said each cation-exchange chromatography is POROS 50HS cationic exchange; Said chelating chromatography is a Chelating Sepharose FF chelating chromatography, and said gel chromatography is the Superdex75 chromatography.
6. like the protein preparation method of claim 1,2,3,4 or 5 described alpha interferon analogues; It is characterized in that said supernatant adopts 1MNaCl to dilute; Extension rate is 20-30, and the substratum of using in fermentation culture described in the step (1) is made up of initial substratum of fermentation and the feed supplement that stream adds, and the initial substratum of said fermentation is the M9 substratum; Said feed supplement comprises as the glucose of carbon source with as the yeast powder of nitrogenous source; Be thermal induction inducing described in the step (2), said thermoinducible temperature is 39.0-43 ℃, and the said thermoinducible time is 3-4 hour.
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