CN101109012B - Representation of vampire plasmin activator alpha 2 in yeast and manufacturing method thereof - Google Patents

Abstract

The invention relates to expression of a vampire bat plasmin activator alpha 2 in yeast and a manufacturing method thereof, especially a method highly expressing in yeast and producing vampire bat saliva plasmin activator alpha2 (DSPA alpha2) protein recombinant. The invention comprises a DSPA Alpha2 gene that is optimized by using a codon, a special yeast fermentation condition to improve the biomass yield, and six groups of histidine tag coding sequence added before the 5' end of DSPA alpha2 gene to facilitate the rapid isolation and purification of the DSPA alpha2 protein recombinant process. The DSPA alpha2 can activate the human plasmin and turn the plasmin into active plasmin, which causes the further hydrolysis of the fibrinous. Therefore, it is quite promising for the recombinant protein to be developed into the protein medicines to cure various acute human thrombosis diseases.

Description

Expression and the production method of vampire plasmin activator alpha 2 in yeast

Technical field

The present invention relates to the genetically engineered field, relate to particularly in yeast the method for expressing and producing vampire salivary plasminogen activator α 2 (DSPA α 2) recombinant protein, DSPA α 2 genes that are optimized comprising the son that accesses to your password, specific saccharomycetes to make fermentation incubation growth condition with improve its biological yield and before 5 ' end of DSPA α 2 genes interpolation 6 histidine-tagged encoding sequences to make things convenient for the fast purifying of DSPA α 2 recombinant proteins.

Background technology

So-called thrombus (thrombus) is exactly interior some major organs (such as heart, brain, limbs and lung etc.) of human body or some formed elementss (thrombocyte, scleroproein, red corpuscle etc.) in other position Endovascular blood have formed " mechanicalness " part and more stable when being sticked together mutually embolus, can stop and delay passing through smoothly of blood, it is different with normal physiological hemostasis, is a kind of pathologic result of hemostatic mechanism excessive activation.

Thrombus is very large to the harm of human body, be mainly manifested in the following aspects: (1) obstructing arterial blood vessel: when not yet total blockage lumen of vessels of thrombus, can cause the local organs ischemic and cause atrophy, such as total blockage or cause that essential amount of blood supply is not enough and lack effective side and prop up circulation time, can cause the ischemic necrosis of local organs.For example, cerebral artery thrombosis may cause cerebral infarction, and the heart coronary artery thrombus may cause myocardial infarction, and thromboangiitis obliterans then can cause suffers from limb gangrene etc.Behind the venous thrombosis, if fail to set up effective side Zhi Xunhuan, then cause local extravasated blood, oedema, hemorrhage, in addition downright bad, can cause hemorrhagic infarct such as mesenteric venous thrombosis.(2) embolism: before thrombus was not firmly adhered with vessel wall, can coming off in whole or in part of thrombus formed embolus, with the blood flow operation, when arriving distal end portion, can cause embolism.Include bacterium such as embolus, can cause septic infarct or the embolic abscess of embolism tissue.(3) heart valve distortion: heart valve thrombus organization, can cause valvular adhesion, cause valvular stenosis, such as then scar contraction of proliferation of fibrous tissue in the machine process, valvular inadequacy be can cause, rheumatic endocarditis and subacute bacterial endocarditis seen.(4) microcirculatory popularity microthrombusis, i.e. DIC: can cause extensively hemorrhage and shock of general.This shows that thrombus is very dangerous to human body, such as untimely treatment, just may cause death or disable.Therefore, exploitation has prevention and treats the protein thrombolysis new drug of a new generation of various thrombotic diseases, is very significant to protect mankind health.

Develop the thrombolysis new drug, at first just needing what is understood is " fibrinolytic system " or " fibrinolytic system ".It is a kind of proteolysis system with different physiological roles in people or the mammalian body, its Main Function is to prevent the fibrinous excessive formation of Endovascular, therefore, fibrinolytic system also is the basis of many protein thrombolysis new drug developments not only with the formation of thrombus be dissolved with substantial connection simultaneously.

Fibrinolytic system can make the fibrin clot local or a property crossed that produces in the human body obtain at any time dissolving (scleroproein that deposits on the vessel wall of namely degrading), thereby prevents thrombosis; In addition, it also have guarantee body of gland excretion road unobstructed, remove wound or the local fibrin clot of inflammation to promote the function of wound healing.Moreover, fibrinolytic system also occurs relevantly with cell migration, growth and embryo, also demonstrates very consequence in the development of malignant tumour and transfer process.

Fibrinolytic system mainly is comprised of four parts such as Profibrinolysin, plasminogen activator, plasminogen activator inhibitor and plasmin deactivation thing, wherein the composition of fundamental sum core is Profibrinolysin, it is without any fibrinolytic at first, but in vivo, under the effect of outer activator, can be transformed into can fibrinolytic plasmin, plasminogen activator inhibitor and plasmin deactivation thing then play opposite effect, and they mainly play regulating effect to the excessive activation of fibrinolytic system.

Vampire (Desmodus rotundus) biology take the blood of livestock and other large mammals as food that is one way of life in South and North America, its saliva having prevents the special role that other animal blood solidifies.At first, people are separated to some from the saliva of vampire can thrombolytic protein matter, but afterwards more deep research finds that it is exactly bat salivary plasminogen activator (Salivary PlasminogenActivivator, DSPA).This thrombolytic effect of bat saliva is the result of natural selection, and this is so that vampire is more suitable for getting zoophagous fresh blood.Bat DSPA has 85% homology approximately with human plasminogen activator (t-PA) aspect aminoacid sequence, therefore, can demonstrate extremely low immunogenicity in intravenous drip behind human body ^[1]What be worth to propose especially is, different from other protein thrombolytic drug, bat DSPA has high selectivity to scleroproein, and in the situation that does not have scleroproein to exist, it only keeps very low biological activity; But when having scleroproein to occur, it can be rapidly with it in conjunction with and activity improved tens thousand of times rapidly.Exactly because this specific character is arranged, make it be unlikely to when thrombus to cause that the excessive activation because of whole fibrinolytic system causes scleroproein, fibrinogenic a large amount of consumption and thrombin degraded, and and then cause the risk of irregular bleeding.So in all its bearings, vampire DSPA meets people to the expectation of novel thrombolytic protein medicaments.DSPA α 2 can activate human plasminogen, make it change activated plasmin into, further cause thus fibrinous fast hydrolyzing, so this recombinant protein is hopeful to be developed to the protein medicaments that can treat various acute thrombus class diseases into a kind of very much from now on.

1991, the people such as Kratzschmar ^[2]From vampire, be separated to first the cDNA of four kinds of different DSPA of coding, respectively called after DSPA α 1, DSPA α 2, DSPA β and DSPA γ.Sequence homology relatively finds, these four kinds of bat DSPA are respectively by different coded by said gene, namely are not to seize and process by compiling after the same genetic transcription.DSPA α 1 and DSPA α 2 sequences are the longest, the polypeptide chain of all encoding, their maturation protein (containing 441 amino-acid residues) comprises four functional domains, is respectively one and refers to district (Finger), an epidermal growth factor subarea (EGF), a Kringle district (K) and a serine protease district (P).The albumen of DSPA β and DSPA γ coding is all smaller, and maturation protein only contains 395 and 358 amino-acid residues; In addition, the hypoproteinosis Finger district of DSPA β coding, hypoproteinosis Finger and two districts of EGF of DSPA γ coding.The people such as Bringmann find that bat DSPA is extremely sensitive to scleroproein when research, compare with people t-PA, and DSPA lacks plasmin susceptibility shearing site ^[3]People t-PA exists with the strand zymogen forms, just can change activated double chain form into after shearing at avtive spot, and bat DSPA exists with single stranded form, does not need other proteolytic enzyme cutting.When having fibrin polymer to exist, the biological activity of DSPA α 1 or DSPA α 2 increases sharply, and will exceed 102,100 times than without scleroproein the time, and people t-PA only increases 550 times ^[4]In addition, confirm also that after deliberation the Finger district is to bat DSPA and fibrinous in conjunction with extremely important.Lack in the DSPA family DSPA β in Finger district and DSPA γ to scleroproein in conjunction with undertighten, and be subjected to scleroproein stimulation degree also less.

DSPA α 1 is because of content in bat saliva and the highest active concern that is subject to a lot of scholars, and is therefore also many for its research, once expressed restructuring DSPA α 1 such as people such as Petri in Chinese hamster ovary celI, and expression amount reaches 60mg/L; Utilize simultaneously baculovirus in expressed in insect cells, expression amount reaches 10mg/L ^[5]The people such as Schiermeyer have also expressed this albumen in tobacco, its expression amount is up to and contains 38 μ g recombinant proteins in every gram blade material ^[6]To the research of active height and specificity also very strong DSPA α 2 at present also seldom, only have the people such as early stage Kratzschmar in bhk cell, to express above-mentioned four kinds of multi-form DSPA fibrinolysin activators, but regrettably, the expression amount of recombinant protein is not high in this expression system ^[7]

Although DSPA is the very tempting thrombolytics of a kind of prospect, because it is present in a kind of common and not dangerous vampire, obviously be unpractical by artificial extraction.Obtain cheap and effective bat DSPA thrombolysis class medicine, it is very necessary to set up engineered scale operation platform.

The latest developments of genetically engineered research field make and utilize some yeast cell as bio-reactor and efficiently expressing exogenous gene becomes possibility, and what be worth especially proposing is pichia pastoris phaff (Pichia pastoris) expression system.Up to now, people utilize this expression system the foreign protein genes of successful expression surpassed 300 kinds ^[8], comprising from the extensive gene in source such as bacterium, fungi, prokaryotic organism, plant, animal and human's class.As bio-reactor, its superiority also is: (1) yeast is unicellular organism, and genetic background, physiological characteristic are comparatively clear.Its growing nutrient requires simple, and cell density is high, easy handling, and also fermentation technique is comparatively ripe, technique is simple, with low cost; (2) in protein induce expression process take methyl alcohol as sole carbon source, without not secreting toxic substance in resistance mark, the culturing process, toxicity is little than bacterium, has good security; (3) utilize methanol induction alcohol oxidase promoter (AOX1) to carry out the expression of recombinant protein, no matter be in born of the same parents or be secreted into and all might realize outside the born of the same parents efficiently expressing, and background is expressed seldom when using secretion type expression, product is easy to purifying; (4) yeast is eukaryote, can carry out a lot of typical higher eucaryote protein translation post-treatment and modifications, such as the formation of glycosylation, acyl, phosphorylation and protein cleavage, folding, disulfide linkage etc. ^[9～11]

Before the present invention, also the codon had a preference for according to yeast of nobody carried out optimizing to the gene of coding bat DSPA α 2 so that it can be more stable and more efficiently at the yeast expression in vivo, greatly increased thus the biological yield of DSPA α 2 albumen of recombinating; Also nobody adds 6 histidine-tagged encoding sequences in order to help this recombinant protein fast purifying in Downstream processing before 5 ' end of DSPA α 2 genes; The fermentation culture conditions of recombination yeast was not carried out optimization yet, thereby for extensive, industrial fermentation are produced this medicinal recombinant protein and laid the foundation, and this recombinant protein is through partially or completely being used for the treatment of human various acute thrombus class diseases behind the purifying.

Summary of the invention

The technical problem that (one) will solve

One of purpose of the present invention is to confirm that the gene of encoding D SPA α 2 is under the prerequisite that does not change aminoacid sequence, if all encoding sequence is after the codon of having a preference for according to yeast is optimized, the new sequence of gene not only can be expressed in yeast and be produced, and its expression efficiency is greatly improved than the gene of the DSPA α 2 of not codon optimization, thereby the biomass that is secreted into external restructuring DSPA α 2 albumen is also increased greatly, according to a preferential embodiment, the gene of DSPA α 2 after codon optimized is wanted and can be efficiently expressed in pichia spp and produce, and that DSPA α 2 recombinant proteins that produce can be secreted into born of the same parents is outer and have a biological activity as the native protein.

Two of purpose of the present invention is to add a label construction to DSPA α 2, greatly facilitates also and can significantly enough reduce production costs for the Downstream processing of this recombinant protein brings thus.According to a preferential embodiment, the encoding sequence of 6 histidine-tagged structures is added to 5 ' end of the gene of the DSPA α 2 after codon optimized, it not only can not affect the efficiently expressing and producing in pichia spp of DSPA α 2 genes, and DSPA α 2 recombinant proteins that produce can also be secreted into outside the born of the same parents, in addition, with 6 histidine-tagged structures in the situation of not cutting away, do not affect its biologic activity.

Three of purpose of the present invention provides the suitableeest recombination yeast incubation growth and abduction delivering condition, according to a preferential embodiment, the suitableeest incubation growth and the abduction delivering condition of relevant recombinant yeast pichia pastoris are provided especially, make thus the increment of this recombination yeast and the secretory volume of recombinant protein DSPA α 2 albumen all reach as much as possible maximization, in order to lay the foundation for the large-scale industrial production of this recombination yeast, low-cost fermentation and purifying.

(2) technical scheme

The present invention utilizes yeast expression system to produce DSPA α 2, and this recombinant protein has the biological activity the same with native protein, namely can plasminogen activation, change it into activated plasmin and solution fibrin promptly.According to a preferential embodiment, the invention provides exactly the method that greatly improves DSPA α 2 recombinant proteins expression efficiency in pichia spp, method and the condition that the method for DSPA α 2 recombinant protein fast purifyings is provided and the high density fermentation of recombination yeast is provided.

The present invention utilizes yeast expression system to produce DSPA α 2 recombinant proteins, if with it through partially or completely being used for the treatment of various acute heart brain embulus diseases behind the purifying.For example, with certain dosage intravenous drip or be filled into and suffer from acute myocardial infarction or the cerebral infarction patient body, patient's symptom is eased rapidly DSPA α 2 recombinant proteins behind the purifying.

DSPA α 2 recombinant proteins of indication of the present invention are expressed in yeast and are produced, at first be the gene of encoding D SPA α 2 by complete synthetic out, comprising gene order codon optimized mistake or that pass through optimization, and all contain 6 histidine-tagged structural coding sequences at 5 ' end of gene, then, they are respectively inserted on the Yeast expression carrier with α-factor signal peptide sequence.On this plasmid vector, also contain an inducible promoter, this promotor is positioned at the upstream of DSPA α 2 and 6 histidine-tagged encoding sequences, and it handles the expression of DSPA α 2 genes in yeast cell.This plasmid vector is after linearizing, can pass through electro fusion method or LiCl method transformed yeast cell, and then stable integration is to yeast chromosomal, and through after inducing, DSPA α 2 recombinant proteins of expression are secreted in the substratum under the guiding of signal peptide this recombination yeast in the fermentation culture process.

The invention provides the method that greatly improves DSPA α 2 recombinant proteins biological yield in yeast cell, according to a preferential embodiment, the invention provides the method that has greatly improved DSPA α 2 recombinant proteins biological yields in Pichia pastoris, and the known effect that under native state, has plasminogen activation of this recombinant protein; Perhaps more definite theory, DSPA α 2 recombinant proteins of the present invention are the same with these DSPA α 2 recombinant proteins that are derived from vampire saliva or other conventional expression system generation, can be widely used in field of medicaments as thrombolytics.

Recombinant protein of the present invention can be the whole of DSPA α 2 native proteins.Yet in some specific embodiments, expressed DSPA α 2 recombinant proteins also may be the part of this native protein.Sometimes, DSPA α 2 recombinant proteins can have the protein gene of different biological function such as coli heat-sensitive toxin B subunit with another one expresses respectively simultaneously in same yeast cell or is stitched together and form the fusion rotein co expression, and purpose is in order conveniently to purify or to improve its biological activity.The fusion of albumen can be passed through protein translation post-treatment, covalently bound mode, perhaps by the DNA recombinant technology gene just is stitched together mutually before accurate translation, and these two kinds of technology is technology that the expert of the art knows already.

In yeast cell, the height of DSPA α 2 protein gene expression efficient, the height of the extraction processing and utilization of this recombinant protein and the related products cost of producing after being directly connected to, according to a preferential scheme of implementing, for improving the expression amount of DSPA α 2 recombinant proteins in yeast cell, the gene of encoding D SPA α 2 recombinant proteins can be optimized first according to the codon that yeast is had a preference for, in order to make this foreign gene more stable in yeast cell, efficient is higher when being transcribed into mRNA and translating into albumen.In addition, for fast purifying DSPA α 2 recombinant proteins, also can carry out amalgamation and expression with other albumen, according to a preferential embodiment, 5 ' end at DSPA α 2 genes adds 6 Histidine encoding sequences, just because of this change, make these DSPA α 2 recombinant proteins be easier to obtain by affinity chromatography the purifying of height, and then make this recombinant protein can more can be widely used in field of medicaments.

To the relevant item with other of the method used in the present invention structure of some plasmid vectors very importantly, they are at the yeast cell that obtains indication of the present invention and express in the processed and applied of recombinant products and play an important role.The plasmid vector that is used for transformed yeast cell is by the dna sequence dna of gene or derivatives thereof after codon optimized, encoding D SPA α 2 and handle this gene or a inducible promoter that dna sequence dna is expressed at said yeast cell forms.In some specific embodiments, plasmid vector also comprises a selectivity or marker gene, is mainly used in screening and the detection of recombinant yeast cell.In some specific embodiments, inducible promoter of the present invention is alcohol oxidase gene promoter (AOX1).As mentioned above, according to some specific embodiment, plasmid vector of the present invention is used for transforming pichia spp, its coded foreign protein is DSPA α 2, furtherly, coded DSPA α 2 recombinant proteins of plasmid vector can plasminogen activation, solution fibrin rapidly after forming plasmin.Perhaps more definite theory, DSPA α 2 recombinant proteins of the present invention are the same with these DSPA α 2 albumen that are derived from vampire saliva or other conventional expression system generation, can be widely used in field of medicaments as thrombolytics.

The present invention also provides the conversion of yeast cell and the screening method of recombinant yeast cell, and it may further comprise the steps: the synthetic of (1) DSPA α 2 genes, wherein relate to that the codon of having a preference for according to yeast is optimized this gene codon and add 6 Histidine encoding sequences at its 5 ' end; (2) make up a plasmid expression vector, the gene (comprise that gene codon is optimised, change or mutually merge etc. with other genes) that wherein contains the gene or derivatives thereof of encoding D SPA α 2 recombinant proteins, and this gene or derivatives thereof is placed under the manipulation of an inducible promoter; (3) by electro fusion method or LiCl method, with above-mentioned plasmid expression vector DNA transformed yeast cell; (4) select the yeast cell list bacterium colony of conversion, be inoculated into respectively one by one on MM and the MD solid plate substratum, those MD substratum growth normal but the growth of MM flat board extremely slowly or the yeast list bacterium colony of not growing fully be exactly the recon that contains foreign gene.

According to a specific embodiment, the invention provides through the recombinant yeast pichia pastoris incubation growth condition of optimization and the method for quickly purifying of DSPA α 2 recombinant proteins, it has comprised following four-stage: (1) yeast culture, after 22-24 hour cultivation of process, yeast thalline weight in wet base will reach about 95-100g/L; (2) carbon source of feeding, after cultivating 4 hours, the weight in wet base of yeast thalline will arrive about 190-200g/L; (3) abduction delivering adds methyl alcohol, induces yeast cell to efficiently express DSPA α 2 recombinant proteins and it is secreted in the substratum; (4) protein purification, fermented liquid through centrifugal, get the steps such as supernatant liquor, ammonium sulfate precipitation and affinity chromatography, the purity of DSPA α 2 recombinant proteins is more than 99%.

The present invention has only mentioned pichia yeast expression system when introducing yeast expression system in detail, yet, as the yeast expression system that the expert of the art knew already, many primary yeast expression systems can utilize method provided by the invention to transform, express and produce.Therefore, all these yeast expression systems all should be included within the claim scope of the present invention.

In following example, to describe in detail, used plasmid expression vector is a conformability plasmid expression system in the yeast conversion method that the present invention describes, according to a preferential embodiment, plasmid expression vector used in the present invention is pPIC9K.

The present invention includes following integral part: the synthetic of (1) DSPA α 2 genes wherein comprises respectively gene coded sequence codon optimized mistake or that pass through optimization.With regard to codon optimized, be the codon of having a preference for according to yeast, the dna nucleotide sequence of synthetic encoding D SPA α 2 full genes, wherein in building-up process, added 6 Histidine encoding sequences at this gene 5 ' end simultaneously.Above-mentioned product is inserted directly in the T site of interstitial granules among the PCF-T, selects through the Lan Bai system, can obtain to contain the bacterial clone (recon) of external source Insert Fragment, exactness and the integrity of gene that sequential analysis obtains; (2) utilize the DNA recombinant technology to make up the yeast plasmid expression vector.They contain respectively without codon optimized and DSPA α 2 protein genes (in contrast) transformed and codon through optimizing and improved DSPA α 2 protein gene or derivatives thereofs (comprise with the mutual fusion of other gene fragments etc.); (3) screening of high expression level yeast recon.Through electro fusion method or LiCl method, yeast plasmid expression vector after the above-mentioned restructuring is imported respectively the yeast recipient cell, select the yeast cell list bacterium colony of conversion at RDB solid plate substratum, be inoculated into respectively one by one on MM and the MD solid plate substratum, those MD substratum growth normal but the growth of MM flat board extremely slowly or the yeast list bacterium colony of not growing fully be exactly the recon that contains foreign gene.In order to screen the high expression level restructuring yeast strains, the yeast recon that obtains is seeded in one by one carries out abduction delivering in the abduction delivering substratum, under identical culture condition, its abduction delivering product is carried out fibrinolytic activity identify, therefrom can pick out the highest restructuring yeast strains of expression efficiency; (4) provide the recombination yeast the most suitable growth to cultivate and the abduction delivering condition.The high density fermentation of recombination yeast is divided into yeast culture, carbon source is fed and the abduction delivering three phases, all gives the suitableeest incubation time and envrionment conditions in each stage.After the steps such as that fermented liquid passes through respectively is centrifugal, ammonium sulfate precipitation and affinity chromatography, can obtain purity and reach DSPA α 2 recombinant proteins more than 99%.

Selection DSPA α 2 genes are expressed in yeast and production is that therefore, this albumen might can be widely used in field of medicaments as thrombolytics because this albumen can generate plasmin also and then solution fibrin by plasminogen activation.Although DSPA α 2 has tempting application prospect, its mass-producing and suitability for industrialized production problem are never solved well, and select pichia yeast expression system to be because it is a present the most frequently used external source expression of recombinant proteins system.

(3) beneficial effect

Adopt method of the present invention, Effective Raise the expression amount of DSPA α 2 recombinant proteins, and simplified the purifying process of recombinant protein, be suitable for commercial scale production DSPA α 2 recombinant proteins.

Description of drawings

In order to describe some preferential embodiment of the present invention in detail, and for referencial use with corresponding drawing:

Contrast before and after Fig. 1 DSPA α 2 gene codon optimizations and the transformation, N represents the gene order before codon optimized and the transformation; M represents codon optimized and improved gene order;

Fig. 2 codon is through optimizing the structure of DSPA α 2 gene clonings and Yeast expression carrier thereof;

Fig. 3 codon is without the structure of optimizing DSPA α 2 gene clonings and Yeast expression carrier thereof;

Fig. 4 utilizes fibrin plate method detection resources from the DSPA of different recombinant pichia yeast strains α 2 expression of recombinant proteins situation: CK+:t-PA (identify institute available from Chinese medicine, working concentration is 10IU/mL) (positive control); CK-1: the yeast recon abduction delivering product (one of negative control) through the pPIC9K Plasmid Transformation is processed; CK-2: process (one of negative control) through the BMMY nutrient solution; 1～7: process without the abduction delivering product of the restructuring yeast strains of DSPA α 2 gene transformation of optimizing through codon; 8～17: the abduction delivering product of the restructuring yeast strains of DSPA α 2 gene transformation is processed after codon optimized and change;

Fig. 5 SDS-PAGE detects PP-DSPA α 2M yeast recon (contain codon optimized after DSPA α 2 genes) at the expression of different abduction delivering incubation time DSPA α 2 recombinant proteins: 1～6: be respectively the situation of cultivating 24,48,72,96,120 and 144 hours.7: be the yeast recon abduction delivering situation (negative control was cultivated 144 hours) of pPIC9K Plasmid Transformation.8: be standard molecular weight marker (Amersham Biosicences product);

Fig. 6 SDS-PAGE detects PP-DSPA α 2M high density fermentation abduction delivering product and reaches by DSPA α 2 recombinant proteins behind centrifugal collection fermented supernatant fluid, ammonium sulfate precipitation and the affinity chromatography: 1 is standard protein molecular weight marker (Amersham Biosicences product); 2 be and PP-DSPA α 2M high density fermentation after abduction delivering product (abduction delivering 96 hours); 3 is DSPA α 2 recombinant proteins behind the purifying.

Embodiment

The below describe some preferential embodiments, but application of the present invention is not limited only to this take Pichia anomala expression DSPA α 2 as example.Just some the special preferential embodiments that further describe of the present invention are according to the patent application requirement and in order to explain and illustrate the content of this patent.Obviously, do not deviating within the spirit and scope of the present invention, can on this basis, do further improvement and variation.

Embodiment 1:

1. the synthetic of bat DSPA α 2 genes after codon optimized

Although bat and yeast all belong to eukaryote, they are still existing certain difference aspect the gene codon preference, and this species diversity might have influence on DSPA α 2 genes and stability and the expression efficiency of transcription product in yeast.For improving the biological yield of DSPA α 2, gene order according to the vampire saliva DSPA α 2 that had already announced (is seen GenBank, accession number is M63988), under the prerequisite that does not change this Argine Monohydrochloride sequence (see sequence table 1), the codon of having a preference for according to yeast ^[12]Manually design and synthesized the dna encoding sequence of new vampire DSPA α 2 maturation proteins, meanwhile, for the ease of the structure of later yeast plasmid expression vector and the purifying of DSPA α 2 recombinant proteins, in the process of these DSPA α 2 encoding sequences of synthetic, restriction enzyme site SnaB I (TACGTA) and 6 histidine-tagged structured coding sequence C ACCACCACCACCACCAC (seeing 2 in the sequence table) before this first codon of gene 5 ' end GCT, have been added; Behind 3 ' end terminator codon TAA of this gene, increased restriction enzyme site Not I (GCGGCCGC) (said gene splicing and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing).DSPA α 2 mature protein genes after codon optimized with transform before compare, changed 251 nucleotide bases wherein, altogether relate to 216 codons, and G+C content becomes 46.9% by original 53.8%, Fig. 1 is seen in contrast before and after the genetic modification.

2. DSPA α 2 gene clonings after codon optimized

Bat DSPA α 2 gene DNA fragments behind above-mentioned synthetic codon optimized are directly inserted and are connected in the T site in the PCF-T plasmid (available from TransGenic company, product description is seen in operation), the method that provides according to the said firm, by the white screening system of indigo plant, obtain containing the bacterial clone pCFDSPA α 2M (seeing Fig. 2) of DSPA α 2 genes after codon optimized, then, by nucleotide sequence analysis, the gene of determining encoding D SPA α 2M is correct and complete (seeing 2 in the sequence table) (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

3. the structure of Yeast expression carrier p9KDSPA α 2M

During DSPA α 2 gene after synthetic is codon optimized, because in its 5 ' end and 3 ' terminal sequence, introduced respectively SnaB I and Not I restriction enzyme site, so pCFDSPA α 2M plasmid DNA is behind SnaB I and Not I double digestion, codon optimized and improved DSPA α 2M gene can be cut down, pass through agarose gel electrophoresis, and then this dna fragmentation of separable acquisition, again its orientation is inserted into subsequently in the corresponding site of plasmid pPIC9K (SnaB I and Not I double digestion), just is built into yeast inducible expression plasmid vector p9KDSPA α 2M (seeing Fig. 2).

Plasmid pPIC9K is available from American I nvitrogen company, and it is an efficient yeast inducible expression carrier, under the inducing of methyl alcohol, but efficiently expressing exogenous gene.Because before this expression vector multienzyme is cut the site, contain α-factor signal peptide-coding sequence, with the foreign protein genes amalgamation and expression after, bootable external source recombinant protein is to the yeast exocytosis.In the process outside being secreted into born of the same parents, this signal peptide sequence can fully be cut down by yeast Kex2 or Ste13 gene expression product, thereby can not have influence on the biological activity of DSPA α 2 recombinant proteins fully.

Embodiment 2:

1. the synthetic of bat DSPA α 2 genes do not optimized of codon

According to the gene coded sequence (seeing GenBank, M63988) of the vampire saliva DSPA α 2 that had already announced, synthetic the dna encoding sequence of DSPA α 2 maturation proteins.For the ease of the structure of later yeast plasmid expression vector and the purifying of DSPA α 2 recombinant proteins, in synthetic DSPA α 2 maturation protein dna encoding sequences, before holding first codon GCA, added 5 ' of this gene restriction enzyme site SnaB I (TACGTA) and 6 histidine-tagged structured coding sequence C ACCACCACCACCACCAC (seeing 3 in the sequence table); Behind 3 ' end terminator codon TAA of this gene, increased restriction enzyme site Not I (GCGGCCGC) (said gene splicing and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing).

2. bat DSPA α 2 gene clonings do not optimized of codon

Bat DSPA α 2 gene DNA fragments that the codon of above-mentioned synthetic is not optimized directly insert and are connected in the T site in the PCF-T plasmid, by the white screening system of indigo plant, obtain containing the bacterial clone pCFDSPA α 2 (seeing Fig. 3) that codon is not optimized DSPA α 2 genes, then, by nucleotide sequence analysis, the gene of determining encoding D SPA α 2 is correct and complete (seeing 3 in the sequence table) (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

3. the structure of Yeast expression carrier p9KDSPA α 2

When DSPA α 2 gene that the synthetic codon is not optimized, because in its 5 ' end and 3 ' terminal sequence, introduced respectively SnaB I and Not I restriction enzyme site, so pCFDSPA α 2 plasmid DNA are behind SnaBI and Not I double digestion, DSPA α 2 genes of codon can not being optimized cut down, pass through agarose gel electrophoresis, and then this dna fragmentation of separable acquisition, be inserted into subsequently (SnaBI and Not I double digestion) in the plasmid pPIC9K, just be built into yeast inducible expression plasmid vector p9KDSPA α 2 (seeing Fig. 3).

Embodiment 3:

1.p9KDSPA the preparation of α 2 and p9KDSPA α 2M plasmid DNA

At first use alkaline lysis ^[13]From Bacillus coli cells DH5 α (available from U.S. GIBCO company), extract respectively p9KDSPA α 2 and p9KDSPA α 2M plasmid DNA, then with 1-2 doubly excessive restriction enzyme Sal I respectively enzyme cut above-mentioned plasmid DNA, make it Total Linearization, can utilize agarose gel electrophoresis to detect enzyme and cut whether fully.Then with phenol and chloroform respectively the above-mentioned enzyme of extracting cut product, the ethanol precipitation after lyophilize, is dissolved in precipitation in the aseptic deionized water again, saves backup in-20 ℃ of refrigerators.

2. the conversion of yeast cell

The single bacterium colony of picking Pichi strain GS115 (available from American I nvitrogen company), be inoculated in 5mLYPD substratum (1% yeast extract, 2% Tryptones, 2% glucose) in, 30 ℃ of shaking culture are spent the night, therefrom take out the 0.5mL nutrient solution, be inoculated in the 500mL YPD substratum, 30 ℃ of shaking culture are to OD _600nmAbsorbance value is about 1.4-1.5,4 ℃ of lower centrifugal 2 minutes collection yeast cell of 4000rpm.In the sterilized water of 500mL ice bath, then 4 ℃ of 4000rpm collected yeast cell in lower centrifugal 2 minutes again with the yeast cell Eddy diffusion of precipitation.Repeat once above-mentioned washing process.Yeast cell with the 1mol/L sorbyl alcohol Eddy diffusion of 25mL ice bath precipitation, as above centrifugal collection yeast cell, again with the yeast cell of the 1mol/L sorbyl alcohol Eddy diffusion of 0.5mL ice bath precipitation, therefrom take out 80uL yeast competent cell, fully mix with the linearization plasmid carrier DNA (4-5ug) of above-mentioned preparation respectively, then transfer in the aseptic electric shock cup of 0.2cm behind the ice bath.Utilize electric shock instrument Micropulser ^TM(BioRad company product) imports linearizing plasmid DNA in the yeast competent cell, and employed shock parameters is 0.8kv, 11.5uF.After electric shock is finished, the 1mol/L Sorbitol Solution USP that adds immediately the 0.8mL ice bath in the electric shock cup, fully behind the mixing, this bacterium liquid is applied to RDB[1.34%Yeast Nitrogen Base With AmmoniumSulfate without amino acids (YNB) (Sigma company product) with the volume of every plate 200uL, the 1mol/L sorbyl alcohol, 1% glucose, 0.00004%Biotin, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, 1.5% agar] on the solid plate substratum, the dull and stereotyped juxtaposition of being inverted is cultured in 30 ℃ of incubators and transforms recon and occur.

With the yeast list bacterium colony (recon) that newly grows on the aseptic toothpick picking RDB solid plate substratum, difference dibbling MM (1.34% YNB, 0.00004%Biotin, 0.5% methyl alcohol, 1.5% agar) dull and stereotyped and MD (1.34%YNB, 0.00004%Biotin, 2% glucose, 1.5% agar) the solid plate substratum; Cultivated 2 days for 30 ℃, consistent with the MD growth at MM, its phenotype is Mut ⁺Normal in MD growth, poor growth or long on MM, phenotype is Mut ^s

3. the screening of high expression level yeast strain

Picking MD flat board growth normal but on the MM flat board poky transformant, be inoculated in 5mL BMGY[1% yeast extract, 2% Tryptones, 100mmol/L potassium phosphate buffer (pH 7.0), 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V)] in the liquid nutrient medium (this substratum is take glycerine as carbon source), 30 ℃ of shaking tables were cultivated 2 days, and the centrifugal 2min of 4000rpm removes supernatant; With 1mL BMMY[1% yeast extract, 2% Tryptones, 100mmol/L potassium phosphate buffer (pH 7.0), 1.34%YNB, 0.00004%Biotin, 0.5% methyl alcohol] liquid nutrient medium Eddy diffusion thalline (this substratum with methyl alcohol as foreign protein genes induced expression thing), 30 ℃ of inducing culture 4 days; 4 ℃ of lower centrifugal 5min of 10000rpm, collection contains the supernatant liquor that might contain DSPA α 2 recombinant proteins.This supernatant liquor can be directly used in the mensuration of fibrinolytic.

The mensuration of DSPA α 2 recombinant protein fibrinolytics adopts the fiber flat band method, and the making of fibrin plate is carried out with reference to the people's such as Fish method substantially ^[14]Take by weighing the 0.75g agarose in 50mL PBS (8g NaCl, 0.2gKH ₂PO ₄, 2.9g Na ₂HPO ₄12H ₂O, 0.2gKCl, adding distil water is to 1L, pH7.4) in, heat fused treats that it is cold really to 55 ℃～60 ℃, add 21mL and contain the PBS of 50 μ g Fibrinogens (Sigma company product) and zymoplasm (the Sigma company product of 150 μ L 200U/mL, contain Profibrinolysin) solution, pour into rapidly behind the mixing in three sterilization plates, punch at the fiber flat board with punch tool after cooling.Add 100 μ L yeast fermentation supernatant liquors in every hole, simultaneously with the positive contrast of standard substance t-PA (available from Chinese drug inspection office).Flat board places 37 ℃ of incubators, observations behind the 12h.

With the pichia spp methanol induction culture that turns pPIC9K empty plasmid expression vector as negative control, found that, transform its methanol induction culture supernatant of some restructuring yeast strains that obtains through p9KDSPA α 2M plasmid DNA and really can form transparent circle at the fiber flat board, be that expressed DSPA α 2 recombinant proteins of yeast can plasminogen activation, it can be changed into plasmin and and then make fibrin degradation.Can reach a conclusion thus, in yeast expressed DSPA α 2 recombinant proteins be have bioactive.

But it is worthy of note especially, if contain DSPA α 2 genes (transforming the restructuring yeast strains that obtains through p9KDSPA α 2 plasmid DNA) that codon is not optimized in the restructuring yeast strains, its methanol induction culture supernatant all can not form transparent circle (seeing Fig. 4) on the fiber flat board, this may be since DSPA α 2 expression of recombinant proteins amounts cross low due to.The above results shows that these two kinds of recombination yeasts are that there were significant differences in the quantity of secretion DSPA α 2 recombinant proteins, this means gene codon through optimization and transform after really can greatly improve DSPA α 2 genes in yeast cell expression efficiency and be secreted into biological yield outside the born of the same parents (multiple of raising is excessively low because of the yeast recon exogenous protein expression amount that contains modifying gene not, so can't measure the concrete multiple of raising).Single its methanol induction culture of yeast strain by the pPIC9K Plasmid Transformation can not form transparent circle (negative control).In addition, some restructuring yeast strains that contains DSPA α 2 genes after codon optimized can not form transparent circle, and this may fail to express relevant with DSPA α 2 genes that import.

The yeast recon that can form transparent circle is picked out, and with its called after PP-DSPA α 2M (containing codon optimized rear DSPA α 2 genes).

Embodiment 4:

1. the preparation of seed liquor

Choose PP-DSPA α 2M yeast list bacterium colony from the RDB solid plate, be inoculated in the 10mL YPD liquid nutrient medium, 30 ℃ of shaking culture are spent the night, transfer in 100mL YPD liquid nutrient medium with 10% inoculum size, 30 ℃ of shaking culture 24 hours, transfer in 1L BMGY substratum with 10% inoculum size again, 30 ℃ of shaking culture 24 hours, then with it as seed liquor.

2. the high density fermentation of recombination yeast in the 20L fermentor tank

This fermenting process can be divided into following three phases: 1) the yeast culture stage: held 10L basis fermention medium [10 * BasalSalts (2.67% phosphoric acid in 20L fermentor tank (Shanghai Baoxing biological plant Engineering Co., Ltd product), 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide)+4% glycerine], adding first 28% ammoniacal liquor before inoculation makes the pH value of this substratum maintain (ammoniacal liquor also can be used as the nitrogenous source of yeast bulk-growth simultaneously) about 7.5, again in following ratio, in every liter of basic fermention medium, add 4.37mL trace salt solution PTM1 (0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganous sulfate, 0.02% Sodium orthomolybdate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid).The seed liquor that ratio inoculation in 10% prepares before this, 30 ℃ of aeration-agitations were cultivated about 24 hours.Carry out in the process in this stage, along with the growth of thalline, the dissolved oxygen amount in the substratum will reduce gradually by 100%, and after the carbon source in the substratum runs out of, dissolved oxygen amount will be increased to more than 80% once again, and the weight in wet base of thalline will reach 90-95g/L this moment.2) feed the carbon source stage: by peristaltic pump flow feeding liquid, feed supplement liquid is 50% glycerine (wherein containing 12mL PTM1/L), and the stream dosage is 18.15mL/hr/L.30 ℃ of aeration-agitations were cultivated about 4 hours, adjusted air flow in this stage and made dissolved oxygen amount all the time greater than 20%, and the thalline weight in wet base will reach about 180-190g/L this moment.3) the abduction delivering stage: stream adds methyl alcohol (containing 12mL/LPTM1), make the concentration of methyl alcohol maintain all the time about 0.3%, dissolved oxygen amount is all the time greater than 20%, 30 ℃ of aeration-agitations were cultivated 144 hours, descended centrifugal 10 minutes through 4 ℃ of 5000rpm every 24 hours peek milliliter fermented liquids, get the 30uL fermented supernatant fluid and carry out the SDS-PAGE electrophoresis detection, discovery has the observable protein band of naked eyes, molecular weight is about about 47KD, matches with the molecular weight of DSPA α 2 recombinant proteins in inferring.In addition, find from electrophorogram that after 96 hours, the expression amount of DSPA α 2 recombinant proteins reaches climax (seeing Fig. 5) at inducing culture.

3. the purifying of recombinant protein

After treating that a fermentation period all finishes, leave and take the 500mL fermented liquid directly carries out next round as seed liquor (inoculum size is 5%) fermenting process.Similar operation adds up to carry out 3 to be taken turns, and takes turns in the fermenting process every, all the increment of thalline and the expression amount of DSPA α 2 recombinant proteins is measured.In addition, take turns after fermenting process finishes fully every, also get a little bacterium liquid and be coated on the YPD solid plate substratum, and any 10 yeast lists of picking bacterium colony therefrom, and fully according to the people's such as Cai Chuanqi method ^[15]Its genomic dna of rapid extraction also carries out PCR to it and detects, found that, the biomass of thalline, the expression amount of the speed of growth and DSPA α 2 recombinant proteins is taken turns kept stable in the fermenting process at each, in addition, the detected result of PCR confirms that also PP-DSPA α 2M has good genetic stability (seeing Table 1).

The genetic stability of table 1.PP-DSPA α 2M bacterial strain and the mensuration of exogenous protein expression stability

After a fermentation period finished, except staying the 500mL fermented liquid as the seed liquor, remaining fermented liquid descended centrifugal 10 minutes through 4 ℃ of 5000rpm, left and took supernatant.Add respectively therein solid ammonium sulfate to saturation ratio and reach 80%, at room temperature stirred 1 hour, then 5000rpm is at room temperature centrifugal 20 minutes, collecting precipitation, to precipitate Eddy diffusion in 10ml 100mM Tris-HCl (pH8.0) damping fluid, and be transferred in the dialysis tubing, then 100mM Tris-HCl (pH8.0) damping fluid for 1L is dialysed under 4 ℃, to remove remaining ammonium sulfate, in 24 hours dialysis time, change dialyzate outside three times.After dialysis finished, sucking-off was by dialysate, then according to ProBond in dialysis tubing ^TMThe method that specification sheets provides in the Purification System test kit (Invitrogen company) is progressively carried out purifying.The albumen elutriant of collecting is carried out SDS-PAGE analysis and the detection of gel imaging instrument, and result's demonstration has obtained the single band consistent with the target protein molecular weight, and its purity of protein can reach more than 99% (sees Fig. 6).Again utilize the fibrin plate method to measure its activity, and compare before the purifying, specific activity has improved 2.3 times.

Utilize Lowry method (protein determination kit is available from Sigma company, and article No. is P5656) to measure the content (concrete grammar is seen the test kit specification sheets) of DSPA α 2 recombinant proteins behind the purifying, be about 25mg/L (mean values of three high density fermentations).

Reference

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Sequence table

Claims (5)

1. method that efficiently expresses and produce the vampire plasmin activator alpha 2 by recombination yeast, it comprises the synthetic of DSPA α 2 genes, the structure of expression vector, the screening of high expression level yeast recon, the fermentation of recombination yeast and the expression of recombinant protein, purifying, it is characterized in that, in the synthetic step of DSPA α 2 genes, the gene of coding recombinant protein is codon optimized DSPA α 2 genes of having a preference for according to yeast, and it is synthetic to add 6 histidine-tagged encoding sequences before 5 ' end of DSPA α 2 genes, through optimization and add 6 DSPA α 2 gene DNA sequences after histidine-tagged shown in the nucleotide sequence among the SEQ ID NO:2; The condition of while optimum combination saccharomycetes to make fermentation and recombinant protein abduction delivering, be specially: a: yeast culture: with basic fermention medium fermentation, before inoculation, add first 28% ammoniacal liquor the pH value of this substratum is maintained about 7.5, then in every liter of basic fermention medium, add 4.37mL trace salt solution PTM1; By volume 10% ratio inoculation seed liquor, 30 ℃ of aeration-agitations were cultivated about 24 hours; B: the carbon source of feeding: by peristaltic pump flow feeding liquid, feed supplement liquid is 50% glycerine that contains 12mL PTM1/L, and the stream dosage is 18.15mL/hr/L, and 30 ℃ of aeration-agitations were cultivated about 4 hours, adjusts air flow and makes dissolved oxygen amount all the time greater than 20%; C: abduction delivering: stream adds the methyl alcohol that contains 12mL/L PTM1, and methanol concentration is maintained about 0.3% all the time, and dissolved oxygen amount was cultivated 144 hours greater than 20%, 30 ℃ of aeration-agitation all the time; Wherein, basic fermention medium: 10 * Basal Salts+4% glycerine; PTM1:0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganous sulfate, 0.02% Sodium orthomolybdate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid; Wherein, 10 * Basal Salts is 2.67v/v% phosphoric acid, 0.093w/v% calcium sulfate, 1.82w/v% vitriolate of tartar, 1.49w/v% sal epsom and 0.413w/v% potassium hydroxide.

2. the expression plasmid carrier of the vampire plasmin activator alpha 2 of recombinating, it is characterized in that it be the dna sequence dna shown in SEQ ID NO:2 is inserted into pPIC9K or other can abduction delivering and secrete the plasmid vector and make up of recombinant protein outside born of the same parents and obtain.

3. a pichia pastoris engineered strain of expressing restructuring vampire plasmin activator alpha 2 is characterized in that it comprises expression vector claimed in claim 2.

4. pichia pastoris engineered strain according to claim 3 is characterized in that its host cell is Pichi strain GS115.

5. the application of method according to claim 1 in the medicine of the various acute thrombus diseases of preparation treatment.

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