CN1842600B - A process for proteolytic cleavage and purification of recombinant proteins - Google Patents

Abstract

The present invention relates to improved methods for protein purification of high-value heterologous proteins by providing fusion proteins suitable for affinity purification and improved and economical methods of proteolytic cleavage of fusion proteins. The methods are useful for large-scale production of purified recombinant proteins from plants, plant-derived tissue or plant cells. The invention aims to reduce the cost and improve the quality of downstream processing of heterologous proteins produced in plants and other biological production systems.

Description

The method of proteolytic cleavage and purification of recombinant proteins

Invention field

The invention belongs to biological chemistry and albumen technical field and relate to especially by transgenic plant material and separating and the improving one's methods of purifying heterologous protein.In addition, the invention provides the improved and economic mode of the proteolytic cleavage of fusion rotein.

Background technology

Albumen based on bio-pharmaceuticals demonstrates important prospect (referring to summary " RecombinantProtein Drugs " Ed.P.Buckel 2001) in the more specificity that anti-serious disease is provided and tissue specificity or cell-specific pharmacological agent.

A lot of instance of the prior art has shown and utilizes mikrobe such as bacterium and zooblast to produce this bio-pharmaceutical; Wherein Regular Insulin is important instance.

Many instances have shown and utilize transgenic plant or plant cell cultures to be used to express and the heterologous polypeptide or the bio-pharmaceutical of production high value in document.This working method based on plant can be called the molecule farming.

Valuable proteic production can be through utilizing plant as producing biological more economical generation.Compare such as protokaryon production system, animal cell culture etc. with most of production systems, use plant can reduce significantly as the cultivation cost that host living beings is used for protein production based on bio-reactor.Yet for above-mentioned all production systems, the purifying of heterologous protein remains necessary and expensive task.Therefore, for for the production system of plant, consumed most production cost in the processing of the production middle and lower reaches of high value heterologous protein.

Protein purification with separate be through utilize gene engineering in various host living beings, accumulate and the proteic downstream processing that produces in critical process.By the host living beings purifying protein possibly be ten minutes effort, complicated and expensive.Various stratographic analysis strategies are used to be used for separating and the purifying target protein by producing host living beings commercially.Physical chemistry difference between that the stratographic analysis strategy can be dependent on pollution or intrinsic protein and the purpose heterologous protein, different such as aspect size, solubleness, electric charge, hydrophobicity and the affinity.

The combination of stratographic analysis strategy comprises multiple step, needs the chromatography substrate and the necessary hardware of some costlinesses to comprise post, controls unit or the like, and in each step, follows the loss of product productive rate, and therefore causes economic loss.Except that comprising the stratographic analysis step, downstream processing generally includes multi-filtering and centrifugation step.As a result, purifying and downstream cost of processing possibly become the proteic restriction of purifying based on the biotechnology product.As being used for a large amount of products based on proteic lesser value, such as industrial albumen, the downstream cost of processing possibly limit using and selling of they, causes producing coarse and uncomfortable product.For most of biotechnology products, the purifying cost is undoubtedly the major portion of production cost.

Influence separates the cost of the specific chromatography substrate of purpose by separating contaminants because their production relates to complicated combination chemical action is expensive.During can causing in the purification process process by matrix, the chemical complexity of these matrix carries out the unnecessary leaching of part and other material; Wherein necessary precaution is measured, and monitoring or going out thing by removal drop in the target protein has increased expensive downstream cost of processing.

The purification process that affinity chromatography is the most powerful is because it based on the specificity affinity between reagent and the ligands specific, simulates the interaction of native protein-part usually.Can utilize some dissimilar affinity sorbent materials, some is a high degree of specificity to specific protein, other be incorporated into the albumen type except that specific protein.

In most cases, affinity chromatography depends on the existence that is connected in the specific marker of purpose heterologous protein through recombinant DNA technology.When these marks are used for the purifying of purpose allos product, need be fallen by cracking.The high degree of specificity proteolytic enzyme of the amino acid backbone of this cracking through being utilized in specificity cleavage site scinderin is realized, also promptly imports in the stage between mark and the target protein the clone.The split product of the needs of protein isolates enzyme and generation has each other increased complicacy, the number of related purification step and the tooling cost of utilizing this locus specificity proteolytic cleavage effectively.Usually limit industrial application with utilizing this specific protease cost related, therefore limited the utilization of these effective purification techniques based on the affinity chromatography of mark.More economical and effectively utilize the mode of specific protease to make bioprocess technology industry become the production cost that maybe and possibly reduce purification of recombinant proteins usually.

Under many circumstances, the affinity chromatography meaning is used for the proteic absorption agent that specificity selects to be incorporated into part with fixed ligands.Utilize the combining to comprise of part and affinity sorbent material the cyanogen bromide that combines chemical action such as sorbent material-, tolylsulfonyl-, or vinyl sulfone(Remzaol (vinylsulfone)-activation.The part that is incorporated into base for post matter possibly be or possibly not be dietary protein origin.The former instance does, but is not limited to, and has the immobilization albumin A or the Protein G of gamma globulin affinity, so the antagonist purifying is useful, and gp is had the lectin of affinity.As the latter's instance, the immobilization gsh that is incorporated into matrix combines to comprise the fusion rotein of the sweet skin S-of paddy Guang transferring enzyme structural domain.Fixed metal affinity chromatography (IMAC) is based on metal-chelating ligand and matrix immobilization and depend on the metals ion that is fixed on the post and the formation of the faint dative bond between the basic group on the albumen (mainly being histidine residues).Commercially available cloning vector provides the possibility of in frame, cloning the cDNA with a series of histidine residues-His-mark, the fusion rotein that the enough IMAC purifying of its ability generate.Although be widely used for proteic small-scale purifying, IMAC is nonspecific optionally method, because the natural histidine residues in contaminating protein possibly cause combining (Scopes 1993) in IMAC.Mark that some are dissimilar or binding domains are useful in the commercially available expression vector that produces fusion rotein, and wherein mark/binding domains is incorporated into fusion rotein the part that is attached on the base for post matter.

Available these matrix-Fas lignand systems obtain protein bound high degree of specificity.In above-mentioned situation, comprise that complicated combination chemical action is fixed to part on the inert base.Therefore, the cost of affinity matrix becomes the restriction of the industrial scale applications of these effective technologies usually.

In addition, because as far as the chromatography method of other types of great majority, the stability that part is incorporated into matrix becomes a difficult problem and leaching is the problem of especially considering.In the biological activity protein purge process of many sensitivities, the heavy metal leaching among the IMAC can cause unacceptable and serious pollution, and possibly make by purified proteins inactivation (Scopes 1993).

Usually, albumen is that the so strong destruction part-protein bound elution requirement that makes need make by the fierce condition of the valuable protein part sex change of purifying with the affinity that combines of its part.These non--limitative examples is to discharge antibody by albumin A-affinity matrix wash-out antibody at low pH place sex change cause post.Because the risk of purifying protein loss of activity does not hope in the protein purification process, to comprise denaturing step, increase the folding again additional step of albumen, and for the required activation analysis subsequently of folded protein product, and increase cost.

In order to utilize chromatography to carry out large scale purification by plant based on affinity; Hope very much the purge process that development is simpler and more more economical than present commercial method, its should have less combination chemical action and with the specification of quality of pharmaceutical industry standard contradiction not.

Polysaccharide and polysaccharide be conjugated protein be used in combination the design that is used for the affinity chromatography step (referring to, for example, Boraston etc., 2001).

People's such as Shani USP 6; 331; 416 have described expression has the method that is incorporated into the recombinant protein of cellulosic polysaccharide binding domains in the host plant cell wall; And the protein purification process of utilizing this albumen not have the cellulosic affinity of host plant of insufficient definition to carry out, generation can with solubility contaminating protein isolated cells wall-albumen composition.Bonded intensity can make possibly need fierce protein denaturation condition by discharging albumen in the Mierocrystalline cellulose host plant material, and said condition is to being had negative interaction by the proteic activity of purified recombinant.The complicacy that relates to and above-mentioned antibody-albumin A wash-out are similar.

Glucide binding domains cbm9-2 from Thermotoga maritima (Thermotogamaritime) zytase 10A (Winterhalter etc., 1995:Mol.Microbiol, 15 (3), 431-444).The CBM9-2 genomic dna sequence can belong to the IX family of CBM-s and have many attracting characteristics that are used for high-resolution affinity purification available from GenBank accession number Z46264 and its; Comprise and utilize the non-denaturing elution requirement of 1M glucose as elutriant; And be used for amorphous and high degree of specificity affinity (Boraston etc. crystalline cellulose; 2001:Biochemistry 40,6240-6247).

Shown with the proteic important prospect of the mode scale operation of economy based on plant production albumen, as shown (summary is referring to Hammond 1999) by the instance in document.

With with the relevant cultivation cost of plant molecular farming than compare reduction significantly with traditional method based on bio-reactor.

Extensively generally acknowledge need a kind of effective, simple and economically by the downstream working method of transgenic plant material purifying heterologous protein.In addition, this downstream processing need comprise that the gentleness that is used for target protein, non-denaturing condition (the specificity affinity purification method that especially, has gentle elution requirement) are with the biological activity of guaranteeing target protein and improve productive rate.Do not receive the protein purification process of above-mentioned restriction can reduce significantly with by the relevant production cost of plant production bio-pharmaceutical, and can be used in that its downstream processing of purifying is limited and be complicated and expensive and valuable heterologous protein.

Summary of the invention and purpose

Main purpose of the present invention provides plant, available from the improvement method for purifying proteins that produces highly valuable heterologous protein in the tissue of plant or the vegetable cell, and by the affinity labelling that merges such as the proteolyze method of for example separating the efficient and cost-effective of purpose heterologous protein with CBM that albumen merges.

The object of the invention is to reduce the quality of cost and the downstream processing that improves the heterologous protein that in plant, produces.

The key character of purge process is by cell wall fragment and derives from the inherent target protein that separates of other solid substance that does not have the abundant plant that defines as the CBM-fusion rotein, because CBM-fusion rotein of the present invention debond is in these components.This can carry out separately or carry out simultaneously with the affinity chromatography step before affinity chromatography.

Aspect first, the invention provides the method that is used for comprising the solubility heterologous fusion proteins of Mierocrystalline cellulose coupling unit (CBM) by the transgenic plant of expressing said fusion rotein or transgenic plant cells production and purifying, this method comprises:

Broken transgenic plant material;

Extracting liq is made an addition to vegetable material, produce solubility and insoluble plant mixtures of material thus, so that obtain protein extract in vegetable material extraction soluble fusion protein to the liquid phase by said fragmentation;

Said protein extract by comprising said purpose fusion rotein separates the insoluble plant material that comprises cell-wall material and solid substance;

Said protein extract is contacted with the polysaccharide matrix that is incorporated into said fusion rotein;

Have the matrix of bonded fusion rotein with one or more suitable aqueous solution such as one or more damping fluid flushing, that is, gradient capable of using or a series of different washings wash in a step; With

The condition that is discharged by matrix through the said fusion rotein of adjustment influence is by said polysaccharide matrix wash-out fusion rotein.

Usually; Transgenic plant or vegetable cell are selected from dicotyledons and monocotyledons; And in preferred embodiments, said vegetable cell or transgenic plant are from tobacco, Semen Brassicae campestris, soybean, clover, lettuce, barley, corn, wheat, oat and paddy rice.

Separating step comprises and is selected from expanded bed adsorption (EBA) in certain embodiments, the filling type chromatography, and deposition is filtered, the method for centrifugal or its arbitrary combination.

The affinity integrating step that fusion rotein is incorporated into polysaccharide matrix preferably includes chromatographic step.

Yet; In specific useful embodiment; Said do not have fully to define the solid substance of plant origin by target protein CBM fusion rotein isolated cell-wall fragment and other; And CBM fusion rotein affinity and polysaccharide matrix affine combine to utilize and have suitablely, and the expanded bed adsorption chromatography (EBA) of cheap polysaccharide matrix is carried out in single effective purification step.These characteristic innovations have also improved the downstream Economics of Processing.

In the useful embodiment of the present invention, polysaccharide matrix comprises Mierocrystalline cellulose, and preferably includes the fiber rope of pharmaceutically compatible.The pharmaceutical grade Mierocrystalline cellulose that this CBM-fusion rotein bonded clearly limits can allow the various high-end applications of purifying heterologous protein.The cellulosic material that is used as the useful pharmaceutically compatible of polysaccharide matrix comprises Avicel ^TM(FMC Corporation, PA, the U.S.).

The polysaccharide matrix that is used for affinity chromatography of the present invention does not need the complicated combination chemical action or the immobilization of potential leaching part.Polysaccharide matrix provides the support and the rigidity of structure when constituting affinity adsorbent itself.Therefore, more economically can be used in heterologous protein available from plant with protein purification safety.

Another advantage of the present invention is the different polysaccharide matrixes that said processing can be used for different mass according to the different end-uses of the heterologous protein of purifying, for example is used in for example agricultural, chemical industry or the pharmaceutical industry.The affinity adsorbent of being processed by the polysaccharide of pharmaceutical grade is that loose material and the price in the pharmaceutical industry is markedly inferior to any commercially available affinity chromatography medium.Therefore, processing of the present invention capable of using prepares very high-quality affinity matrix economically, can process the high value albumen from vegetable material in downstream, more economical ground.

Further advantage of the present invention is in case be incorporated into the polysaccharide chromatography substrate available from the CBM-fusion rotein of plant; And after flushing matrix is removed the endogenous plant albumen of all pollutions; Non-denaturing capable of using; Mild conditions is generally neutral or acidic conditions and preferably adds soluble-carbohydrate (sugar) by post wash-out fusion rotein, and said condition has kept proteic activity of the fusion partner of any CBM of being connected in and structure.Sugar will discharge the fusion rotein that all combine CBM basically with the binding site of Mierocrystalline cellulose competition CBM and suitable concentration.

The preferred CBM-s that is used for the inventive method and is blended in the purpose heterologous protein has the purpose binding characteristic to allow to be incorporated into strongly fully the combination CBM fusion rotein that suitable polysaccharide matrix obtains high yield, and the CBM-s that discharges through aforesaid mild conditions.CBM9-2 has come to light and has had these purpose characteristics.Utilize other CBM-s also within the scope of the invention with this specific character.This CBM-s can have the sequence of the CBM of purpose characteristic through the coding of retrieving in the obtainable gene database, for example is considered to and be that the similar motif of motif among the important CBM9-2 is found for binding characteristic.Simultaneously, the CBM-s of existence can modify its binding characteristic to obtain appropriate C BM-s of the present invention with point mutation technology well-known in the art.

Fusion rotein by polysaccharide affinity matrix wash-out after, it can randomly carry out one or more further purifying or separating step, depends on the required form and the albumen of use.

In useful embodiment, transgenic plant or vegetable cell comprise the nucleotide sequence of the CBM that encodes, and preferred CBM is heat-staple and at high temperature keeps solubility.The heat-staple albumen of here representing of term is in the also promptly about temperature more than 25 ℃ of high temperature, and about more than 37 ℃ usually, comprises keeping solubility, correctly folding and active under 40 ℃ of-100 ℃ of range temperature.

The gene of this preferred CBM of encoding can comprise thermophilic bacterium available from thermophile, algae and fungi and import in host plant or the vegetable cell with the mode that expression comprises the fusion rotein of said CBM.Term is thermophilic to be meant that here biological optimum growth temp is above 40 ℃.Preferred CBM is through the zytase 10A genes encoding from Thermotoga maritima (Thermotoga maritima), and the zone in the host plant of optimized encoding CBM or the vegetable cell is the zone of said gene.The zone of said coding CBM can comprise sequence SEQ IDNO:1 in specific embodiment, or the nucleotide sequence of coding same acid sequence, or coding and said aminoacid sequence have the sequence of the aminoacid sequence of sequence identity basically.

Need heating to comprise the protein extract to 37 ℃ of soluble fusion protein and the temperature in 100 ℃ of scopes in certain embodiments of the present invention in the course of processing; For example; Temperature in 50-80 ℃ of scope is such as by the for some time in 1 minute to 120 minutes the scope.For this purpose, heat-staple CBM is such as being particularly useful from thermophilic source.In this kind heat-processed, the part of endogenous vegetable-protein maybe inactivation and/or sex change and can therefore easily separating with protein extract.Said heating and extracting possibly preferably comprise with the expanded bed adsorption of polysaccharide matrix separates the procedure of processing that solid substance and affinity combine said CBM fusion rotein simultaneously by the extract of heating.

In the specific embodiment of the present invention; Said heterologous fusion proteins comprises proteolytic enzyme (polypeptide of expression protein decomposing activity); It is Mammals enteropeptidase (EK) or its enteropeptidase-active part in specific useful embodiment, for example, and ox EK or ox EK catalyst structure domain (EKc).The useful EKc that is used for this purpose is by the nucleic acid sequence encoding shown in the SEQ ID NO:2.

In the highly useful embodiment of the present invention, said fusion rotein comprises CBM and through comprising the proteolyze cleavage site, the purpose heterologous polypeptide that is preferably blocked by the amino acid projection of specific protease identification and the proteoclastic cleavage site of cracked.Owing to have a this site, CBM can by easily with fusion rotein in the fusion partner cracking fall to obtain not follow the purpose heterologous protein of CBM.

Therefore, at related aspect, the invention provides the method for purifying purpose heterologous protein, this method comprises

(a) fusion rotein that is blended in the CBM that is blocked by the proteolyze cleavage site that comprises like above definition is provided,

(b) promoting that through said proteolytic enzyme said fusion rotein contacts with the functional protein enzyme that is blended in CBM under the condition of proteolytic cleavage, coming from the CBM of purpose heterologous protein cracking fusion,

(c) therein CBM-proteolytic enzyme and free CBM be incorporated into said polysaccharide matrix and wherein the purpose heterologous protein do not remain under the condition on the said polysaccharide matrix; As here limit; With CBM-proteolytic enzyme, the solution of free CBM and purpose heterologous protein contacts with polysaccharide matrix

(d) separate not-bonded purpose heterologous protein by polysaccharide matrix,

(e) wash polysaccharide matrix with one or more suitable aqueous solution with bonded CBM-proteolytic enzyme and CBM,

(f) condition that is discharged by matrix through the said CBM-proteolytic enzyme of adjustment influence is by matrix wash-out CBM-proteolytic enzyme; With

(g) randomly restore the CBM-proteolytic enzyme of said wash-out, keep the affinity of itself and said polysaccharide matrix, make restorative CBM-proteolytic enzyme can be used further to the repetition of method subsequently.

Step in conjunction with fusion rotein, flushing and wash-out is preferably carried out as stated.

In preferred embodiments, the proteolytic enzyme that is blended in CBM is enteropeptidase, preferably as stated.This kind CBM-fusion protease, EK or other type can suitably produce and purifying through described method here.

Will be understood that aforesaid method can combine to prepare with aforesaid method and the purification of soluble heterologous fusion proteins valuably, thus obtain not contain the purified form that merges CBM at first as CBM expressing fusion protein and isolating purpose heterologous protein.

Other advantage of method is promptly utilized the same protein enzyme of the batch process that is recycled and reused for the target protein that is expressed as the CBM fusion rotein for restoring the possibility of used proteolytic enzyme, makes described method more economically here.These are easy to be implemented, and do not have the proteolyze cleavage site because the CBM-fusion protease is designed, and make to hinder CBM by the cracking of CBM proteolytic enzyme own.In addition, as indicated above, this recovery makes it become possibility through the mild conditions that is used for wash-out, does not damage the protease activities of wash-out thus.

Thus; A further advantage of the invention is it provides and can restore method expensive, the specific proteins lytic enzyme; The extra thus heterologous protein that reduced is such as especially, but is not limited to the cost available from the affinity purification in the proteic downstream processing of genetically modified plant, vegetable material and vegetable cell.

The present invention successfully overcome be used for such as but be not limited to agricultural, chemical industry and produce the shortcoming of relevant downstream processing based on the extensive heterologous protein of proteic drug manufacture.Especially; Provide with less procedure of processing by novel method such as the biomaterial separation of C BM-fusion rotein of plant cellulose material; Said procedure of processing has been utilized safety and the more cost effective affinity chromatography principle that can be used for pharmaceutical industry; Keep the active gentle elution requirement of high value heterologous protein, and comprise the procedure of processing of the specificity high value proteolytic enzyme in can restored method.

Description of drawings

Fig. 1 is the synoptic diagram of preferred embodiment of the proteolytic cleavage of CBM protease purification method and CBM fusion rotein.

Fig. 2 has shown the analytical results available from the SDS-PAGE of embodiment 1.Swimming lane 1: the big tick marks of molecular weight; Swimming lane 2: the CBM9-2 of purifying; Swimming lane 3: from the solid substance of the flushing of the barley seed of milling; Swimming lane 4: the supernatant after biomaterial interacts, swimming lane 5: with the flushing first time that low salt buffer carries out, swimming lane 6: the 5th with high-salt buffer carries out is washed.

Fig. 3 shows according to the inventive method by the barley seed extract success purifying CBM9-2 that mills, like the detailed description in embodiment 2.The figure illustrates and have the elution profile that the cellulosic expanded bed of 200mL absorbs (EBA) post.

Fig. 4 shows the result available from embodiment 3.This diagram has shown the ELISA reading of control sample and has comprised and has been blended in CBM9-2 and according to the least reading of the sample of the purpose heterologous protein of the small scale purification of described purge process here.This post has shown (a) elution buffer, (b) from the fusion rotein extract of transgenic seed, and the ELISA value of the protein determination that (c) extracts by non--transgenic seed.

Detailed Description Of The Invention

To specifically describe the present invention below.

Only if definition is arranged in addition, all technology of here using have identical with the implication of person of ordinary skill in the field's common sense of the present invention and use with scientific term.

Term " polypeptide " is meant any polymer of amino acid here, monomeric or polymer, and do not relate to the polymer of amino acid of length-specific.Thus, for example, term peptide, oligopeptides, albumen and enzyme are included in the definition of polypeptide.This term also comprises having expresses the back and modifies such as for example glycosylation, acetylize, the polypeptide of phosphorylation etc.

Term " purpose heterologous polypeptide " or " desired polypeptides " here are meant and are used for any polypeptide of utilizing method of the present invention or compsn to express at plant-cell or plant tissue.As the instance of non--restriction, medicinal polypeptide (for example, being used for pharmaceutical use) or industrial polypeptide (for example, enzyme) can prepare according to the present invention.

Term " downstream processing " is meant separation of biotechnology product and purifying is formed the form that is suitable for its purpose purposes.

Term " fusion partner " is meant the heterologous protein that is connected with CBM here.

Term " CBM fusion rotein " is meant the molecule that comprises the CBM that is connected in heterologous protein, and it is understood that not have the molecule of proteolyze cleavage site in context, only if having said in addition.

Term " can be operatively connected " the functional connection that is meant between the promotor (expression of nucleic acid control sequence, or serial transcription factor binding site point) and second nucleotide sequence, and wherein promotor instructs transcribing corresponding to the nucleic acid of second sequence.

Term " sex change " is meant proteic natural structure and the ruined state of protein-active subsequently, and albumen is unfolded or folding improperly its natural three-dimensional structure that changed.

Term " expression " and " production " are meant the biosynthesizing of gene product, comprise transcribing and translating of said gene product.

" molecule farming " is meant that the operation that utilizes open-air or any kind of plant in provision for sealing in their tissue, expresses and produce heterologous protein.

Therefore term " transgenic " is meant and imports the non-natural nucleotide sequence and changed its genotypic any cell that clone is organized plant part, organ or biological and have a filial generation of non-natural nucleic acid.Usually, import to the non-natural nucleotide sequence in the genotype through genetic engineering method or import in the genotype of paternal cell or plant by this method and import in the filial generation through sexual hybridization or vegetative propagation subsequently.

Transient expression is meant through the gene of moment importing and in biology, expresses, is not inserted in the biological genome.Therefore, transient expression usually receives the restriction of time, and transient gene does not pass to next son generation.

" sequence identity basically " is represented at least 50% sequence identity in context; And more preferably at least 60% such as at least 70 sequence identity; Such as at least 80% with at least 90% sequence identity preferably, sequence identity such as at least 95% or 99%.The algorithm that is used for sequential analysis is known in the art, and such as BLAST, Altschul etc. describe in J.Mol.Biol. (1990) 215:403-10.Usually, with respect to, for example, the default value of " getting sub matrix " and " breach point penalty " is provided with being used for comparison.

Term " conversion " or " conversion " be meant nucleotide sequence imported in the DNA genome of host living beings, and with nucleic acid fragment to be imported to the technology of using in the host cell irrelevant.

" thermophilic " is meant that biological optimum growth temp is above 45 ℃.

Term " GMP " (high-quality production standard) is meant the mode that produces bio-pharmaceutical and other medicine and medical device.GMP requires to comprise standard operating procedure, aseptic condition, material and equipment and professional's affirmation.

But the unifacial leaf and the dicotyledons of genetic manipulation can be used for the present invention.Preferred plant is a monocotyledons, is barley more preferably, and is most preferably barley (Hordeum vulgaris).But the plant of genetic transformation is expressed for importing, stable maintenance, and the plant that is delivered to dDNA sequence in the filial generation that produces subsequently, and the dDNA sequence comprises the dna sequence dna of coding region.Genetic manipulation and method for transformation have been used to utilize weedicide to comprise for example weeding peptide or basta or microbiotic, produce barley plants such as Totomycin as selected marker.

Method

Other purpose of the present invention, advantage and new feature are conspicuous according to the following example to those skilled in the art, it is not the purpose that is used to limit.In addition, the present invention as above-described each different embodiment and aspect and as in the following example, find experimental support in that the claim part is desired.

Although only specifically described the preferred embodiment of the invention, can expect to produce described a large amount of improvement of the above embodiment of the present invention and variation for a person skilled in the art, only otherwise deviate from the spirit and scope of the present invention.

Fig. 1 has explained the synoptic diagram of CBM-protease purification process, or in this respect, any CBM-fusion rotein, this method is described below:

(1) parent material that is used for this method is transgenic plant, and the material available from transgenic plant or transgenic plant cells includes but not limited to, the suspension culture of vegetable cell and the undifferentiated cell of callus.In multiple embodiments of the present invention, said material can be the proteic plant tissue of moment expressing heterologous source.This material is the transgenic line of expressing the heterologous gene that is operably connected to the CBM open reading frame with the mode of regulation and control; Its by one of skill in the art known method be directed in the vegetable cell; Such as; But be not limited to the conversion of the conversion of Agrobacterium-mediation or particle bombardment-mediation or the conversion of plant viral vector-mediation.Parent material is preferably selected based on the gratifying expression level of fusion rotein, is starting the method for the invention before by the analysis and judgement of carrying out RNA or protein level through those skilled in the art.

(2) known method realizes broken transgenic line by one of skill in the art, and it produces the homogenizing plant tissue and the vegetable cell of dry or wetting state.Can be selected from the method in various suitable vegetable materials source.Therefore, for seed, milling is the good method of broken transgenic plant tissue, and for leaf and soft chlorenchyma, can adopt such as, but be not limited to the Wei Lin whisking appliance, the equipment of Sorvall Omnimixer or Poltron homogenizer is realized.Leaf and softer tissue can also be frozen drying and be used for homogenizing subsequently and mill.The equipment that is used for broken plant tissue and vegetable cell is commercially available and amplifies in proportion as required easily.Extract proteic ordinary method by plant origin and be described in G.Paul Powell that S.Roe edits and publishes etc., Protein purification applications, the 2nd edition, in (2001).Simple and the successful method of being extracted soluble proteins by plant origin is to add simple damping fluid class low salt buffer to broken, in the plant tissue that stirs, and thorough mixing.For anti-oxidant brown China, be enough to optimization usually such as interpolation polyvinylpyrrolidine (1%w/v) and organize purifying so that by chelating resol in the plant tissue, otherwise can spinoff be arranged heterologous protein to be purified by most plants.Proteolyze is not the problem that always causes plant origin to have.If, yet, consider proteolyze, can be with protease inhibitor, such as, for example, Serine-, halfcystine-with the metalloprotein enzyme inhibition factor makes an addition to and extracts in the damping fluid.The fragmentation of vegetable material can be carried out existing or lack under the situation of buffered soln.Extract solution can comprise or not comprise reductive agent such as, but be not limited to 2 mercapto ethanol or WR 34678 (DTT).Solubility vegetable-protein and CBM-fusion rotein are present in the liquid phase together

(3) liquid and vegetable material to mix for the water-soluble fusion rotein that extracts in the liquid phase be essential.The liquid that adds can comprise or not comprise the buffer reagent of regulating preferred pH in about 5.2 to 8.3 scopes, and it can comprise or not comprise the reductive agent or the sequestrant of target protein described in any (2).With its simple form, liquid can be water.Behind liquid and broken and the vegetable material thorough mixing that stirs, liquid phase comprises the CBM-fusion rotein now.

(4) (A) depend on level of homogenization to a certain extent, the broken vegetable material and the mixture of extracting liq can directly apply to expanded bed adsorption (EBA) post.In this method, mixture is used to post as the flow through expanded bed of affinity adsorbing base of polysaccharide characteristic of liquid flow.In liquid flow was flowed through the process of post, the fusion rotein in the liquid phase was exposed to polysaccharide matrix and being selected property of the selective affinity absorption through CBM and polysaccharide asborbent medium.Particle such as, but be not limited to, cell wall fragment and other solid substance are with any solubility vegetable-protein EBA post of in fluxion, flowing through.

4 (B) randomly, the known by one of skill in the art the whole bag of tricks capable of using of the most of solids in the mixture is before the affinity integrating step and liquid separation, these methods include, but are not limited to deposition, filter centrifugal and sedimentation.As indicated above, abandon solid and will comprise the heating steps 4 (C) that the liquid of CBM-fusion rotein is used to choose wantonly or directly apply to affinity integrating step (5).

4 (C) are at affinity step (5) optionally heating mixture or liquid before; But the CBM-fusion rotein can be used as additional purification step under the situation that keeps solubility under the temperature that increases therein, but and solubility vegetable-protein variability and deposition and monocotyledons proteolytic enzyme maybe be through heating by inactivations.For reaching this purpose, consider the heating means of the characteristic of CBM-fusion rotein, can be included in the course of processing for some time in 2 minutes to 60 minutes scopes of scope internal heating of 50 ℃ to 100 ℃.In these embodiments, the CBM in thermophilic source is especially useful.

(5) matrix combines affinity.Comprise plant origin the CBM-fusion rotein the liquid protein extract with contact with polysaccharide matrix that CBM has an affinity.Contact may be implemented in a variety of ways, such as, but be not limited to; Chromatography column with the polysaccharide matrix filling; Wherein liquid is to fill or the expansible mode post of flowing through, and it is meant the density of polysaccharide matrix, or it can batch mode be realized; Wherein polysaccharide matrix mixes in suitable containers with liquid, reclaims the CBM-fusion rotein of matrix and absorption subsequently.Polysaccharide matrix can be Mierocrystalline cellulose source, such as, but be not limited to, Avicel, or it can be the xylanoic source, such as insoluble xylan.The research particularly in the new publication of (2001) such as Boraston of the binding specificity of CBM9-2 and thermodynamics.Yet the inventor finds surprisingly; Compared with prior art; According to the method that the present invention here describes, the CBM9-2 debond is in the vegetable cell wall fraction but be easy to dissolving, and keeps for the good binding specificity such as the polysaccharide matrix that uses at affinity adsorption step (5).As here describe, this surprising characteristic has introduced several advantages for the downstream processing of the CBM-fusion rotein of plant origin, has improved the downstream method for processing greatly thereby provide.

(6) flushing matrix.The polysaccharide affinity adsorbent that is incorporated into the CBM-fusion rotein can use several column volumes (if affinity matrix places chromatography column; The term of relative quantity) the aqueous solution (for example water) or damping fluid; Such as, but be not limited to phosphate buffered saline (PBS) or wash based on the damping fluid of Tris-.For improving the efficient of rinse step; The compsn of dcq buffer liquid can through such as but be not limited to; The mode that progressively changes the gradient of the progressively change separate each column volume or salt concn or stain remover is regulated, and is used for discharging faint but the contaminating protein of non-specific binding by matrix.

(7) discharge fusion rotein by matrix.Has the CBM-s of purpose of description binding characteristic here through utilization; By affinity matrix wash-out CBM-fusion rotein can through the CBM-fusion rotein is exposed to competition sugar such as, but be not limited to glucose, semi-lactosi; Lactose, SANMALT-S and cellobiose are achieved.Arbitrary these or other similarly sugar or its combination can appropriate vol, such as in 1mM to 1M range of concentrations, adding in the elution buffer such as for example, phosphate buffered saline (PBS) or based on the damping fluid of Tris-to carry out elution step.Sugar concentration can be for example in the scope of 25mM to 1M, in the scope such as 50-500mM.These sugar are commercially available low price containerization chemicals, have further reduced the overall cost of downstream of the present invention processing.

(8) separation/purification of fusion rotein.Be further purified/separation of C BM-proteolytic enzyme is useful or needed in some instance.This further separation is available arbitraryly to well known to a person skilled in the art that usually red, orange, green, blue, yellow (ROGBY) realizes, such as, but be not limited to ion exchange chromatography or steric exclusion chromatography.

(9) final product.Final product is to be the CBM-proteolytic enzyme of highly purified form in this case, prepares to be used for the preparation and the packing of its final form in case of necessity.

(10) comprise the separation of the fusion rotein of proteolyze cleavage site.The method of the purifying CBM-proteolytic enzyme of (1-9) describing can be to be used for the preferred method that purifying is connected in any heterologous protein of CBM.This CBM-fusion rotein possibly contain between CBM and purpose heterologous protein or possibly not contain the proteolyze cleavage site.In the above-described preferred embodiment of the present invention, purification process can be used for cracking CBM-fusion rotein and separate the purpose heterologous protein by CBM-proteolytic enzyme effectively with cracked CBM together with CBM-proteolytic enzyme.Further separate the CBM-fusion rotein that contains the proteolyze cleavage site before in proteolytic cleavage step (11) and possibly be or possibly not need, or possibly be useful in some cases.It is useful that the separation of these fusion roteins possibly contain the damping fluid of the CBM-fusion rotein with proteolyze cleavage site for change, makes its condition to the proteolytic cleavage reaction that helps step (11) of adjustment.These separation can well known to a person skilled in the art that usually red, orange, green, blue, yellow (ROGBY) realizes with arbitrary, such as, but be not limited to ion exchange chromatography or steric exclusion chromatography.

(11) come proteolyze separation of C BM by the heterologous protein fusion partner.In this stage of this method, the CBM-fusion rotein that will contain the proteolyze cleavage site is exposed to the specific protease that is blended in CBM but does not have cleavage site, and this proteolytic enzyme can be discerned cleavage site and by purpose heterologous protein separation of C BM.The specific protease that connects CBM is preferably selected from the group of cleavage site-specific protease, such as enteropeptidase, and factor Xa, zymoplasm or the like.Scission reaction is accomplished the CBM that afterreaction solution comprises release, residual uncracked CBM-fusion rotein, CBM-proteolytic enzyme (for example, CBM-EK), and the purpose heterologous protein that discharges.

(12) affinity that combines of CBM-proteolytic enzyme and free CBM and matrix.The solution that described in preceding text step (5), will contain all these components with contact with polysaccharide matrix that CBM has an affinity.All components that contain CBM will be adsorbed on the affinity matrix and the highly purified purpose heterologous protein that discharges is recovered in the flow.

(13) the further separation of heterologous protein.Although the purpose heterologous protein is highly purified, it possibly be that need or useful being further purified in some cases/separating.These further separation can be chromatographic step, such as, for example, ion exchange chromatography or steric exclusion chromatography.

(14) final product.Final product is to be the CBM heterologous protein of highly purified form, prepares to be used for preparation of its final form in case of necessity (for example, lyophilize) and packing.

(15) this step of flushing matrix can be carried out as step (6) is said.

(16) release of CBM-proteolytic enzyme and CBM.Utilize the described elution requirement of above-mentioned steps (7), from solution, reclaim CBM-proteolytic enzyme.The recovery of these solution comprises the neutralization of condition and/or removes reagent (for example sugar) that its influence discharges CBM by polysaccharide affinity matrix.Said reagent can be dialysed by for example using ultrafiltration in the solution with CBM proteolytic enzyme, and steric exclusion chromatography and ion exchange chromatography are under certain condition removed.If desired, can also be such as some these method of steric exclusion chromatography by residual CBM and CBM-fusion rotein purifying CBM-proteolytic enzyme.

(17) recovery of CBM-proteolytic enzyme.In case CBM-proteolytic enzyme restores the state of said polysaccharide matrix being recaptured affinity that is returned to, it can be introduced the proteolytic cleavage of a new round that comes to be used to contain like step (11) the CBM-fusion rotein of proteolyze cleavage site in this method once more.Although the CBM-protein enzyme solution of reconstruct possibly contain the not cracking CBM-fusion rotein from the residual CBM of last reaction and trace, they will not disturb the purifying of cracked heterologous protein fusion partner, because they will be total to-purifying with CBM-proteolytic enzyme.Therefore, effective recovery of high value specific C BM-proteolytic enzyme improved novel method of the present invention efficient and economy and can scale operation and the albumen that produces of purification of Recombinant, limited the expensive excessively of former downstream processing thus.

Other purpose of the present invention, advantage and new feature are conspicuous according to the following example to those skilled in the art, it is not the purpose that is used to limit.In addition, the present invention as above-described each different embodiment and aspect and as in the following example, find experimental support in that the claim part is desired.

Embodiment

Embodiment 1 biomaterial repercussion study: CBM9-2 and the barley seed of milling

In the Retsch mill, the exsiccant barley seed is ground to form fine powder carefully.The low salt buffer (the 50mM potassium phosphate buffer of pH 7.02) that the seed of milling of 1g is used for through 5ml extracts water soluble ingredient from seed; As the method for all water soluble ingredients of removing sample, but said water soluble ingredient interfere biomaterial Study of Interaction.Vortex mixed thing and overturning was guaranteed the thorough mixing of liquid and the barley seed material of milling in 5 minutes.

After the mixing, came the solid substance granulation in 4 minutes at the centrifugal sample of 5000 * g.After centrifugal, abandon supernatant.This process repeats 3 times with low salt buffer, abandons supernatant after each centrifugal.Use high-salt buffer (50mM potassium phosphate buffer pH 7.02,1M NaCl) to repeat flushing process 3 times then, abandon supernatant as previously mentioned.The flushing solid substance that produces is most vegetable cell-wall fragment and insoluble starch.With the solid substance that low salt buffer carries out 3 flushing balance flushings as stated, obtain to help CBM9-2 affinity in the affinity chromatography to be incorporated into the same terms of cellulose matrix.The typical sample of the solid substance before the biomaterial repercussion study is used for SDS-PAGE analysis (swimming lane 3).Will be through Mierocrystalline cellulose (Avicel ^TMThe bacteriogenic CBM9-2 of 10 μ l of)-affinity column (O.D.280nm 0,394) purifying is used for SDS-PAGE (swimming lane 2) subsequently.The CBM9-2 of purifying is repeated (4x) dilution and concentrated as the method for proof from any bonded glucose of CBM9-2 desorption at ultrafiltration module in advance, so that the Mierocrystalline cellulose of recoverin combines the affinity characteristic.

The CBM9-2 of the purifying of 2ml is made an addition at room temperature vibrate incubation 60 minutes of flushing, equilibrated solid substance and mixture available from the barley seed of milling.Behind the incubation, with 5000xg rotation mixture 10 minutes, and subsequently with 13.000xg centrifugal clarification supernatant 5 minutes.To be used for SDS-PAGE from the interactional 10 μ L clarified supernatant of biological material and analyze (swimming lane 4).The deposition that will comprise the barley seed solid substance of milling subsequently is as stated with low salt buffer flushing 5 times and subsequently with high-salt buffer flushing 5 times.The 1st the low salt buffer flushing (swimming lane 5) of 10 μ L and the 5th low salt buffer flushing (swimming lane 6), the SDS-PAGE that is used for subsequently analyzes.For by all the bonded CBM9-2 of barley seed solid substance wash-out that mill, with centrifugal 5 minutes of 5000xg with remove supernatant (eluate) before with the elution buffer (50mMKPO of 1ml ₄In 1M glucose, pH 7.02) make an addition to solid substance and mix incubation 15 minutes.The eluate sample of preparing 10 μ l is used for SDS-PAGE and analyzes (swimming lane 7).The typical sample of the solid substance behind biomaterial repercussion study and the wash-out is used for SDS-PAGE analysis (swimming lane 8).Preparation is used for sample that the biomaterial of SDS-PAGE interact to measure utilizes PhastSystem (AmershamPharmacia Biotech) to carry out SDS-PAGE on 12.5%SDS-PAGE gel (PhastGels homogeneity 12,5).Electrophoresis accomplish the back with Coomassie blue R-250 to gel-colored, and decolour.The result is shown in Fig. 2.

These results show that CBM9-2 is not incorporated into cell-wall or other insoluble solid from the barley seed of milling available from plant significantly.

Embodiment 2

Purifying from the CBM9-2 of the extract of the barley seed of milling

Utilize commercially available shredder (Aarslev Maskinfabrik, Erhvervsvangen 11,5792Aarslev DK) is milled into meticulous powder with barley seed.With the barley meal that produces with barley meal: the volume ratio that damping fluid was respectively 2: 3 is soaked in low salt buffer (50mM potassium phosphate buffer pH 7.02).Liquid and powder are thoroughly mixed in container and allow 4 ℃ of depositions of spending the night.CBM9-2 by bacteria purification is added in barley seed-supernatant.Second day, the supernatant (100ml) that will contain the CBM9-2 peak value offered and contains Mierocrystalline cellulose (Avicel ^TM) Streamline 25 (Amersham Biotech) chromatography column.With the flow velocity of 184cm/h, 5 column volume high-salt buffers (the 50mM KPO of 1MNaCl is used in the mode charging of expanded bed then ₄Solution, pH 7.02), the low salt buffer of 5 column volumes (50mM KPO then ₄, pH 7.02) wash.(settler=20cm) also carries out wash-out with the 300ml elution buffer (the 50mM KPO4 solution of 1M glucose, pH 7.02) of 92cm/h to allow expansion post bed deposition.Elution requirement produces and contains the proteic small peak of CBM9-2 (referring to Fig. 3).

This shows the above-described method of utilizing; At first, CBM9-2 remain in the solution with from the cell-wall fragment of the barley seed of milling and other fully the solid substance of definition take off absorption, secondly; It is possible utilizing polysaccharide affinity chromatography of the present invention to catch CBM9-2 by the barley seed extract of milling, the 3rd; Can be through utilizing the pharmaceutical grade Mierocrystalline cellulose (Avicel) that better limits carry out and the 4th as matrix; Can carry out affinity chromatography step, the 5th through expanded bed mode of the present invention; CBM9-2 by barley seed-extract purifying can avoid any denaturing step by the matrix wash-out under mild conditions of the present invention.Can be used for by the transgenic purification of seed CBM9-2-fusion rotein of milling with identical condition as indicated above and method.Suitable method is described in pendent " by the non-denaturing process of plant purification of recombinant proteins " that the applicant applies for simultaneously and here is incorporated herein by reference in full.

Described polysaccharide affinity chromatography also effectively is used for reacting the CBM of back by target protein protein isolates enzyme-CBM and cutting like proteolytic cleavage of the present invention.

Embodiment 3

From what mill, single barley seed extract purifying is connected in the heterologous protein of CBM9-2.

In the small-scale form of purification process mentioned above, expression is connected in CBM9-2 the purpose heterologous protein the transgene barley plant single seed respectively homogeneous turn to fine powder.With the seed powder that produces with the barley powder: the volume ratio that damping fluid was respectively 1: 7 is soaked in low salt extraction damping fluid (50mM potassium phosphate buffer pH 7.02,0,01% polyvinylpyrrolidine).Liquid thoroughly mixes 1 hour with single seed powder, and at room temperature with 5 minutes interval vibration water-soluble protein is extracted in the liquid phase.

In whizzer, rotated extract mixtures 10 minutes with 6000rpm.Collect from the supernatant (extract) of individual seed and be applied to be full of Mierocrystalline cellulose (Avicel ^TM) micropore filter plate (MSHVN45, Millipore ^TM) on.Extract is made an addition at room temperature stirred at interval in the Mierocrystalline cellulose of corresponding micropore and continue 15 minutes with per 3 minutes.After 15 minutes, through vacuum manifold (Multiscreen resist vacuum manifold-MAVM0960R, Millipore ^TM) vacuum will be applied to microwell plate to discharge the liquid from Mierocrystalline cellulose (flowing through) in each hole.As preceding text are described Mierocrystalline cellulose is carried out rinse step: 5 posts (being the plain micropore of fiberfill fibers) volume high-salt buffer (the 50mM KPO of 1M NaCl ₄Solution, pH 7.02), the low salt buffer of 5 column volumes (50mM KPO then ₄, pH 7.02).Elution buffer (the 50mM KPO of 1M glucose with 250 μ l ₄Solution, pH 7.02) carry out wash-out.To the micropore of fresh microwell plate, collect eluate through using vacuum.Eluate is used for high degree of specificity ELISA CBM9-2 analyzes, also promptly based on the specific polyclonal antibody of anti-CBM9-2.ELISA carries out as follows; The antigenic solution (0.1-5ng albumen) of the suitable dilution of 50 μ L is applied to each hole (Cat.no.442404) of Nunc-Immuno 96 microwell plates.Micropore covers with scotch tape and 37 ℃ of incubations 1 hour.Antigenic solution is shifted out micropore also with 100 μ lTBS flushing 3 times.With the lock solution closed porosity in the every hole of 100 μ l, cover micropore and 37 ℃ of incubations 1 hour with scotch tape.After abandoning lock solution, wash micropore 5 times with the 100 μ lTBS that contain 0.01% tween.The primary antibody of anti-CBM9-2 is suitably diluted interpolation (for example 1/3000) to be covered and 37 ℃ of further incubations 1 hour in the TBS of 1%BSA (every hole 50 μ L) and with scotch tape.Shift out antibody-solutions and wash micropore 10 times with the TBS that contains 0.01% tween.Secondary antibodies (with the HRPO bonded) is 1/3000 dilution in the TBS (the every hole of 50 μ l) of 1%BSA.Micropore covers with scotch tape and 37 ℃ of incubations 1 hour.Discard behind the solution and contain the TBS flushing 8x of 0.01% tween and wash 2x, with 100 μ l TMB One solution (Promega with the TBS of no tween with 100 μ l ^TM) be added into every hole and incubation microwell plate 30 seconds-5 minutes at room temperature.When blue color is launched, add the 0.2M sulfuric acid of 100 μ 1.Its colour changed into yellow, and the 450nm place measures absorbancy in the microwell plate reader.

Elisa assay result from the single seed extract of individual transgene barley seed is shown among Fig. 4.Be used in the past prove that the specific elisa assay of CBM9-2 showed, at first; Can obtain and prove the accumulation of heterologous fusion proteins in the individual seed; Isolating work mentioned above with heterologous fusion proteins of cleavage site, even still supported the scale ability of purge process on a small scale the time; It shows that heterologous fusion proteins is not exposed to Denaturation in the chromatography method process; Owing to do not related to any renaturation step by specific antibody identification behind the wash-out, given prominence to advantage like the present invention's other harsh affinity chromatography method of discussing at preceding text with respect to some; Be applicable to the heterologous fusion proteins of any CBM9-2 of being connected in and be not limited at above-described purification process and catch CBM-proteolytic enzyme.

Embodiment 4

The purifying of CBM-proteolytic enzyme and determination of activity

Have the locus specificity proteolytic enzyme that connects the CBM9-2 mark in order to produce, method is following:

Make up agrobacterium tumefaciens strains A GLO; Make it contain the composition promotor of carrying by before the enteropeptidase cDNA; The signal sequence of target endoplasmic reticulum (ER) and albumen maintained the binary plasmid of the expression construct that the stick signal among the ER forms, wherein enteropeptidase cDNA is equivalent to the cDNA of CBM9-2 by connection.This agrobacterium strains is cultivated under selection condition in the YEB substratum, at first in 10ml 28 ℃ cultivate 2 days until OD ₆₀₀Be 0.8.28 ℃ 2-3 days with a spot of culture 1: 50 dilution for the 500ml culture that contains 20 μ M Syringylethanones and vibrate to OD strongly ₆₀₀Nm is 2.5.With 6000rpm rotation bacterium 10 minutes and be resuspended in the MS-solution and (contain 55g/l sucrose) to OD ₆₀₀Be 2.5.Add Syringylethanone (10mM), final concentration is 20uM.Bacterial suspension maintains room temperature 1 hour and adds tween 20 (10%), and final concentration is 0.005%.

For transient expression CBM-proteolytic enzyme in lettuce plant, with lettuce plant be immersed into contain the Agrobacterium bacterial suspension the bowl in 15 seconds.Plant places vacuum and applies 0.4 barometric point 20 minutes subsequently, and open inlet mouth is compensatory pressure promptly.In the bowl that tap water is housed, wash the excessive bacterium on the blade face through continuous impregnating off.Lettuce plant places 22 ℃ of incubators to cultivate 4 days at 8 hours 16 hours/nights of daytime.

Through excision leaf texture results plant and freezing subsequently and remain on-86 ℃.Utilize mortar and liquid nitrogen homogenizing plant until obtaining very fine powder.Through add respectively 1.2: 1 (volume: volume) low-salt extraction damping fluid and lettuce powder extract powdery lettuce leaves material, protein extract at room temperature between or stirred 30 minutes.

Extract subsequently with centrifugal 20 minutes of 6000rpm by liquid phase separation solid matter and cell wall fragment.Abandon supernatant and rotation once more as stated.As stated clarified supernatant is filled into and contains cellulose matrix (Avicel ^TM) the filling column on.After CBM-proteolytic enzyme specificity is connected in cellulose matrix and washes respectively with the high salt of 5 column volumes and less salt dcq buffer liquid, in gentleness, under the non-denaturing condition with the 1M glucose solution of single peak value by post wash-out CBM-proteolytic enzyme.

Utilize micropore thickener (Ultrafree-15-Biomax-5) to concentrate the peak product subsequently.

Utilize specific synthetic substrate to measure enterokinase activity: synthetic substrate: Gly-Asp-Asp-Asp-Asp-Lys-β-naphthalene amino acid (GD according to standard method (Grant & Hermon-Taylor, 1979) ₄K-na); 37 ℃ of condition determinations.Reaction volume is 1.5ml.Reaction mixture comprises: 25 μ l10mM GD ₄K-na (0.5mM), the 100mMTris-HCl of 125 μ l, pH 8.4 (25mM), 50 μ l 100%DMSO (10%), the 100mM CaCl of 50 μ l ₂(10mM), (20-100) μ l enteropeptidase, the μ l of 250 μ l zero(ppm) water-(20-100).By λ _Ex=337nm and λ _EmThe speed that β-naphthylamine forms is measured in the increase of the fluorescence the between=420nm.Monitor 5 minutes continuously.

The result of determination of activity shows that the CBM-enteropeptidase of said generation and purifying is active; Enterokinase activity and blank 0,0001cps/min/ μ g relatively is measured as 442,7cps/min/ μ g.

This embodiment shows at first; CMB-proteolytic enzyme can produce in plant, and moment produces in lettuce under situation, and above-mentioned said purification process capable of using successfully separates the proteolytic enzyme with purifying CBM-.It is functional completely that it shows that further the CBM9-2 affinity marker of fusion rotein has; CBM-proteolytic enzyme effectively is connected in cellulose matrix and it can be eluted by matrix under the gentle elution requirement that the present invention describes, and the proteolytic enzyme of wash-out is proved to be to have active fully.The purified product of enzymatic activity itself provides the evidence of the non-denaturing characteristic of purge process, and like the enzymic activity especially responsive to Denaturation partially or completely, it causes active loss easily.This shows that effectively all component of the present invention is functional and their characteristic and performance can easily be used it with the above-mentioned method of the present invention, produces the downstream processing economy of the heterologous protein that has improved main specificity, any source and the method for efficient.

Reference

Altschul etc., J.Mol, Biol. (1990) 215:403-10.

Boraston etc., (2001) Biochemistry 40, pp.6240-6247.

" Recombinant Protein Drugs " Ed.P.Buckel-from series-Milestones in Drug Therapy of Contributors (2001), Birkhauser Verlag, Basel2001.

Grant?D.A.，Hermon-Taylor?J.，Biochim.Biophys.Acta?567(1979)，.207-15.

" the Plant bioechnology of Hammond (1999); New products andapplications " Eds.Hammond, McGarvey & Yusibov, Springer Verlag, NY1999.

IEX，Principles?and?Methods?Series，nr.18-1114-21-AmershamPharmacia?Biotech.

" Downstream processing ofproteins " Ed.M.A.DesaiHumana Press N.J. of Kalyanpur M. (2000)

The Proteinpurificatioh applications of Paul G.PaulPowell G. ", Ed.by S.Roe, second edition (2001).

" Protein Purification:Principles andPractice " 3rd ed.Springer-Verlag NY. of R.K.Scopes R.K. (1993)

Shani etc., USP 6,331,416.

Winterhalter etc., (1995) Mol.Microbiol.15 (3), 431-444.

Sequence table

<110>ORF?Liftaekni?hf.

 

< 120>method of proteolytic cleavage and purification of recombinant proteins

 

(A?PROCESS?FOR?PROTEOLYTIC?CLEAVAGE?AND?PURIFICATION?OF?TO?PURIFY

RECOMBINANT?PROTEINS)

 

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<170>PatentIn?version?3.1

 

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<400>1

gtggccaccg?ccaagtacgg?caccccagtg?atcgacgggg?agatcgacga?gatctggaac 60

accaccgagg?agatcgagac?caaggccgtg?gccgtgggga?gcctcgacaa?gaacgccacc 120

gccaaggtgc?gcgtgctctg?ggacgagaac?tacctctacg?tgctcgccat?cgtgaaggac 180

ccagtgctca?acaaggacaa?cagcaacccc?tgggagcaag?acagcgtgga?gatcttcatc 240

gacgagaaca?accacaagac?cggctactac?gaggacgacg?acgcccaatt?ccgcgtgaac 300

tacatgaacg?agcaaacctt?cgggaccggc?gggagcccag?cccgcttcaa?gaccgccgtg 360

aagctcatcg?aggggggcta?catcgtggag?gccgccatca?agtggaagac?catcaagcca 420

accccaaaca?ccgtgatcgg?cttcaacatc?caagtgaacg?acgccaacga?gaaggggcaa 480

cgcgtgggga?tcatcagctg?gagcgaccca?accaacaaca?gctggcgcga?cccaagcaag 540

ttcgggaacc?tccgcctcat?caag 564

 

<210>2

<211>705

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<213>Homo?sapiens

 

<400>2

atcgtcggcg?ggagcgattc?cagggagggc?gcatggccat?gggtcgtggc?actctacttc 60

gatgatcaac?aagtctgcgg?ggcatccctg?gtgagcaggg?attggctcgt?gtccgcagca 120

cattgcgtgt?acggcaggaa?catggagcca?tccaagtgga?aggcagtgct?cggcctgcat 180

atggcatcca?acctcacctc?cccacaaata?gagaccaggt?tgatcgatca?aatcgtcata 240

aacccacatt?acaacaagcg?gaggaagaac?aacgacatcg?caatgatgca?tctcgagatg 300

aaggtgaact?acaccgatta?catacaacca?atctgcttgc?cagaggagaa?ccaagtgttc 360

ccaccaggga?ggatctgctc?catcgcaggc?tggggcgcac?tcatatacca?agggtccacc 420

gcagatgtac?tgcaagaggc?agacgtgcca?ctcctctcca?acgagaagtg?ccaacaacaa 480

atgccagagt?acaacatcac?cgagaacatg?gtgtgcgcag?gctacgaggc?aggcggggta 540

gattcctgcc?aaggcgattc?cggcgggcca?ctcatgtgcc?aagagaacaa?caggtggctc 600

ctggcaggcg?tgacctcctt?cggctaccaa?tgcgcactcc?caaaccggcc?aggggtgtac 660

gcacgggtgc?caaggttcac?cgagtggata?caaagcttcc?tccat 705

Claims (17)

1. the method for purifying purpose heterologous protein comprises

(a) fusion rotein is provided, said fusion rotein comprises the said heterologous protein that is blended in the CBM that is blocked by the proteolyze cleavage site,

(b) under the condition that promotes said protease protein hydrolytic rupture, said fusion rotein is contacted with the functional protein enzyme that is blended in CBM, from purpose heterologous protein cracking CBM,

(c) therein CBM-proteolytic enzyme and free CBM be incorporated into comprise cellulosic polysaccharide matrix and wherein the purpose heterologous protein do not remain under the said condition that comprises on the cellulosic polysaccharide matrix; With CBM-proteolytic enzyme; The solution of free CBM and purpose heterologous protein comprises that with said cellulosic polysaccharide matrix contacts

(d) by comprising that cellulosic polysaccharide matrix separates not-bonded purpose heterologous protein,

(e) wash polysaccharide matrix with one or more suitable aqueous solution with bonded CBM-proteolytic enzyme and CBM,

(f) condition that is discharged by matrix through the said CBM-proteolytic enzyme of adjustment influence is by matrix wash-out CBM-proteolytic enzyme; With

(g) randomly restore the CBM-proteolytic enzyme of said wash-out, keep itself and the said affinity that comprises cellulosic polysaccharide matrix, make restorative CBM-proteolytic enzyme can be used further to repeat subsequently method step (a)-(g) limit,

Wherein said CBMs can be incorporated into reversiblely and comprise cellulosic polysaccharide matrix and discharge from said matrix through non-sex change elution requirement, said CBM also basically debond in insoluble cell walls vegetable material.

2. the process of claim 1 wherein the proteolytic enzyme of the said CBM of being blended in from enteropeptidase, marmor erodens proteolytic enzyme, factor X and zymoplasm.

3. the method for claim 2, wherein said proteolytic enzyme is Mammals enteropeptidase or its enterokinase activity part.

4. the method for claim 3, wherein said EK comprises the catalyst structure domain of ox EK, the catalyst structure domain of wherein said ox EK is by the nucleic acid sequence encoding shown in the SEQ ID NO:2.

5. the method for claim 1; The heterologous protein that the proteolytic enzyme of the wherein said CBM of being blended in and said is blended in the CBM that blocks through the proteolyze cleavage site individually through produce comprise the Mierocrystalline cellulose coupling unit with purifying the method for solubility heterologous fusion proteins by transgenic plant of expressing said fusion rotein or transgenic plant cells acquisition, comprise step:

(a) broken transgenic plant material;

(b) extracting liq is made an addition to vegetable material, produce solubility and insoluble plant mixtures of material thus, obtain protein extract so that extract by the vegetable material of said fragmentation in fusion rotein to the liquid phase of solubility;

(c) by separatin non-soluble vegetable material in the said protein extract that comprises said purpose fusion rotein, comprise cell-wall material and solid substance;

(d) with said protein extract with have comprising of binding affinity cellulosic polysaccharide matrix with CBM and contact;

(e) with the flushing of one or more suitable aqueous solution have the bonded fusion rotein matrix and

(f) condition that is discharged by matrix through the said fusion rotein of adjustment influence is by said polysaccharide matrix wash-out fusion rotein.

Therefore obtain the solubility heterologous fusion proteins of purifying substantially.

6. the method for claim 5, wherein separating step (c) comprises and is selected from expanded bed adsorption, the filling type chromatography, deposition is filtered, the method for centrifugal or its arbitrary combination.

7. the method for claim 5, wherein the said contact in step (d) is carried out in chromatography column.

8. the method for claim 5; In comprising, combine, as by said protein extract isolated cell-wall material and solid substance and with the affine method that is attached on the polysaccharide matrix of said CBM-fusion rotein simultaneously with step (c) with (d) with the process step of the expanded bed adsorption of polysaccharide matrix.

9. each method of claim 1-8, wherein said Mierocrystalline cellulose is the Mierocrystalline cellulose of pharmaceutically compatible.

10. the method for claim 9, wherein said Mierocrystalline cellulose is Avicel ^TM

11. each method of claim 1-9, the recovery of the CBM-proteolytic enzyme of wherein said wash-out comprises neutralization, and/or influences the reagent that CBM discharges by removing in the CBM-proteolytic enzyme elutriant from said polysaccharide matrix.

12. the method for claim 11, wherein said recovery comprise neutralization or remove soluble-carbohydrate from elutriant, said soluble-carbohydrate disturbs said CBM-proteolytic enzyme to comprise combining of cellulosic polysaccharide matrix with said.

13. the method for claim 12, wherein said soluble-carbohydrate are sugar.

14. each method of claim 1-13, the said fusion rotein that wherein comprises said purpose heterologous protein are expressed by transgenic plant or vegetable cell and are reclaimed or plant, transient expression in plant tissue or the vegetable cell.

15. the method for claim 14, wherein said transgenic plant or vegetable cell are selected from dicotyledons and monocotyledons.

16. the method for claim 15, wherein said vegetable cell or transgenic plant are selected from: tobacco, Semen Brassicae campestris, soybean, clover, lettuce, barley, corn, wheat, oat and paddy rice.

17. the method for claim 1; Said CBM that wherein is blended in said heterologous protein and the said CBM that is blended in said proteolytic enzyme be heat-staple and keep solubility at elevated temperatures, and the two is by the CBM from the regional code of the zytase 10A gene of Thermotoga maritima for wherein said CBMs.

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