CN1842600B - Method 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 for proteolytic cleavage of fusion proteins. These methods can be used for large-scale production of purified recombinant proteins from plants, tissues obtained from plants or plant cells. The present invention is aimed at reducing costs and improving the quality of downstream processing of heterologous proteins produced in plants and other biological production systems.

Description

Method for proteolytic cleavage and purification of recombinant proteins

field of invention

The present invention belongs to the field of biochemistry and protein technology and relates to improved methods for isolating and purifying heterologous proteins especially from transgenic plant material. Furthermore, the present invention provides an improved and economical way of proteolytic cleavage of fusion proteins.

Background technique

Biopharmaceutical-based proteins show significant promise in providing more specific and tissue-specific or cell-specific drug treatments against serious diseases (see review "Recombinant Protein Drugs" Ed. P. Buckel 2001).

Many examples in the prior art have shown the use of microorganisms such as bacteria, and animal cells to produce such biopharmaceuticals; among them insulin is an important example.

Many examples in the literature have shown the use of transgenic plants or plant cell cultures for the expression and production of high value heterologous polypeptides or biopharmaceuticals. This plant-based production method can be called molecular farming.

The production of valuable proteins can be produced more economically by using plants as production organisms. Compared to most production systems based on bioreactors, such as prokaryotic production systems, animal cell cultures, etc., the cultivation costs for protein production using plants as host organisms can be significantly reduced. However, purification of heterologous proteins is still a necessary and expensive task for all the production systems mentioned above. Thus, for plant-based production systems, downstream processing in the production of high-value heterologous proteins consumes most of the production costs.

Protein purification and isolation are key processes in the downstream processing of proteins accumulated and produced in various host organisms by utilizing genetic technology. Purification of proteins from host organisms can be laborious, complicated and expensive. Various chromatographic strategies are used commercially for the isolation and purification of proteins of interest from production host organisms. Chromatographic strategies can rely on physicochemical differences between contaminating or endogenous proteins and heterologous proteins of interest, such as differences in size, solubility, charge, hydrophobicity, and affinity.

Combination of chromatographic strategies involves multiple steps, requires several expensive chromatographic substrates and necessary hardware including columns, control units, etc., and is accompanied by a loss of product yield in each step, and thus results in an economical loss. Downstream processing often includes multiple filtration and centrifugation steps in addition to chromatographic steps. As a result, the cost of purification and downstream processing can be a limitation in the purification of proteins based on biotechnology products. As with the bulk of the lower value protein-based products, such as industrial proteins, the cost of downstream processing can limit their use and sale, resulting in crude and unsuitable products. For most biotechnology products, purification costs are undoubtedly a major part of production costs.

Affected by the separation of pollutants The cost of specific chromatography matrices for separation purposes is expensive due to the complex binding chemistry involved in their production. The chemical complexity of these matrices can lead to unnecessary leaching of ligands and other substances from the matrices during purification methods, where the necessary preventive assays, monitoring, or removal of leachates from the protein of interest add to an already costly process. The cost of downstream processing.

Affinity chromatography is the most powerful purification method because it is based on specific affinities between reagents and specific ligands, often mimicking natural protein-ligand interactions. A number of different types of affinity adsorbents are available, some are highly specific for particular proteins and others bind to protein types other than specific proteins.

In most cases, affinity chromatography relies on the presence of specific tags attached to the heterologous protein of interest by recombinant genetic techniques. When these tags are used for purification of heterologous products of interest, they need to be cleaved off. This cleavage is achieved by utilizing a highly specific protease that cleaves the amino acid backbone of the protein at a specific cleavage site, ie introduced during the cloning stage between the tag and the protein of interest. The need to efficiently separate proteases and cleavage products produced from each other increases the complexity, number of purification steps involved, and the processing cost of utilizing such site-specific proteolytic cleavage. The costs associated with utilizing such specific proteases often limit the industrial application of label-based affinity chromatography, thus limiting the availability of these efficient purification techniques. A more economical and efficient way of utilizing specific proteases enables the bioprocessing industry and often reduces the production costs of purified recombinant proteins.

In many cases, affinity chromatography implies the use of an immobilized ligand as an absorbent for the specific selection of proteins bound to the ligand. Binding of ligands to affinity sorbents involves the use of binding chemistries such as cyanogen bromide-, tosyl-, or vinylsulfone-activation of the sorbent. Ligands bound to the column matrix may or may not be of protein origin. Examples of the former are, but not limited to, immobilized protein A or protein G which have an affinity for gamma-globulin and are therefore useful for antibody purification, and lectins which have an affinity for glycoproteins. As an example of the latter, immobilized glutathione bound to a matrix binds a fusion protein comprising a glutathione S-transferase domain. Immobilized metal affinity chromatography (IMAC) is based on the immobilization of metal-chelating ligands to the matrix and relies on the interaction between metal ions immobilized on the column and basic groups (mainly histidine residues) on the protein. Formation of weak dative bonds. Commercially available cloning vectors offer the possibility to clone in frame a cDNA with a series of histidine residues - His - tag, which enables purification of the resulting fusion protein with IMAC. Although widely used for small-scale purification of proteins, IMAC is a non-specific but selective method because native histidine residues in contaminating proteins may cause binding in IMAC (Scopes 1993). A number of different types of tags or binding domains are useful in commercially available expression vectors to produce fusion proteins where the tag/binding domain binds the fusion protein to a ligand bound to the column matrix.

High specificity of protein binding can be achieved with these matrix-ligand systems. In the above cases, complex binding chemistry is involved to immobilize the ligand to the inert substrate. Therefore, the cost of affinity matrices often becomes a limitation for industrial-scale application of these effective techniques.

Furthermore, as with most other types of chromatographic methods, the stability of ligand binding to the matrix becomes a problem and leaching is a particular concern. During the purification of many sensitive biologically active proteins, heavy metal leaching in IMAC can lead to unacceptable and severe contamination and may inactivate the purified protein (Scopes 1993).

Often, the binding affinity of a protein for its ligand is so strong that elution conditions that disrupt ligand-protein binding require drastic conditions that partially denature the valuable protein being purified. A non-limiting example of these is the need for denaturation at low pH to release antibody from the column and elution of antibody from protein A-affinity matrices. Including a denaturation step in the protein purification process is undesirable due to the risk of loss of activity of the purified protein, adding an additional step for protein refolding and subsequent activity analysis required for the folded protein product, as well as increasing costs.

To enable large-scale purification from plants using affinity-based chromatography, it would be highly desirable to develop a simpler and more economical purification process than current commercial methods, which should have less binding chemistry and be of the same quality as pharmaceutical industry standards. The requirements are not contradictory.

Polysaccharides and polysaccharide-binding proteins can be used in combination for the design of affinity chromatography steps (see, eg, Boraston et al., 2001).

U.S. Patent 6,331,416 to Shani et al. describes a method for expressing a recombinant protein having a polysaccharide-binding domain that binds to cellulose in the cell wall of a host plant, and making use of this protein's lack of a poorly defined affinity for host plant cellulose A purification process that produces cell wall-protein complexes that can be separated from soluble contaminating proteins. The strength of the binding may be such that release of the protein from the cellulosic host plant material may require severe protein denaturing conditions that have a negative effect on the activity of the purified recombinant protein. The complexity involved is similar to the antibody-Protein A elution described above.

The carbohydrate binding domain cbm9-2 is from Thermotoga maritime xylanase 10A (Winterhalter et al., 1995: Mol. Microbiol, 15(3), 431-444). The CBM9-2 genomic DNA sequence is available from GenBank Accession No. Z46264 and it belongs to the IX family of CBM-s and has many attractive properties for high-resolution affinity purification, including the use of 1M glucose as the eluent. - Denaturing elution conditions, and highly specific affinity for amorphous and crystalline cellulose (Boraston et al., 2001: Biochemistry 40, 6240-6247).

Plant-based protein production shows significant promise for large-scale production of proteins in an economical manner, as has been shown by examples in the literature (for review see Hammond 1999).

The cultivation costs associated with plant molecular farming are significantly lower than with traditional bioreactor-based methods.

There is a widely recognized need for an efficient, simple and economical downstream processing method for the purification of heterologous proteins from transgenic plant material. Furthermore, this downstream processing needs to include mild, non-denaturing conditions for the protein of interest (in particular, a specific affinity purification method with mild elution conditions) to ensure the biological activity of the protein of interest and increase the yield. A protein purification process that is not limited above can significantly reduce the production costs associated with the production of biopharmaceuticals from plants and can be used to purify heterologous proteins whose downstream processing is limited and complex and expensive and valuable.

Summary and Purpose of the Invention

The main object of the present invention is to provide improved protein purification methods for the production of highly valuable heterologous proteins in plants, tissues obtained from plants, or plant cells, and to isolate heterologous proteins of interest from fused affinity tags such as, for example, CBM fused to the protein. Efficient and economical proteolysis method for proteins.

The present invention aims at reducing costs and improving the quality of downstream processing of heterologous proteins produced in plants.

An important feature of the purification process is the isolation of the protein of interest as a CBM-fusion protein from cell wall fragments and solids from other poorly defined plants, since the CBM-fusion protein of the invention does not bind to these components. This can be done alone prior to the affinity chromatography or simultaneously with the affinity chromatography step.

In a first aspect, the present invention provides a method for producing and purifying a soluble heterologous fusion protein comprising a cellulose binding module (CBM) from a transgenic plant or transgenic plant cell expressing said fusion protein, the method comprising:

Crushing of genetically modified plant material;

adding an extraction liquid to the plant material, thereby producing a mixture of soluble and insoluble plant material, such that a soluble fusion protein is extracted from said disrupted plant material into a liquid phase to obtain a protein extract;

isolating insoluble plant material including cell-wall material and solids from said protein extract comprising said fusion protein of interest;

contacting the protein extract with a polysaccharide matrix bound to the fusion protein;

washing the matrix with the bound fusion protein with one or more suitable aqueous solutions, such as one or more buffers, i.e. washing may be performed in one step using a gradient or series of different washes; and

The fusion protein is eluted from the polysaccharide matrix by adjusting conditions that affect the release of the fusion protein from the matrix.

Typically, the transgenic plant or plant cell is selected from dicotyledonous and monocotyledonous plants, and in preferred embodiments, the plant cell or transgenic plant is from tobacco, rapeseed, soybean, alfalfa, lettuce, barley, corn, wheat, Oats and rice.

In certain embodiments the separation step comprises a method selected from expanded bed adsorption (EBA), packed chromatography, precipitation, filtration, centrifugation, or any combination thereof.

The affinity binding step of binding the fusion protein to the polysaccharide matrix preferably comprises a chromatography step.

However, in certain useful embodiments, the separation of cell-wall fragments and other solids of poorly defined plant origin by the CBM fusion protein of interest, and the affinity binding of the CBM fusion protein to the polysaccharide matrix can be exploited Expanded bed adsorption chromatography (EBA) with a suitable, inexpensive polysaccharide matrix is performed in a single efficient purification step. These features revolutionize and improve the economics of downstream processing.

In an advantageous embodiment of the invention, the polysaccharide matrix comprises cellulose, and preferably pharmaceutically compatible cellulose cords. The well-defined pharmaceutical-grade cellulose to which this CBM-fusion protein incorporates may allow purification of heterologous proteins for various high-end applications. Useful pharmaceutically compatible cellulosic materials for use as polysaccharide matrices include Avicel ^(TM) (FMC Corporation, PA, USA).

The polysaccharide matrices used in the affinity chromatography of the present invention do not require complex binding chemistry or immobilization of potential leaching ligands. The polysaccharide matrix provides structural support and rigidity when constituting the affinity sorbent itself. Therefore, more economical and safe protein purification can be used for heterologous proteins obtained from plants.

A further advantage of the present invention is that the processing can be used for different polysaccharide matrices of different qualities depending on the different end uses of the purified heterologous protein, for example in agriculture, chemical industry or pharmaceutical industry. Affinity sorbents made of pharmaceutical grade polysaccharides are bulk materials within the pharmaceutical industry and are significantly less expensive than any commercially available affinity chromatography media. Thus, very high quality affinity matrices can be produced economically using the process described in the present invention, enabling more economical downstream processing of high value proteins from plant material.

A further advantage of the present invention is that once the plant-derived CBM-fusion protein is bound to a polysaccharide chromatography matrix and the matrix is washed to remove any contaminating endogenous plant proteins, non-denaturing, mild conditions, typically neutral or acidic, can be utilized. Conditions and preferably the addition of soluble carbohydrates (sugars) elute the fusion protein from the column under conditions that preserve the activity and structure of any fusion partner protein attached to the CBM. The sugar competes with cellulose for the binding site of the CBM and the appropriate concentration will release essentially all of the fusion protein bound to the CBM.

Preferred CBM-s for use in the methods of the invention and fused to a heterologous protein of interest are those that have the desired binding properties to allow sufficiently strong binding to a suitable polysaccharide matrix to obtain high yields of bound CBM fusion proteins, and that can be achieved by gentle binding as described above. Conditionally released CBM-s. CBM9-2 has been found to have these purpose properties. It is also within the scope of the invention to utilize other CBM-s having such properties. Such CBM-s can be discovered by searching available gene databases for sequences encoding CBMs with properties of interest, eg, motifs similar to those in CBM9-2 thought to be important for binding properties. Meanwhile, existing CBM-s can be modified by point mutation techniques well known in the art to modify their binding properties to obtain suitable CBM-s of the present invention.

After the fusion protein has been eluted from the polysaccharide affinity matrix, it may optionally be subjected to one or more further purification or isolation steps, depending on the desired form and the protein used.

In useful embodiments, the transgenic plant or plant cell includes a nucleic acid sequence encoding a CBM, preferably the CBM is thermostable and remains soluble at elevated temperatures. The term thermostable here means that the protein remains soluble, properly folded and active at elevated temperatures, ie temperatures above about 25°C, and usually above about 37°C, including temperatures in the range of 40°C to 100°C.

Genes encoding such preferred CBMs can be obtained from thermophilic organisms, including thermophilic bacteria, algae and fungi, and introduced into host plants or plant cells in such a manner as to express fusion proteins comprising said CBMs. The term thermophilic here refers to an organism whose optimal growth temperature exceeds 40°C. Preferably the CBM is encoded by the xylanase 10A gene from Thermotoga maritima, preferably the host plant or the region within the plant cell encoding the CBM is a region of said gene. The region encoding the CBM may comprise the sequence SEQ ID NO: 1 in a specific embodiment, or a nucleic acid sequence encoding the same amino acid sequence, or a sequence encoding an amino acid sequence having substantial sequence identity with the amino acid sequence.

Certain embodiments of the present invention require heating the protein extract comprising the soluble fusion protein to a temperature in the range of 37°C and 100°C, for example, a temperature in the range of 50-80°C, such as from 1 minute to 120 minutes during processing. within a period of time. Thermostable CBMs such as those from thermophilic sources are especially useful for this purpose. During such heating, a part of the endogenous vegetable protein may be inactivated and/or denatured and may thus be easily separated from the protein extract. The heated extraction may preferably be subjected to a processing step comprising simultaneous separation of solids and affinity binding of the CBM fusion protein from the heated extract by expanded bed adsorption with a polysaccharide matrix.

In particular embodiments of the invention, said heterologous fusion protein comprises a protease (polypeptide expressing proteolytic activity), which in a particularly useful embodiment is mammalian enterokinase (EK) or an enterokinase-active portion thereof, For example, bovine EK or bovine EK catalytic domain (EKc). A useful EKc for this purpose is encoded by the nucleic acid sequence shown in SEQ ID NO:2.

In a highly useful embodiment of the invention, the fusion protein comprises a CBM and a heterologous protein of interest truncated by an amino acid overhang comprising a proteolytic cleavage site, preferably a proteolytic cleavage site recognized and cleaved by a specific protease. peptide. Because of this site, the CBM can be easily cleaved away from the fusion partner in the fusion protein to obtain the target heterologous protein without CBM.

Accordingly, in a related aspect, the invention provides a method of purifying a heterologous protein of interest comprising

(a) providing a fusion protein comprising a CBM as defined above fused to a CBM truncated by a proteolytic cleavage site,

(b) contacting the fusion protein with a functional protease fused to the CBM under conditions that promote proteolytic cleavage by the protease to cleave the fused CBM from the heterologous protein of interest,

(c) CBM-protease, free CBM, as defined herein, are bound to the polysaccharide matrix under conditions in which the CBM-protease and free CBM are bound to the polysaccharide matrix and wherein the heterologous protein of interest is not retained on the polysaccharide matrix. and the solution of the heterologous protein of interest is contacted with the polysaccharide matrix,

(d) separating un-bound heterologous protein of interest from the polysaccharide matrix,

(e) washing the polysaccharide matrix with bound CBM-protease and CBM with one or more suitable aqueous solutions,

(f) eluting the CBM-protease from the matrix by adjusting conditions affecting the release of said CBM-protease from the matrix; and

(g) optionally reconstituting the eluted CBM-protease to maintain its affinity for the polysaccharide substrate so that the reconstituted CBM-protease can be reused for subsequent repetitions of the method.

The steps of binding the fusion protein, washing and elution are preferably performed as described above.

In a preferred embodiment, the protease fused to the CBM is enterokinase, preferably as described above. Such CBM-fusion proteases, EK or other types, can be suitably produced and purified by methods described herein.

It will be appreciated that the methods described above may be advantageously combined with the methods described above to prepare and purify soluble heterologous fusion proteins, thereby obtaining a heterologous protein of interest originally expressed and isolated as a CBM fusion protein in a purified form free of fusion CBM.

A further advantage of the method is the possibility to reconstitute the protease used, ie to utilize the same protease repeatedly used in the mass production of the protein of interest expressed as a CBM fusion protein, making the method described here more economical. These are easily achieved because the CBM-fusion protease is designed without a proteolytic cleavage site such that cleavage of the CBM by the CBM protease itself is blocked. Furthermore, as mentioned above, this recovery is made possible by the mild conditions used for elution, thereby not compromising the activity of the eluted protease.

Thus, a further advantage of the present invention is that it provides a method capable of recovering expensive, specific proteolytic enzymes, thereby additionally reducing heterologous proteins such as, but not limited to, those obtained from genetically modified plants, plant material and plant Cost of affinity purification in downstream processing of cellular proteins.

The present invention successfully overcomes the disadvantages of downstream processing associated with large-scale heterologous protein production for applications such as, but not limited to, agriculture, chemical industry, and protein-based pharmaceutical production. In particular, new methods are provided for isolating CBM-fusion proteins from biological materials such as plant cellulosic materials with fewer processing steps utilizing the principles of affinity chromatography which are safe and more economical for the pharmaceutical industry , mild elution conditions that maintain the activity of high-value heterologous proteins, and include processing steps that enable recovery of specific high-value proteases in the method.

Description of drawings

Figure 1 is a schematic diagram of a preferred embodiment of the CBM protease purification process and proteolytic cleavage of CBM fusion proteins.

FIG. 2 shows the analysis results of SDS-PAGE obtained in Example 1. FIG. Lane 1: Size markers, Lane 2: Purified CBM9-2, Lane 3: Washed solids from milled barley seeds, Lane 4: Supernatant after biomaterial interaction, Lane 5: 1st wash with buffer, lane 6: 5th wash with high salt buffer.

Figure 3 shows the successful purification of CBM9-2 from milled barley seed extract according to the method of the present invention, as detailed in Example 2. The figure shows the elution profile of an expanded bed absorption (EBA) column with 200 mL of cellulose.

Figure 4 shows the results obtained from Example 3. The graph shows ELISA reads for a control sample and minimal reads for a sample comprising a heterologous protein of interest fused to CBM9-2 and purified on a small scale according to the purification procedure described here. The column shows (a) elution buffer, (b) fusion protein extracts from transgenic seeds, and (c) ELISA values determined for proteins extracted from non-transgenic seeds.

Detailed description of the invention

The present invention will be specifically described below.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood and used by one of ordinary skill in the art to which this invention belongs.

The term "polypeptide" herein refers to any polymer of amino acids, monomeric or polymeric, and does not refer to polymers of amino acids of a particular length. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. The term also includes polypeptides with post-expression modifications such as, for example, glycosylation, acetylation, phosphorylation, and the like.

The term "heterologous polypeptide of interest" or "polypeptide of interest" herein refers to any polypeptide for expression in a plant-cell or plant tissue using the method or composition of the invention. As non-limiting examples, pharmaceutical polypeptides (eg, for pharmaceutical use) or industrial polypeptides (eg, enzymes) may be prepared according to the invention.

The term "downstream processing" refers to the isolation and purification of biotechnological products into a form suitable for their intended use.

The term "fusion partner" refers here to the heterologous protein linked to the CBM.

The term "CBM fusion protein" refers to a molecule comprising a CBM linked to a heterologous protein, and in this context it is understood as a molecule without a proteolytic cleavage site, unless stated otherwise.

The term "operably linked" refers to a functional linkage between a promoter (a nucleic acid expression control sequence, or a series of transcription factor binding sites) and a second nucleic acid sequence, wherein the promoter directs the transcription of the nucleic acid corresponding to the second sequence.

The term "denatured" refers to a state in which a protein's native structure and subsequent protein activity is disrupted, and the protein is unfolded or improperly folded to alter its native three-dimensional structure.

The terms "expression" and "production" refer to the biosynthesis of a gene product, including the transcription and translation of said gene product.

"Molecular farming" refers to the manipulation of plants of any kind using the open field or in closed facilities to express and produce heterologous proteins in their tissues.

The term "transgenic" refers to any cell, cell line, tissue plant part, organ or organism and its progeny in which a non-native nucleic acid is present, into which a non-native nucleic acid sequence has been introduced, thereby altering its genotype. Typically, the non-native nucleic acid sequence is introduced into the genotype by genetic engineering methods or by such methods into the genotype of the parent cell or plant and subsequently passed into the progeny by sexual crossing or vegetative propagation.

Transient expression refers to the expression of a gene in an organism through transient introduction without insertion into the genome of the organism. Thus, transient expression is often limited in time, and the transient gene is not passed on to the next generation.

"Substantial sequence identity" in this context means at least 50% sequence identity, and more preferably at least 60% such as at least 70 sequence identity, such as at least 80% and preferably at least 90% sequence identity, such as at least 95% or 99% sequence identity. Algorithms for sequence analysis are known in the art, such as BLAST, described by Altschul et al. in J. Mol. Biol. (1990) 215:403-10. Typically, relative to, for example, default value settings for "Score Matrix" and "Gap Penalty" will be used for the alignment.

The term "transformation" or "transformed" refers to the introduction of a nucleic acid sequence into the DNA genome of a host organism, regardless of the technique used to introduce the nucleic acid fragment into the host cell.

"Thermophilic" means that the optimum growth temperature of the organism exceeds 45°C.

The term "GMP" (Good Manufacturing Practice) refers to the manner in which biopharmaceuticals and other pharmaceuticals and medical devices are produced. GMP requirements include standard operating procedures, aseptic conditions, substances and equipment, and qualifications by professionals.

Both monocot and dicot plants that can be genetically manipulated can be used in the present invention. Preferably the plant is a monocot, more preferably barley, and most preferably barley (Hordeum vulgaris). A genetically transformable plant is a plant that introduces, expresses, stably maintains, and transmits to subsequently produced progeny non-native DNA sequences, including those of the coding region. Genetic manipulation and transformation methods have been used to generate barley plants using herbicides including eg herbicides or basta or antibiotics such as hygromycin as selectable markers.

method

Other objects, advantages, and novel features of the present invention will be apparent to those skilled in the art from the following examples, which are not intended to be limiting. In addition, the present invention as described above in each of the various embodiments and aspects and as claimed in the claims section finds experimental support in the following examples.

Although only the preferred embodiment of the present invention has been specifically described, those skilled in the art can expect numerous modifications and variations of the above-described examples of the present invention without departing from the spirit and scope of the present invention.

Figure 1 illustrates a schematic of the purification process of a CBM-protease, or for that matter, any CBM-fusion protein, the method is described as follows:

(1) The starting material for this method is a transgenic plant, material obtained from a transgenic plant or a transgenic plant cell, including but not limited to, a suspension culture of plant cells and undifferentiated cells of a callus. In various embodiments of the invention, the material may be a source of plant tissue transiently expressing a heterologous protein. The material is a transgenic material that expresses a heterologous gene operably linked to the CBM open reading frame in a regulated manner, which has been introduced into plant cells by methods known to those skilled in the art, such as, but not limited to, Agrobacterium -mediated transformation or particle bombardment-mediated transformation or plant viral vector-mediated transformation. The starting material is preferably selected based on a satisfactory expression level of the fusion protein, as judged by analysis of RNA or protein levels by those skilled in the art before initiating the method of the present invention.

(2) Breaking up the transgenic material by methods known to those skilled in the art, which produces homogenized plant tissues and plant cells in a dry or wet state. A variety of methods suitable for the source of plant material can be selected. Thus, for seeds, milling is a good method of disrupting transgenic plant tissues, while for leaves and soft green tissues equipment such as, but not limited to, Waring mixers, Sorvall Omnimixer or Poltron homogenizers can be used. accomplish. Leaves and softer tissues can also be freeze-dried and subsequently used for homogenization and milling. Equipment for disrupting plant tissue and plant cells is commercially available and easily scaled up as needed. General methods for the extraction of proteins from plant sources are described in G. Paul Powell et al., Protein purification applications, 2nd edition, edited by S. Roe, (2001). A simple and successful method of extracting soluble proteins from plant sources is to add a simple buffer such as a low-salt buffer to crushed, homogenized plant tissue and mix well. For antioxidant brown blooms, additions such as polyvinylpyrrolidine (1% w/v) are usually sufficient to optimize purification from most plant tissues to sequester phenolic resins from plant tissues that could otherwise have adverse effects on the heterologous protein to be purified. Proteolysis does not always cause the problems that plant sources have. If, however, proteolysis is a concern, protease inhibitors such as, for example, serine-, cysteine- and metalloprotease inhibitors can be added to the extraction buffer. Disintegration of plant material can be performed in the presence or absence of a buffer solution. The extraction solution may or may not contain a reducing agent such as, but not limited to, 2-mercaptoethanol or dithiothreitol (DTT). Soluble vegetable protein and CBM-fusion protein are present together in the liquid phase

(3) The mixing of the liquid and the plant material is necessary for the extraction of the water-soluble fusion protein in the liquid phase. The added liquid may or may not contain a buffer to adjust the pH preferably in the range of about 5.2 to 8.3, it may or may not contain any reducing or chelating agent for the protein of interest described in (2). In its simple form, the liquid can be water. After the liquid has been thoroughly mixed with the broken and homogenized plant material, the liquid phase now contains the CBM-fusion protein.

(4)(A) Depending somewhat on the level of homogenization, the mixture of crushed plant material and extraction liquid can be applied directly to an expanded bed adsorption (EBA) column. In this method, the mixture is applied to a column as a liquid stream flowing through an expanded bed of an affinity adsorption matrix of polysaccharide character. During the passage of the liquid stream through the column, the fusion protein in the liquid phase is exposed to the polysaccharide matrix and is selectively adsorbed by the selective affinity of the CBM to the polysaccharide absorbent medium. Particles such as, but not limited to, cell wall fragments and other solids, pass through the EBA column in a flowing liquid, along with any soluble vegetable proteins.

4(B) Optionally, the majority of solid particles in the mixture can be separated from the liquid prior to the affinity binding step by various methods known to those skilled in the art, including, but not limited to, sedimentation, filtration, centrifugation and settlement. The solids were discarded and the liquid containing the CBM-fusion protein was either used for the optional heating step 4(C) or applied directly to the affinity binding step (5) as described above.

4(C) Optional heating of the mixture or liquid prior to the affinity step (5), but can be used as an additional purification step in cases where the CBM-fusion protein remains soluble at increased temperature, while soluble vegetable proteins may denature and precipitated and monocotyledonous proteases may be inactivated by heating. For this purpose, the heating method considering the characteristics of the CBM-fusion protein may include heating in the range of 50°C to 100°C for a period of time ranging from 2 minutes to 60 minutes during processing. In these embodiments, CBM of thermophilic origin are especially beneficial.

(5) Substrate binding affinity. A liquid protein extract comprising a plant-derived CBM-fusion protein is contacted with a polysaccharide matrix that has an affinity for the CBM. Contacting can be achieved in a variety of ways, such as, but not limited to, a chromatography column packed with a polysaccharide matrix, where liquid flows through the column in a packed or expanded manner, which refers to the density of the polysaccharide matrix, or it can be achieved in a batch fashion, where The polysaccharide matrix is mixed with liquid in a suitable container, and the matrix and the adsorbed CBM-fusion protein are subsequently recovered. The polysaccharide matrix may be of cellulose origin, such as, but not limited to, Avicel, or it may be of xylanoic origin, such as insoluble xylan. The binding specificity and thermodynamics of CBM9-2 have been specifically studied in a recent publication by Boraston et al. (2001). However, the inventors have surprisingly found that, in contrast to the prior art, CBM9-2 does not bind to plant cell wall components but is readily soluble in accordance with the methods described herein in accordance with the present invention, while remaining resistant to, for example, affinity adsorption Good binding specificity of the polysaccharide matrix used in step (5). As described here, this surprising property introduces several advantages to the downstream processing of plant-derived CBM-fusion proteins, thus providing a method for greatly improved downstream processing.

(6) Rinse the matrix. The polysaccharide affinity adsorbent bound to the CBM-fusion protein can be used with several column volumes (relative volume terms if the affinity matrix is placed in a chromatography column) of aqueous solution (e.g. water) or buffer, such as, but not Washing is limited to phosphate-buffered saline or Tris-based buffers. To increase the efficiency of the wash step, the composition of the wash buffer can be adjusted by means such as, but not limited to, stepwise changes in the volume of each column or gradients in salt concentration or detergent for the release of weak but Contaminating proteins that bind nonspecifically.

(7) The fusion protein is released from the matrix. By utilizing CBM-s with the binding properties of interest described here, elution of CBM-fusion proteins from affinity matrices can be achieved by exposing the CBM-fusion proteins to competing sugars such as, but not limited to, glucose, galactose, lactose, maltose and cellobiose were achieved. Any of these or other similar sugars or combinations thereof may be added to an elution buffer such as, for example, phosphate-buffered saline or a Tris-based buffer in a suitable amount, such as in a concentration range of 1 mM to 1 M, for elution step. The sugar concentration may be, for example, in the range of 25 mM to 1M, such as in the range of 50-500 mM. These sugars are commercially available as low priced bulk chemicals, further reducing the overall cost of the downstream processing of the present invention.

(8) Separation/purification of fusion protein. Further purification/isolation of the CBM-protease is beneficial or required in some instances. This further separation can be achieved by any chromatographic method generally known to those skilled in the art, such as, but not limited to, ion exchange chromatography or size exclusion chromatography.

(9) Final product. The final product in this case is the CBM-protease in highly purified form, ready for formulation and packaging in its final form if necessary.

(10) Isolation of fusion proteins comprising proteolytic cleavage sites. The method of purifying CBM-protease described in (1-9) may be the preferred method for purifying any heterologous protein linked to CBM. Such CBM-fusion proteins may or may not contain a proteolytic cleavage site between the CBM and the heterologous protein of interest. In the preferred embodiments of the present invention described above, the purification method can be used in conjunction with the CBM-protease to cleave the CBM-fusion protein and efficiently isolate the heterologous protein of interest from the CBM-protease and the cleaved CBM. Further isolation of the CBM-fusion protein containing the proteolytic cleavage site prior to the proteolytic cleavage step (11) may or may not be required, or may be beneficial in some cases. Isolation of these fusion proteins may be useful to modify the buffer containing the CBM-fusion protein with a proteolytic cleavage site such that it is adjusted to conditions that favor the proteolytic cleavage reaction of step (11). These separations can be achieved by any chromatographic method generally known to those skilled in the art, such as, but not limited to, ion exchange chromatography or size exclusion chromatography.

(11) Proteolytic isolation of CBM from heterologous protein fusion partners. At this stage of the method, the CBM-fusion protein containing the proteolytic cleavage site is exposed to a specific protease fused to the CBM but without the cleavage site, which is able to recognize the cleavage site and separate the CBM from the heterologous protein of interest. The specific protease linked to the CBM is preferably selected from the group of cleavage site-specific proteases, such as enterokinase, factor Xa, thrombin and the like. After the cleavage reaction is completed, the reaction solution includes released CBM, residual uncleaved CBM-fusion protein, CBM-protease (eg, CBM-EK), and released target heterologous protein.

(12) Binding affinity of CBM-protease and free CBM to matrix. A solution containing all of these components is contacted with a polysaccharide matrix having an affinity for the CBM as described above in step (5). All CBM-containing fractions release the highly purified heterologous protein of interest by adsorption to the affinity matrix and recover it in the liquid stream.

(13) Further separation of heterologous proteins. Although the heterologous protein of interest has been highly purified, in some cases further purification/isolation may be required or beneficial. These further separations may be chromatographic steps such as, for example, ion exchange chromatography or size exclusion chromatography.

(14) Final product. The final product is the CBM heterologous protein in highly purified form, ready for formulation (eg, lyophilization) and packaging in its final form, if necessary.

(15) Rinse the substrate This step can be carried out as described in step (6).

(16) CBM-protease and release of CBM. Using the elution conditions described in step (7) above, the CBM-protease was recovered from the solution. Reconstitution of these solutions includes neutralization of conditions and/or removal of agents (eg, sugars) that affect the release of CBM from the polysaccharide affinity matrix. The reagent can be removed from the solution with the CBM protease, for example by ultrafiltration, dialysis, size exclusion chromatography and under certain conditions ion exchange chromatography. Certain of these methods, such as size exclusion chromatography, can also purify CBM-proteases from residual CBM and CBM-fusion proteins, if desired.

(17) Recovery of CBM-protease. Once the CBM-protease has reverted back to a state where it has regained affinity for the polysaccharide matrix, it can be reintroduced into the process as in step (11) for a new CBM-fusion protein containing a proteolytic cleavage site. Rounds of proteolytic cleavage. Although the reconstituted CBM-protease solution may contain residual CBM from the previous reaction and traces of uncleaved CBM-fusion protein, they will not interfere with the purification of the cleaved heterologous protein fusion partners as they will be together with the CBM-protease Co-purification. Thus, efficient recovery of high-value specific CBM-proteases increases the efficiency and economics of the novel method of the present invention and enables large-scale production and purification of recombinantly produced proteins, thereby limiting previously prohibitive costs of downstream processing.

Other objects, advantages, and novel features of the present invention will be apparent to those skilled in the art from the following examples, which are not intended to be limiting. In addition, the present invention as described above in each of the various embodiments and aspects and as claimed in the claims section finds experimental support in the following examples.

Example

Example 1 Biomaterial Interaction Study: CBM9-2 and Milled Barley Seeds

The dried barley seeds are finely ground to a fine powder in a Retsch mill. 1 g of milled seeds was used to extract water-soluble components from the seeds by 5 ml of low-salt buffer (50 mM potassium phosphate buffer at pH 7.02) as a method of removing all water-soluble components of the sample, which Components may interfere with studies of biomaterial interactions. The mixture was vortexed and inverted for 5 minutes to ensure thorough mixing of the liquid and milled barley seed material.

After mixing, the samples were centrifuged at 5000 xg for 4 minutes to pellet the solids. After centrifugation, discard the supernatant. This process was repeated 3 times with low-salt buffer, discarding the supernatant after each centrifugation. The washing process was then repeated 3 times with high-salt buffer (50 mM potassium phosphate buffer pH 7.02, 1 M NaCl), and the supernatant was discarded as previously described. The resulting wash solids are mostly plant cell-wall fragments and insoluble starch. The washed solids were equilibrated with 3 washes with low salt buffer as described above to obtain the same conditions that favor the affinity binding of CBM9-2 to the cellulose matrix in affinity chromatography. A representative sample of solids prior to biomaterial interaction studies was used for SDS-PAGE analysis (lane 3). 10 μl of bacterially produced CBM9-2 purified by a cellulose (Avicel ^™ )-affinity column (OD280nm 0,394) was used for subsequent SDS-PAGE (lane 2). Purified CBM9-2 was pre-diluted repeatedly (4x) and concentrated in ultrafiltration modules as a proven method to desorb any bound glucose from CBM9-2 in order to restore the cellulose binding affinity properties of the protein.

2 ml of purified CBM9-2 was added to the rinsed, equilibrated solids obtained from milled barley seeds and the mixture was incubated for 60 minutes at room temperature with shaking. After incubation, the mixture was spun at 5000xg for 10 minutes, and the supernatant was then clarified by centrifugation at 13.000xg for 5 minutes. 10 μL of clarified supernatant from the biomaterial interaction was used for SDS-PAGE analysis (lane 4). The pellet comprising milled barley seed solids was then washed 5 times with low salt buffer and subsequently 5 times with high salt buffer as described above. 10 μL of the first low-salt buffer wash (lane 5) and the fifth low-salt buffer wash (lane 6) were used for subsequent SDS-PAGE analysis. To elute all bound CBM9-2 from milled barley seed solids, 1 ml of elution buffer (1 M glucose in ₅₀ mM KPO , pH 7.02) was added to the solids and incubated with mixing for 15 minutes. A 10 μl sample of the eluate was prepared for SDS-PAGE analysis (lane 7). A representative sample of solids after biomaterial interaction studies and elution was used for SDS-PAGE analysis (lane 8). Samples prepared for biomaterial interaction assays by SDS-PAGE were subjected to SDS-PAGE on 12.5% SDS-PAGE gels (PhastGels homogeneous 12,5) using the PhastSystem (AmershamPharmacia Biotech). After electrophoresis, the gel was stained and destained with Coomassie blue R-250. The results are shown in Figure 2.

These results show that CBM9-2 does not bind significantly to plant-derived cell-walls or other insoluble solids from milled barley seeds.

Example 2

Purification of CBM9-2 from extracts of milled barley seeds

Barley seeds were ground to a fine powder using a commercially available grinder (Aarslev Maskinfabrik, Erhvervsvangen 11, 5792 Aarslev, DK). The resulting barley flour was wetted in a low salt buffer (50 mM potassium phosphate buffer pH 7.02) at a volume ratio of barley flour:buffer of 2:3, respectively. The liquid and powder were thoroughly mixed in a container and allowed to settle overnight at 4°C. CBM9-2 purified from bacteria was added to the barley seed-supernatant. The next day, the supernatant (100 ml) containing the CBM9-2 peak was applied to a Streamline 25 (Amersham Biotech) chromatography column containing cellulose (Avicel ^™ ). With the flow velocity of 184cm/h, feed in the mode of expanded bed, then use 5 column volumes of high-salt buffer solution (50mM KPO ₄ solution of 1M NaCl, pH 7.02), then 5 column volumes of low-salt buffer solution (50mM KPO ₄ , pH 7.02) Perform flushing. The expanded bed was allowed to settle (sediment bed = 20 cm) and eluted with 300 ml of elution buffer (1M glucose in 50 mM KPO4, pH 7.02) at 92 cm/h. The elution conditions produced a small peak containing CBM9-2 protein (see Figure 3).

This suggests that using the methods described above; firstly, CBM9-2 remains in solution to desorb cell-wall fragments and other poorly defined solids from milled barley seeds; secondly; and chromatographic capture of CBM9-2 from milled barley seed extracts is possible, third; can be performed by using a well-defined pharmaceutical grade cellulose (Avicel) as a matrix, and fourth; can be performed by the present invention The step of affinity chromatography is carried out in the expanded bed mode described above, and the fifth step; CBM9-2 purified from the barley seed-extract can be eluted from the matrix under the mild conditions described in the present invention to avoid any denaturation step. Exactly the same conditions and methods as described above can be used to purify CBM9-2-fusion proteins from transgenic milled seeds. Suitable methods are described in the co-pending "Non-Denaturing Method for Purification of Recombinant Proteins from Plants" filed concurrently with the applicant and incorporated herein by reference in its entirety.

Said polysaccharide affinity chromatography is also effective for separating protease-CBM and cleaved CBM from the protein of interest after the proteolytic cleavage reaction as described in the present invention.

Example 3

Purification of heterologous proteins linked to CBM9-2 from milled, single barley seed extract.

In a small scale version of the purification method described above, a single seed of a transgenic barley plant expressing a heterologous protein of interest linked to CBM9-2 was homogenized separately to a fine powder. The resulting seed powder was wetted in low-salt extraction buffer (50 mM potassium phosphate buffer pH 7.02, 0,01% polyvinylpyrrolidine) at a volume ratio of barley powder:buffer 1:7, respectively. The liquid was thoroughly mixed with the single seed powder for 1 hour, and the water soluble protein was extracted into the liquid phase with shaking at 5 minute intervals at room temperature.

Spin the extract mixture in a centrifuge at 6000 rpm for 10 minutes. Supernatants (extracts) from individual seeds were collected and applied to cellulose (Avicel ^™ ) filled microwell filter plates (MSHVN45, Millipore ^™ ). The extract was added to the corresponding microporous cellulose and stirred at room temperature for 15 minutes at intervals of 3 minutes. After 15 minutes, a vacuum will be applied to the microplate through a vacuum manifold (Multiscreen resist vacuum manifold - MAVM0960R, Millipore ^™ ) to drain liquid from the cellulose (flow-through) in each well. The cellulose was subjected to a washing step as described above: 5 column (i.e., microwells filled with cellulose) volumes of high-salt buffer (1M NaCl in 50 mM _KPO4 , pH 7.02), then 5 column volumes of low-salt buffer (50 mM _KPO4 , pH 7.02). Elution was performed with 250 μl of elution buffer (1M glucose in 50 mM KPO ₄ , pH 7.02). Collect the eluate by applying vacuum to the wells of a fresh microplate. The eluate was used for a highly specific ELISA CBM9-2 assay, ie based on a specific polyclonal antibody against CBM9-2. ELISA was performed as follows; 50 μL of an appropriately diluted antigen solution (0.1-5 ng protein) was applied to each well of a Nunc-Immuno 96 microwell plate (Cat. no. 442404). Microwells were covered with scotch tape and incubated at 37°C for 1 hour. The antigen solution was pipetted out of the microwells and washed 3 times with 100 μl TBS. The microwells were blocked with 100 μl per well of blocking solution, covered with scotch tape and incubated at 37°C for 1 hour. After discarding the blocking solution, rinse the microwells 5 times with 100 μl TBS containing 0.01% Tween. Primary antibodies against CBM9-2 were added appropriately diluted (eg 1/3000) in 1% BSA in TBS (50 μL per well) and covered with scotch tape and further incubated at 37° C. for 1 hour. The antibody solution was removed and the wells were washed 10 times with TBS containing 0.01% Tween. Secondary antibody (horseradish peroxidase conjugated) was diluted 1/3000 in 1% BSA in TBS (50 μl per well). Microwells were covered with scotch tape and incubated at 37°C for 1 hour. After discarding the solution and washing 8x with 100 μl TBS with 0.01% Tween and 2x with TBS without Tween, add 100 μl TMB One solution (Promega ^™ ) to each well and incubate the microplate at room temperature for 30 seconds-5 minutes . When the blue color develops, add 100 μl of 0.2 M sulfuric acid. The color turns yellow and the absorbance is measured at 450 nm in a microplate reader.

The results of the ELISA analysis of single seed extracts from individual transgenic barley seeds are shown in FIG. 4 . Previous ELISA assays used to demonstrate CBM9-2 specificity demonstrated that, firstly; the accumulation of heterologous fusion proteins in individual seeds was available and demonstrated; the work described above for isolated heterologous fusion proteins with cleavage sites, even The scale capability of the purification process is still supported at small scale; it shows that the heterologous fusion protein is not exposed to denaturation during the chromatographic method and does not involve any refolding steps due to recognition by specific antibodies after elution, highlighting how Advantages of the present invention over some other harsh affinity chromatography methods discussed above; the purification method described above is applicable to any heterologous fusion protein linked to CBM9-2 and is not limited to capturing CBM-proteases.

Example 4

Purification and activity assay of CBM-protease

To generate a site-specific protease with a linked CBM9-2 tag, proceed as follows:

Agrobacterium tumefaciens strain AGLO was constructed to contain an expression construct consisting of a constitutive promoter preceding the enterokinase cDNA, a signal sequence targeting the endoplasmic reticulum (ER), and a retention signal to maintain the protein in the ER Binary plasmid in which the enterokinase cDNA is ligated with the cDNA corresponding to CBM9-2. This Agrobacterium strain was cultured in YEB medium under selective conditions, first in 10 ml at 28° C. for 2 days until OD ₆₀₀ was 0.8. A small amount of culture was diluted 1 :50 to 500 ml culture containing 20 μΜ acetosyringone and shaken vigorously to an _OD600nm of 2.5 for 2-3 days at 28°C. The bacteria were spun at 6000 rpm for 10 minutes and resuspended in MS-solution (containing 55 g/l sucrose) to an _OD600 of 2.5. Acetosyringone (10 mM) was added to a final concentration of 20 uM. The bacterial suspension was maintained at room temperature for 1 hour and Tween-20 (10%) was added to a final concentration of 0.005%.

For transient expression of CBM-protease in lettuce plants, the lettuce plants were submerged for 15 seconds in a bowl containing the Agrobacterium bacterial suspension. The plants were then placed under vacuum and a pressure of 0.4 atmospheres was applied for 20 minutes, opening the air inlet to quickly compensate for the pressure. Wash excess bacteria off leaf surfaces by successive dips into a bowl of tap water. The lettuce plants were grown in an incubator at 22°C for 4 days at 16 hours day/8 hours night.

Plants were harvested by excising leaf tissue and subsequently frozen and kept at -86°C. The plants were homogenized using a mortar and liquid nitrogen until a very fine powder was obtained. Powdered lettuce leaf material was extracted by adding 1.2:1 (vol:vol) low-salt extraction buffer and lettuce powder, respectively, and the protein extract was stirred intermittently for 30 minutes at room temperature.

The extract was then centrifuged at 6000 rpm for 20 minutes to separate solid matter and cell wall fragments from the liquid phase. Discard the supernatant and spin again as above. The clarified supernatant was packed onto a packed bed column containing a cellulose matrix (Avicel ^™ ) as described above. The CBM-protease is specifically bound to the cellulose matrix and washed separately with 5 column volumes of high-salt and low-salt wash buffers, and the CBM-protease is eluted from the column with a single peak of 1M glucose solution under mild, non-denaturing conditions .

The peak product was then concentrated using a microwell concentrator (Ultrafree-15-Biomax-5).

Enterokinase activity was determined according to standard methods (Grant & Hermon-Taylor, 1979) using a specific synthetic substrate: Synthetic substrate: Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide (GD ₄ K-na ); the measurement condition is 37°C. The reaction volume was 1.5 ml. The reaction mixture included: 25 μl of 10 mM GD ₄ K-na (0.5 mM), 125 μl of 100 mM Tris-HCl, pH 8.4 (25 mM), 50 μl of 100% DMSO (10%), 50 μl of 100 mM CaCl ₂ (10 mM), (20-100) [mu]l enterokinase, 250 [mu]l distilled water - (20-100) [mu]l. The rate of β-anthylamine formation was determined by the increase in fluorescence between λ _ex =337 nm and λ _em =420 nm. Monitor continuously for 5 minutes.

The results of the activity assay showed that the produced and purified CBM-enterokinase was active; the enterokinase activity was determined to be 442,7 cps/min/μg compared to the blank 0,0001 cps/min/μg.

This example shows firstly that the CMB-protease can be produced in plants, in this case transiently in lettuce, and that the CBM-protease can be successfully isolated and purified using the purification methods described above. It further demonstrates that the CBM9-2 affinity tag of the fusion protein is fully functional; the CBM-protease is efficiently attached to the cellulose matrix and it can be eluted from the matrix under the mild elution conditions described in the present invention, and elutes The protease was shown to be fully active. The purified product of enzymatic activity itself provides evidence of the non-denaturing nature of the purification process, as enzyme activities are particularly sensitive to partial or complete denaturation, which easily leads to loss of activity. This effectively demonstrates that all components of the invention are functional and that their properties and properties allow them to be readily applied in the methods of the invention described above, resulting in improved primary specificity, economics of downstream processing of heterologous proteins of any origin method of sex and efficiency.

references

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

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

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

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

"Plant bioechnology; new products and applications" by Hammond (1999) Eds. Hammond, McGarvey & Yusibov, Springer Verlag, NY1999.

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

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

Paul G. Protein purificatioh applications by Paul Powell G., Ed. by S. Roe, Second Edition (2001).

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

Shani et al., U.S. Patent 6,331,416.

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

sequence listing

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Claims (17)

1. A method for purifying a heterologous protein of interest, including (a) providing a fusion protein comprising said heterologous protein fused to a CBM truncated by a proteolytic cleavage site, (b) contacting the fusion protein with a functional protease fused to the CBM under conditions that promote proteolytic cleavage of the protease to cleave the CBM from the heterologous protein of interest, (c) CBM-protease, free CBM are bound to the polysaccharide matrix comprising cellulose under the conditions wherein the heterologous protein of interest is not retained on the polysaccharide matrix comprising cellulose, and the CBM-protease, free CBM contacting a solution of a heterologous protein of interest with the polysaccharide matrix comprising cellulose, (d) separating un-bound heterologous protein of interest from a polysaccharide matrix comprising cellulose, (e) washing the polysaccharide matrix with bound CBM-protease and CBM with one or more suitable aqueous solutions, (f) eluting the CBM-protease from the matrix by adjusting conditions affecting the release of said CBM-protease from the matrix; and (g) optionally recovering said eluted CBM-protease to maintain its affinity to said polysaccharide matrix comprising cellulose, so that the recovered CBM-protease can be reused for subsequent repetitions of steps from (a) -(g) the method of qualification, Where said CBMs are capable of reversibly binding to a polysaccharide matrix comprising cellulose and being released from said matrix by non-denaturing elution conditions, said CBM is also substantially unbound to insoluble cell wall plant material. 2. The method of claim 1, wherein the protease fused to the CBM is from enterokinase, tobacco etch virus protease, factor X and thrombin. 3. The method of claim 2, wherein the protease is mammalian enterokinase or an enterokinase active portion thereof. 4. The method of claim 3, wherein said EK comprises the catalytic domain of bovine EK, wherein the catalytic domain of said bovine EK is encoded by the nucleotide sequence shown in SEQ ID NO:2. 5. The method of claim 1, wherein the protease fused to the CBM and the heterologous protein fused to the CBM truncated by a proteolytic cleavage site are separately produced and purified by producing and purifying a soluble heterologous fusion comprising a cellulose-binding module The method for protein is obtained by transgenic plant or transgenic plant cell expressing said fusion protein, comprising steps: (a) crushing genetically modified plant material; (b) adding an extraction liquid to the plant material, thereby producing a mixture of soluble and insoluble plant material, so that the soluble fusion protein is extracted from said crushed plant material into a liquid phase to obtain a protein extract; (c) separating insoluble plant material, including cell-wall material and solids, from said protein extract comprising said fusion protein of interest; (d) contacting the protein extract with a polysaccharide matrix comprising cellulose that has binding affinity for the CBM; (e) washing the matrix with the bound fusion protein with one or more suitable aqueous solutions, and (f) eluting the fusion protein from the polysaccharide matrix by adjusting conditions that affect the release of the fusion protein from the matrix. A substantially purified soluble heterologous fusion protein is thus obtained. 6. The method of claim 5, wherein the separating step (c) comprises a method selected from expanded bed adsorption, packed chromatography, precipitation, filtration, centrifugation or any combination thereof. 7. The method of claim 5, wherein said contacting in step (d) is performed in a chromatography column. 8. The method of claim 5, combining steps (c) and (d) in a process step comprising expanded bed adsorption with a polysaccharide matrix as simultaneous separation of cell-wall material and solids from said protein extract and A method of affinity binding of the CBM-fusion protein to a polysaccharide matrix. 9. The method of any one of claims 1-8, wherein the cellulose is a pharmaceutically compatible cellulose. 10. The method of claim 9, wherein the cellulose is Avicel ^(TM) . 11. The method of any one of claims 1-9, wherein the recovery of the eluted CBM-protease comprises neutralization, and/or removal of substances that affect the release of CBM from the polysaccharide matrix from the CBM-protease eluate. reagent. 12. The method of claim 11, wherein said reconstitution comprises neutralizing or removing from the eluate soluble carbohydrates that interfere with binding of said CBM-protease to said polysaccharide matrix comprising cellulose. 13. The method of claim 12, wherein the soluble carbohydrate is a sugar. 14. The method of any one of claims 1-13, wherein said fusion protein comprising said heterologous protein of interest is expressed and recovered from a transgenic plant or plant cell or expressed transiently in a plant, plant tissue or plant cell. 15. The method of claim 14, wherein the transgenic plant or plant cell is selected from the group consisting of dicots and monocots. 16. The method of claim 15, wherein said plant cell or transgenic plant is selected from the group consisting of tobacco, rapeseed, soybean, alfalfa, lettuce, barley, corn, wheat, oat and rice. 17. The method of claim 1, wherein the CBM fused to the heterologous protein and the CBM fused to the protease are thermostable and remain soluble at elevated temperatures, wherein both of the CBMs is a CBM encoded by a region of the xylanase 10A gene from Thermotoga maritima.

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