JP2008515427A - Methods and compositions for concentrating secreted recombinant proteins - Google Patents

Abstract

  Methods and compositions relating to obtaining concentrated preparations of secreted recombinant proteins are described. This protein is expressed in the form of a fusion protein with a chitin binding domain (CBD). The fusion protein can be concentrated in the presence of chitin. A shuttle vector containing a modified LAC4 promoter, chitinase negative host cells, CBD that can be eluted from chitin under non-denaturing conditions, and sterile chitin that may be magnetized to facilitate recovery of the recombinant protein are also described.

Description

  Chitin, a β-1,4-linked unbranched polymer of N-acetylglucosamine (GlcNAc), is the second most abundant polymer on earth after cellulose. Chitin is found in insect exoskeletons (Merzendorfer, H., et al., J. Exptl. Biol. 206: 4393-4412 (2003), invertebrate crustacean shells and fungal cell walls (Chitin and Chitinases, ed. P). Jolles and R. A. A. Muzzarelli, pub. Birkhauser Verlag: Riccardo, A., et al. “Native, industrial and chief” in Basel, Switzerland (1999). It hydrolyzes the β-1,4-glycoside bond of chitin and is present in prokaryotes, eukaryotes and viruses.Yeast Saccharomyces cerevisiae (Sac In C. cerevisiae), chitinase plays a morphological role in efficient cell separation (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)). Expressing chitinase as a defense against chitin-containing pathogens, in fact, heterologous expression of chitinase genes in transgenic plants has been shown to increase resistance to certain plant pathogens (Carstens, M., et al. Trans. Res. 12: 497-508 (2003); Itoh, Y., et al. Biosci.Biotechnol.Biochem.67: 847-855 (2003); Trans.Res.12: 475-484 (2003)) Chitinases belong to family 18 or family 19 of glycosyl hydrolases based on amino acid sequence similarity (Henrissat, B., et al. Biochem. 293: 781-788 (1993)) Differences between families of chitinase catalytic domain sequences reflect different chitin hydrolysis mechanisms that result in retention of the anomeric configuration of the product (family 18) or inversion (family 19). (Chitin and Chitinases, ed. P. Jolles and R.M. A. A. Muzzarelli, pub. Birkhauser Verlag: Robertus, J., in Basel, Switzerland (1999). D. , Et al. “The structure and action of chitinases”).

  Most chitinases have a modular domain structure that includes separate catalytic and non-catalytic domains that function independently of each other. A region rich in O-glycosylated Ser / Thr can often separate these two domains to help prevent proteolysis or help secrete chitinase (Arakane, Y., Q. et al. Insect Biochem. Mol. Biol. 33: 631-48 (2003)). The non-catalytic chitin binding domain (CBD, also referred to as ChBD) belongs to one of three structural classes (types 1, 2 or 3) based on protein sequence similarity (Chitin and Chitinases, ed. P. Jolles). and R. A. A. Muzzarelli, pub. Birkhauser Verlag: Henrissat, B. "Classification of chitinases modules" in Basel, Switzerland (1999). Depending on the individual chitinase, the presence of CBD promotes chitin hydrolysis by the catalytic domain (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)) or inhibition (Hashimoto, M , Et al., J. Bacteriol., 182: 3045-3054 (2000)).

  Since CBD is small in size (about 5-7 kDa) and has substrate binding specificity and high binding force for chitin, it is used as an affinity tag for immobilizing proteins on the chitin surface (Bernard, M. et al. P., et al. Anal. Biochem., 327: 278-283 (2004); Ferrandon, S., et al. Biochim.Biophys. Acta. 1621: 31-40 (2003)). For example, B.I. B. circulans chitinase A1 type 3 CBD is used to immobilize fusion proteins expressed in bacteria on chitin beads and is mediated by protein splicing mediated by intein (Ferlandon, S., et al. Biochim). Biophys. Acta 1621: 31-40 (2003)) and chitin-coated microtiter dishes (Bernard, MP, et al. Anal. Biochem. 327: 278-283 (2004)). . Eukaryotic protein expression systems can secrete CBD-tagged proteins from eukaryotic cells because they allow biological processes not possible in bacterial systems (eg protein glycosylation, protein folding mediated by chaperones). Is desirable. However, many eukaryotic cells, particularly fungi, compete with the CBD-labeled protein for the chitin binding site, are purified simultaneously with the CBD-labeled protein during the chitin immobilization process, and also degrade the target chitin-coated surface. To secrete endogenous chitinase that complicates the immobilization of CBD-tagged protein to chitin.

  Proteins secreted from the host cells into the surrounding medium are substantially diluted and thus require large amounts of purification, which is expensive and cumbersome. It would be desirable to reduce the cost of separating proteins from secreted media and facilitate separation.

  There are various techniques for purifying proteins from a large amount of medium. These approaches vary in cost, efficiency and time required for purification. For example, proteins in the secretory culture can be collected by precipitation. This technique requires the addition of a large amount of a precipitating agent such as ammonium sulfate, acetone, trichloroacetic acid, followed by centrifugation or filtration. Many of these precipitating agents are toxic or volatile, and all precipitating agents add significant additional costs to protein collection. Furthermore, precipitation can lead to a significant loss of protein function.

  Another approach is chromatography using various resins such as anion / cation exchange resin, hydrophobic interaction resin, size exclusion gel. In order to collect the protein by chromatography, it is necessary to pass all the medium used through the resin at a low flow rate (typically 1-10 ml / min). This can be very time consuming if large volumes of media have to be processed. For example, it takes 333 hours of processing time to pass 100 liters of the used medium through the resin at a flow rate of 5 ml / min. Furthermore, these types of chromatographic resins often do not selectively purify only the target protein and often must be used in conjunction with other methods in a multi-step purification process.

  Affinity chromatography resins that specifically bind to peptide sequences incorporated into the protein structure are often used because the target protein can be selectively purified. In a typical strategy, a peptide sequence (eg, a peptide antibody epitope or hexahistidine sequence) is manipulated into the desired protein sequence. Proteins expressed with these labels interact specifically with the corresponding resin (eg, resin with immobilized antibody or nickel resin for hexahexidine binding). These methods often produce highly purified proteins from small amounts, but have limited cost and performance in practicality in large-scale processes. For example, antibody affinity resins are very expensive. Nickel resins can be purified along with unwanted proteins that accidentally contain histidine residue sequences.

  Magnetic techniques using magnetic carriers, including beads, have been used to purify proteins from cultures (Safarik et al. Biomagnetic Research and Technology 2: 7 (2004)). The problem with this approach is that each magnetic bead reagent needs to be customized to bind to individual secreted proteins. This may require complex chemical reactions that attach the affinity ligand to the beads. This is also an obstacle in terms of efficiency and cost.

  In some cases, the natural affinity between the secreted protein and the substrate is utilized. For example, lysozyme has binding affinity for chitin. As a result, the chicken egg white enzyme can be purified by exposure to chitin (Safarik et al. Journal of Biochemical and Biophysical Methods 27: 327-330 (1993)).

  In one embodiment of the invention, a method is provided for obtaining a concentrated preparation of secreted recombinant protein. The method comprises (a) transforming a host expression cell with a vector comprising DNA encoding a fusion protein comprising CBD and a target protein, (b) expressing the fusion protein in the host expression cell, Secreting from the host-expressing cells, and (c) a secreted fusion protein that can be eluted into the desired buffer volume under non-denaturing conditions by CBD so as to obtain a concentrated preparation of the secreted recombinant protein. Binding to the chitin preparation.

  In another embodiment of the invention, a method for obtaining a concentrated preparation of secreted recombinant protein is provided. This method comprises (a) providing a shuttle vector (the shuttle vector is (i) a plasmid in E. coli and integrated into the genome of a yeast expressing cell; and (ii) And a DNA encoding a fusion protein containing CBD and a target protein.), (B) a chitinase-deficient host expression cell is transformed with a shuttle vector that expresses the fusion protein in a yeast host expression cell, and the chitinase Secreting the fusion protein from the deficient host-expressing cells and (c) binding the secreted fusion protein to the chitin preparation via CBD so as to obtain a concentrated preparation of the secreted protein.

  Both embodiments are illustrated using shuttle vectors. The shuttle vector can be cloned in certain embodiments, although E. It is not expressed in E. coli and can be expressed in host-expressing cells. An example of this type of shuttle vector is a shuttle vector containing a modified LAC4 promoter, further exemplified by pKLAC1.

  Both embodiments are also illustrated using host-expressing cells that are deficient in chitinase. The host expression system can be a yeast cell, eg, a single yeast species selected from Kluyveromyces, Yarrowia, Pichia, Hansenula and Saccharomyces species. . If the yeast cell is a Kluyveromyces species, the yeast cell can be selected from the Kluyveromyces marxianus varieties fragilis or lactis.

  In the above embodiment example, chitin can be added to yeast cells in the medium during or at the end of the culture. If further cultured, the chitin should be sterile. The chitin can be a coating, colloid, bead, column, matrix, sheet or membrane. When the chitin is a bead, the bead may be porous or non-porous. Chitin beads can also be magnetized.

  The fusion protein can be recovered upon binding to magnetized chitin by applying a magnetic force. The binding of the fusion protein to chitin may be reversible so that the fusion protein can be released from the chitin under non-denaturing conditions different from the binding conditions.

  In one embodiment of the invention, the Kluyveromyces cell preparation is characterized by a chitinase negative phenotype that is the result of a mutation in a chitinase gene that expresses a secreted chitinase and has a cell density similar to that of wild-type Kluyveromyces cells. Can grow to Cell density refers to the dry weight of the cells in 48 hours of culture (Colussi et al. Applied and Environmental Microbiology 71: 2862-2869 (2005)).

  Preferably, the Kluyveromyces cell preparation is capable of expressing and secreting the recombinant fusion protein. Expression can be regulated by the LAC4 promoter or variants thereof, eg, expressing proteins in Kluyveromyces It can be regulated using a shuttle vector with a modified LAC4 promoter that does not substantially express the protein in E. coli. An example of a shuttle vector is pKLAC1.

  The Kluyveromyces cell preparation can include a medium that is capable of at least one of growth and maintenance of Kluyveromyces. The medium can also contain sterile chitin. Sterile chitin can be in the form of magnetic beads that can be bound to a magnet placed in the medium or to a magnet in contact with a container containing the medium.

  Described herein are methods for concentrating proteins after the proteins are secreted into the medium from host cells that produce the proteins. This method takes advantage of the binding affinity of CBD for chitin and can be facilitated by using cells that do not secrete chitinase. Chitinase negative cells can be generated as a result of genetic modification or can be naturally occurring. It is desirable that these chitinase-deficient modified cells can grow at similar densities to wild type cells and in yields comparable to wild type cells. The host cell may be transformed with a vector encoding the target gene fused with DNA expressing CBD under an appropriate promoter so that a relatively large amount of the target protein is secreted into the production medium by the host cell. it can. The chitin substrate can be present in the production medium or in a separate reactor that draws the target protein from the mixture. When the CBD fusion protein binds, the secreted recombinant protein is concentrated on the chitin surface. The protein can be further concentrated using any of several techniques. For example, in one embodiment, chitin is magnetized and a magnetic field is applied to the production medium to concentrate chitin beads adjacent to the magnetic surface. Other embodiments include precipitation of chitin beads by centrifugation. The target protein can then be recovered from the concentrated chitin substrate.

  The term “concentration” refers to the ratio of weight to volume after performing the procedure is greater than before the procedure.

Modification of host cells to knockout chitinase expression Preferred host cell environments for the secretion of recombinant CBD-labeled proteins are (i) an environment in which neither chitin binding protein nor chitinolytic activity contaminates the preparation of secreted fusion proteins comprising CBD, (Ii) an environment in which high cell density can be obtained in culture and (iii) an environment in which recombinant proteins can be efficiently secreted. Advantages of host cells that do not secrete chitinase include (i) elimination of competition between the CBD-tagged protein and the endogenous chitinase for the chitin binding site, and (ii) the risk of contamination of the fusion protein immobilized on chitin by the endogenous chitinase. Exclusion, (iii) exclusion of degradation of the target chitin matrix by endogenous chitinase, and the like.

  Suitable host cells include various insect cell culture production systems, mammalian cell lines, yeast production lines, bacterial cells and the like.

  Examples of cells that secrete proteins for production purposes include E. coli. E. coli, Salmonella species, Bacillus species, Streptomyces species, etc., plant cells (eg, Arabidopsis species, yew species, periwinkle species, tobacco species, rice species, soybeans, alfalfa, tomatoes, etc.) Fungal cells (eg, Kluyveromyces spp., Saccharomyces spp., Pichia spp., Hansenula spp., Yarrowia spp., Neurospora spp., Aspergillus spp., Penicillium spp. , Schizosaccharomyces species, Cryptococcus species, Coprinus s) species, Ustylago species, Magnaporte species, Trichoderma species, etc.), insect cells (eg, Sf9 cells, Sfl2 cells, nettle grass cells, Drosophila species, etc.) or mammalian cells (eg, Primary cell lines, HeLa cells, NSO cells, BHK cells, HEK-293 cells, PER-C6 cells, etc.). These cells can be grown in microliter to liter volume cultures.

  Here, a method is described in which a Kluyveromyces species can be used to quickly and easily separate a secreted protein fused with CBD from a mixture. Examples of yeast belonging to the genus Kluyveromyces according to embodiments of the present invention include van der Walt in The Yeasts, ed. N. J. et al. W. Kregervan Rij: Elsevier, New York, NY, p. 224 (1987), and the like. Marxianus var. Lactis (K. lactis), K. lactis Marxianus varieties Marxianus (K. fragilis), K. Marxianus var. Drosophilarum (K. drosophilarum), Walti and other strains classified in the art as Kluyveromyces.

  In host cells that naturally secrete one or more chitinases, chitinase deletion mutants can be made by genetic modification. Genetic modification refers to any one of suppression, substitution, deletion or addition of one or more bases in a target gene. Such modifications may be performed in vitro (on isolated DNA) or in situ, eg by genetic engineering techniques, or by radiating host cells (X-rays, gamma rays, UV, etc.) or various functional groups of DNA bases. Chemicals capable of reacting with, for example, alkylating agents: ethyl methanesulfonate (EMS), N-methyl-N′-nitro-N-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO), bialkylating agents, insertion It can be obtained by exposure to mutagens such as agents. Moreover, the expression of the target gene can be suppressed by modifying a part of the region encoding chitinase and / or the whole or part of the transcription promoter region.

  Genetic modification can also be obtained by gene disruption. An example of chitinase gene disruption is shown in Example 1 for Kluyveromyces lactis. The methods described in the examples can be widely applied to any Kluyveromyces species.

  In Example 1, the chitinase gene encoding KlCts1p An industrial K. strain that preferably lacks a lactis killer plasmid. Destroyed in the Lactis line (GG799). Disruption may be caused by natural K. cerevisiae having a selectable marker gene such as a portion of a chitinase gene, eg, a G418 resistance cassette. This occurred by replacing the first 168 amino acids of the lactisquitinase gene.

  K. Lactis GG799 Δcts1 cells can obtain the same high cell density in culture as wild type cells (Example 2) and do not produce proteins that bind to detectable chitin or degrade chitin (FIG. 3A). ) And abundantly secreted the recombinant protein (FIG. 6). This line was found to be well suited as a host for production of recombinant CBD-labeled protein.

  For production purposes, a chitinase negative mutant host cell is a high cell similar to a wild type cell, although it lacks a secreted protein that binds to or degrades chitin. It is desirable to achieve the density in culture, and it is desirable for these cells to be able to secrete recombinant proteins in abundance. Therefore, chitinase negative host cells should be used for fermentation to efficiently produce purified recombinant proteins linked to CBD, either directly or via industrially useful linker peptides or linking chemical groups. Can do.

Design and use of vectors for expressing and secreting high levels of proteins in yeast Examples of various yeast expression vectors can be found in Muller et al. Yeast 14: 1267-1283 (1998) and U.S. Pat. Nos. 4,859,596, 5,217,891, 5,876,988, 6,051,431, 6,265,186 No. 6,548,285, No. 5,679,544 and US patent application Ser. No. 11 / 102,475.

  The expression vector can be exogenous. For example, YEp24 is an episomal shuttle vector used for gene overexpression in Saccharomyces cerevisiae (New England Biolabs, Inc., Ipswich, Mass.). Other examples of Saccharomyces cerevisiae episomal shuttle vectors are pRS413, pRS414, pRS415 and pRS416. Self-replicating vectors in Kluyveromyces include pKD1 (Falcone et al., Plasmids 15: 248 (1986); Chen et al., Nucl. Acids Res. 14: 4471 (1986)), pEW1 (Chen et al. , J. General Microbiol. 138: 337 (1992)). In addition to the complete pKD1 vector, a smaller vector containing a pKD1 origin of replication and a cis-acting stability locus (CSL) has been constructed; Used for heterologous protein expression in Lactis (Hsieh, et al. Appl. Microbiol. Biotechnol. 4: 411-416 (1998)). Other episomal vectors are also described in K. Replicated in Lactis. Plasmids having both centromere (cen) and self-replicating sequences (ars) are described in K. et al. Used for expression cloning of fungal cDNA in lactis (van der Vlag-Bergmans, et al. Biotechnology Technologies 13: 87-92 (1999)). Further, K.K. Vectors containing the Lactis ARS sequence (KARS) include fungal α-galactosidase (Bergkamp, et al. Curr Genet. 21: 365-70 (1992)) and plant α-amylase (Strasser et al. Eur. J. Biochem. 184). : 699-706 (1989)).

  Other vectors can also be integrated into the host genome. For example, US Pat. Nos. 6,602,682, 6,265,186 and US Patent Application No. 11 / 102,475 for plasmids integrated into the genome of Kluyveromyces.

  The vector is (i) a strong yeast promoter, (ii) a DNA encoding a secretion leader sequence (if secretion of the protein into the medium is desired), (iii) a gene encoding the protein to be expressed, (iv) It should contain at least one of a transcription terminator sequence and (v) a yeast selectable marker gene. These sequence components are typically E. coli. It is assembled in a plasmid vector in E. coli and then transferred to yeast cells to produce the protein. This type of vector is called a shuttle vector.

  The shuttle vector is used for E. coli prior to transformation of host cells. Although preferred because it can be prepared in E. coli, embodiments of the invention are not limited to shuttle vectors.

  For example, DNA fragments that can be integrated into the yeast genome can be constructed by PCR or Helicase-Dependent Amplification (HDA) and introduced directly into yeast cells. Alternatively, the expression vector is E. coli. It can be assembled by cloning steps in bacteria other than E. coli or directly in yeast cells.

Overexpression of proteins in Kluyveromyces, more commonly in yeast, can be achieved by using a shuttle vector containing a DNA fragment with the appropriate sequence to provide high level transcription of the gene of interest when introduced into a yeast host. Construction is necessary. For example, _PLAC4 can function as a strong promoter for protein expression in yeast when present on integrative or episomal plasmids such as pKD1 based vectors, 2 micron containing vectors, centromeric vectors. (If secretion of the protein into the medium is desired) S. Cerevisiae α-MF prepro secretion leader peptide and the like. Other prokaryotic or eukaryotic secretion signal peptides (eg, Kluyveromyces α-mating factor prepro secretion signal peptide, Kluyveromyces killer toxin signal peptide) or synthetic secretion signal peptides can also be used. Alternatively, the secretory leader can be completely removed from the vector to obtain cellular expression of the desired protein.

The shuttle vector can propagate clonal genes in bacteria prior to introduction into yeast cells for expression. However, yeast expression systems that utilize wild-type _PLAC4 are E. _coli . In bacterial host cells such as E. coli, it may be adversely affected by accidental expression of proteins from genes under the control of _PLAC4 . This promoter activity can reduce the cloning efficiency of genes whose translation products are harmful to bacteria.

Although not limited to this, K.K. And substantially retain the ability to function as a strong promoter in Kluyveromyces species exemplified by lactis, a P _LAC4 mutant having a mutation Pribnow box-like sequences can be generated by site-directed mutagenesis . These mutated promoters function in substantially the same way or more than the unmutated Plenowau box-like sequence in wild-type _PLAC4 .

  As used herein, the term “mutation” is intended to include any substitution, deletion or addition of one or more nucleotides in the wild-type DNA sequence.

In one embodiment of the invention, the fungal expression host is a yeast Kluyveromyces species and the bacterial host is E. coli. An E. coli, and a series of _{P LAC4} variants, (a) -198 from -212 region of the promoter, for example, position -201, -203, -204, -207, -209 and -210 is, K. (B) a -133 to -146 region of the promoter, eg positions -139, -140, -141, -142, and -b, characterized in that it does not substantially interfere with the ability of the promoter to function as a strong promoter in lactis. 144 is characterized in that it does not substantially interfere with strong promoter activity, or is capable of incorporating regions (c) -198 to -212 and -133 to -146, and (d) K. Replacing the -1 to -283 region of Lactis P _LAC4 ; A hybrid promoter comprising 283 bp (-1 to -283) of a cerevisiae (Sc) PGK promoter has been produced. These substitutions are described in detail in US patent application Ser. No. 11 / 102,475.

An example of a transcription terminator sequence is TT _LAC4 .

  Yeast selectable marker genes include, for example, genes that confer resistance to antibiotics (eg, G418, hygromycin B, etc.), genes that supplement the auxotrophy of the strain (eg, ura3, trp1, his3, lys2, etc.) or acetamidase (AmdS) gene. Expression of acetamidase in transformed yeast cells allows growth on media lacking a simple nitrogen source but containing acetamide. Acetamidase decomposes acetamide into ammonia. Ammonia can be used by cells as a nitrogen source. The advantage of this selection method is that the number of transformants in cells incorporating multiple tandem integrations of the pKLAC1-based expression vector increases, producing more recombinant protein than single integration (FIG. 5). ).

The vector containing a mutant of _PLAC4 is E. _coli . Inserted into a Koli / Kluyveromyces integrated shuttle vector such as pGBN1 and pKLAC1 (US patent application Ser. No. 11 / 102,475). pGBN1 and pKLAC1 are each integrated into the Kluyveromyces genome after transformation of competent host cells, followed by protein expression.

US patent application Ser. No. 11 / 102,475 is a yeast, more specifically K. et al. Lactis describes a shuttle vector containing the mutant _{P LAC4} for Kluyveromyces exemplified by U.S. Patent No. 4,859,596, the 5,217,891 Patent, the 5,876,988 Patent, This is an improvement over the vectors described in US Pat. Nos. 6,051,431, 6,265,186, 6,548,285, and 5,679,544. This improvement is due to the usefulness of expression of the modified LAC4 in yeast and This is due to the inability to express proteins in E. coli, thus avoiding problems arising from toxicity in bacterial cloning host cells.

Introduction of DNA vectors into host cells Methods for introducing DNA into host cells are well established for eukaryotic and prokaryotic cells (Miller, JF Methods Enzymol. 235: 375-385 ( 1994); Hanahan, D. et al., Methods Enzymol. 204: 63-113 (1991)). In yeast, standard methods for introducing DNA into cells include genetic hybridization, protoplast fusion, transformation with lithium, electroporation, conjugation or literature, eg, Wang et al. Crit Rev Biotechnol. 21 (3): 177-218 (2001), Schenborn et al. Methods Mol Biol. 130: 155-164 (2000), Schenborn et al. Methods Mol Biol. 130: Any other technique described in 147-153 (2000). For transformation of Kluyveromyces yeast, established techniques are described in Ito et al. (J. Bacteriol. 153: 163 (1983)), Durrens et al. (Curr. Genet 18: 7 (1990)), Karube et al. (FEBS Letters 182: 90 (1985)) and patent application EP 361 991.

Properties of CBD including eluting properties CBD as a component of chitinase can be obtained from a number of different sources, such as fungi, bacteria, plants and insects. Although any CBD derived from chitinase can be used in the present invention, CBD separated from chitinase catalytic activity is preferred. Also preferred are CBDs that are dissociable from chitin under non-denaturing conditions that differ from the conditions that allow binding. Not all CBDs can be dissociated from chitin under non-denaturing conditions. For example, as described in US Pat. No. 6,897,285, US Patent Publication Nos. 2005-0196804 and 2005-0196841, B.I. Circulation CBD binds tightly to chitin but is not reversible unless a mutation is introduced into the protein. In contrast, Kluyveromyces species bind tightly to chitin, but can be reversibly dissociated by changing conditions such as NaOH (see Examples 5 and 6).

  Kluyveromyces is an abundantly expressed and secreted endochitinase exemplified herein to be an effective affinity tag that allows reversible immobilization or purification of an alkaline or alkaline resistant protein (KCBD) is produced. KCBD can bind to chitin in the absence of the catalytic domain (see, eg, FIG. 4D) and functions as an affinity tag on proteins heterologously expressed in Kluyveromyces, as shown in FIGS. 4D and 6B. Can be dissociated from chitin with NaOH in the range of about 5 mM to 500 mM. In contrast, B.I. Circulation-derived CBD (BcCBD) cannot do this.

Properties of chitin binding to CBD Synthetic or natural chitin can be used for binding to CBD fusion proteins. Examples of synthetic chitin are acetylated chitosan, polymerized N-acetylglucosamine monosaccharide, polysaccharide or oligosaccharide, or polymerized glucosamine monosaccharide, oligosaccharide or polysaccharide. Glucosamine is subsequently chemically acetylated.

  Examples of natural chitin are chitin derived from crab shells, insect exoskeletons or fungal cell walls, or chitin from sources known in the art.

  The chitin may be fixed on the substrate as shown in FIG. An example of the substrate is a polymer such as plastic. Alternatively, chitin can assemble to form, for example, a suspension, colloid, bead, column, matrix, sheet or membrane. The chitin can be sterilized for addition to the fermentation medium during fermentation and can be used in an unsterile form that binds to the fusion protein at the end of the fermentation process.

A generally applicable technique for concentrating secreted proteins fused with CBD using chitin-coated magnetic beads The chitin substrate that binds to the secreted CBD fusion protein may be magnetized so that the target protein can be easily removed from the medium. Magnetized chitin can be made by combining chitin with a magnetic material. The magnetic material may be a dispersion piece such as iron filings. In a preferred embodiment, the magnetized chitin is in the form of beads, but can also be used as a coating on additional materials that may be inert. In a preferred embodiment, the magnetized material is magnetized chitin, but (a) a property that can be bound to or expressed as a fusion with the target protein and (b) magnetized. Other magnetic materials that have the property of being able to bond to materials that can be used can also be used.

  In one embodiment, magnetized chitin beads (New England Biolabs, Inc., Ipswich, Mass.) Are used to bind the secreted CBD fusion protein. The size of the beads is not critical, but beads formed from sizes less than 200 nm in diameter have the advantage of forming colloids in the medium until they pass through a sterile filter and are magnetically applied (see eg 5,160,726). ). Larger beads can also be used. The chitin beads can be solid (eg, New England Biolabs, Inc., Ipswich, Mass.) Or porous (eg, JP 62151430). Furthermore, the beads can be magnetized by various means, for example, by dispersing iron filings throughout the beads or by forming beads with iron nuclei (by coating iron with chitin). (See, for example, 5,262,176). The magnetized chitin used in the present invention is in the form of beads in a preferred embodiment, but does not exclude other shapes and sizes of the chitin surface.

  Magnetic chitin beads can also be added to the growth medium during or after growth of the cell culture or after removal of the grown cells from the culture (FIG. 9B). Thus, it has been shown in the present invention that magnetized chitin beads can be sterilized without significantly changing their binding properties. When chitin beads are added to the medium during cell growth, it is preferable to sterilize the beads.

  Typically, the maximum amount of CBD labeled protein binding to chitin beads (Figure 9B, steps 4a and 4b) occurs within 1 hour at 4 ° C, although other temperatures and time frames are possible. Proteins immobilized on magnetic chitin beads are collected in a magnetic field (FIG. 9B, steps 5a and 5b), and cells, contaminating proteins and growth media are washed from the beads (FIG. 9B, step 6). The chitin bead-protein complex can then be released from the magnetic field (FIG. 1B, step 7) and the collected protein can be immobilized on the chitin magnetic beads indefinitely (FIG. 9B, step 8b) or can be eluted. When CBD is used as an affinity tag, it can be dissociated from chitin (FIG. 9B, step 8a).

  Therefore, by adding magnetized chitin beads to the medium in the fermentation vessel, a very efficient one-step process for recovering the target protein can be realized. The cells grow in a medium containing magnetized chitin beads so that the secreted CBD-labeled protein binds to the beads during culture and forces magnetic force outside the fermentation vessel, or as desired, from a magnet inside the fermentation vessel. It can be collected directly from the culture medium by a simple step (see Figures 9-11). This approach requires sterilization of the magnetized chitin beads. It was shown here that the yield from sterile chitin beads of size 50-70 μm added at or during the start of fermentation is similar to the protein yield obtained when the beads are added at the end of the fermentation ( FIG. 8).

  A compelling feature of this method of concentrating secreted proteins from a large volume is their universality. The method takes advantage of the ability of the chitin binding domain to bind to chitin when fused with additional proteins. The function of the additional protein is not impaired by the presence of CBD.

  Competitive binding and contamination are avoided by ensuring that cells secreting the fusion protein do not produce a protein that binds chitin as such. CBD binds to chitin with considerable binding power. The desired protein CBD fusion protein can be obtained by mutating the CBD so that binding can be eliminated and release the fusion protein under controlled conditions (US Pat. No. 6,897,286) or by an intein cleavage system or Proteins can be recovered from chitin beads by releasing the protein from the CBD-chitin complex using protease cleavage (WO 2004/053460, US Pat. No. 5,643,758). In addition, if a proteolytic cleavage site exists between the CBD and the desired protein, the CBD tag is released from the desired protein by digestion with a protease (eg, enterokinase, genenase, furin, factor X, etc.). be able to.

  Due to the robust nature of the CBD-chitin interaction, the CBD-labeled protein can be rapidly immobilized on any form of magnetized chitin, eg, beads.

  If the magnetized chitin is in the form of beads, chitin beads containing bound CBD labeled protein can be collected in a magnetic field in seconds. If necessary, the CBD-labeled protein can be dissociated from the magnetized substrate by incubating in an elution buffer (if eluting CBD is used). Advantages of this method include speed improvement, high cost effectiveness, simplicity, and the like.

  Using a magnetic separator that is compatible with common test tubes (eg 96-well microtiter dishes, microcentrifuge tubes, 15 ml falcon tubes, 50 ml falcon tubes, 250 ml nalgene bin, etc.) Collect from 1 liter of medium (FIGS. 3-6). The procedure can be easily expanded so that CBD-labeled proteins can be collected from larger volumes of media. In a preferred embodiment, the magnet is composed of a rare earth metal (eg, neodymium, samarium cobalt, etc.), but other types of magnets (eg, ferrite, ceramics, electromagnets, etc.) can be used.

Use of the expression system Production and separation of the target protein from the mixture is necessary for proteins used as pharmaceuticals, as food, or for industrial use. The list of proteins that are currently being produced mainly by fermentation or that are desirable is enormous. A few examples include superoxide dismutase, catalase, amylase, lipase, amidase, glycosidase, xylanase, laccase, ligninase chymosin, etc., or fragments or derivatives thereof, blood products (serum albumin, alpha- or beta-globin, coagulation factor And, for example, factor VIII, factor IX, von Willebrand factor, fibronectin, alpha-1 antitrypsin, etc., or fragments or derivatives thereof), insulin and mutants thereof, lymphokines such as interleukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF, etc.), TNF, etc., or fragments or derivatives thereof, growth factors (growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, etc., or fragments or derivatives thereof), apolipoprotein and molecular variants thereof, antigenic polypeptide for vaccine production (hepatitis, cytomegalovirus, Epstein Barr virus, herpes virus, etc.), single chain antibody (ScFv), Alternatively, polypeptide fusions such as fusions comprising a biologically active moiety fused to a stabilizing moiety may be mentioned.

All references above and below and US provisional applications 60 / 616,420 and 60 / 690,470 are incorporated herein by reference.
Example

Materials and Methods for the following Examples Yeast strains, culture conditions and transformation conditions Lactis strains and S. cerevisiae S. cerevisiae strains (Table 1) were cultured in a conventional manner at 30 ° C. in YPD medium (1% yeast extract, 2% peptone and 2% glucose) or YPGal medium (1% yeast extract, 2% peptone and 2% galactose). .

  K. Lactis and S. S. cerevisiae transformation was performed by electroporation. K. Lactis transformants were selected by growing on YPD agar containing 200 mg / ml G418. On the other hand, S.M. S. cerevisiae transformants can be transformed into SD medium (0.67% yeast nitrogen base, 2% glucose) or SGal medium (0.67% yeast nitrogen base, containing appropriate supplements necessary to supplement the auxotrophy of the strain. 2% galactose).

Secretion K. Detection and isolation of lactisquitin binding protein Cross-linked with a polyclonal anti-chitin binding domain antibody (α-CBD) against a chitin binding domain from Bacillus circulan skitinase A1 (New England Biolabs, Inc., Ipswich, Mass.) By Western blotting Reactive secretion K. Lactis protein was detected.

  (A) After growth for 48-96 hours, use medium Isolated from a Lactis GG799 culture by centrifugation at 4000 xg for 10 minutes.

  After centrifugation, the proteins in the working medium were separated by SDS-PAGE on a 4-20% Tris-glycine polyacrylamide gel (Daiichi Pharmaceutical Corp., Montvale, NJ), and Protran nitrocellulose membrane (Schleicher & Schuell Bioscience, Keene). , NH). Block the membrane overnight at 4 ° C. with phosphate buffered saline (PBS-T) and 5% skim milk (w / v) containing 0.05% Tween 20, and α-CBD polyclonal antibody (containing 5% skim milk) Probe 1: with horseradish peroxidase conjugated anti-rabbit secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD), 1: 2000 dilution with PBS-T containing 5% nonfat milk) did. The protein-antibody complex was visualized with LumiGlo ™ detection reagent (Cell Signaling Technologies, Beverly, Mass.).

  (B) To isolate secreted proteins bound to chitin, Lactis GG799 cells were grown for 96 hours in 20 ml of YPD medium. Cells were removed from the culture by centrifugation and the working medium was transferred to a new tube containing 1 ml of washed chitin beads (New England Biolabs, Inc., Ipswich, Mass.) And incubated for 1 hour at room temperature with gentle rotation. . Chitin beads were collected by centrifugation and washed with 10 ml of water. Approximately 50 μl of protein-bound chitin beads are removed, boiled in protein loading buffer for 2 minutes to elute the bound protein, and then the eluted protein is separated by SDS-PAGE and Western analysis using α-CBD polyclonal antibody Alternatively, it was subjected to amino terminal protein sequencing.

K. Analysis of chitin binding properties of a chitin binding domain derived from lactiskininase (KlCts1p CBD). KlCts1p obtained from 20 ml of Lactis GG799 working medium was bound to 1 ml of chitin beads as described above. KlCts1p-bound beads were washed with 10 ml of water and resuspended in 1.5 ml of water. Mini columns were prepared by dispensing 100 μl aliquots of KlCts1p-bound beads into individual disposable columns (Bio-Rad Laboratories, Hercules, Calif.). A 1 ml volume (approximately 10 bed volumes) of each of the following buffers was passed through a separate minicolumn: 50 mM Na citrate pH 3.0, 50 mM Na citrate pH 5.0, 100 mM glycine-NaOH pH 10.0, unbuffered 20 mM. NaOH pH 12.3, 5M NaCl and 8M urea. Each column was then washed with 2 ml of water, after which the beads were resuspended in 200 μl of water, transferred to a microfuge tube and collected by brief centrifugation. Proteins still bound to chitin were eluted by boiling the beads for 5 minutes in 50 ml of 3 × SDS-PAGE loading buffer (New England Biolabs, Inc., Ipswich, Mass.). The eluted protein was separated by SDS-PAGE and detected by Western analysis as described above.

  KlCts1p elution profiles with various concentrations of NaOH (0-40 mM) were similarly performed. An aliquot (100 μl) of KlCts1p bound to chitin was prepared as described above and distributed to microfilter cups (Millipore, Billerica, Mass.) Inserted into a 1.5 ml microfuge tube to make a spin column. The flow-through was collected by microcentrifugation at 15,800 × g for 1 minute and discarded. The beads were then resuspended in 100 μl NaOH at each desired concentration (0-40 mM) and the eluate was collected by centrifugation at 15,800 × g for 1 minute. The chitinase activity of each eluate was measured as shown below.

を使用して、ＡＤＨ２−Ｇ４１８含有ベクターｐＧＢＮ２からの破壊ＤＮＡ断片をＴａｑ ＤＮＡポリメラーゼを用いて増幅した。増幅産物を用いて、Ｋ．ラクチスＧＧ７９９細胞を形質転換し、２００ｍｇ／ｍｌ Ｇ４１８を含むＹＰＤ寒天上でコロニーを選択した。ＫｌＣＴＳ１特異的順方向プライマー５’−ＧＧＧＣＡＣＡＡＣＡＡＴＧＧＣＡＧＧ−３’（配列番号３）（組込み部位の上流用）及びＧ４１８特異的逆方向プライマー５’−ＧＣＣＴＣＴＣＣＡＣＣＣＡＡＧＣＧＧＣ−３’（配列番号４）を用いた細胞全体のＰＣＲによって、破壊ＤＮＡ断片をＫｌＣＴＳ１部位に正確に組み込んだ細胞からの約６００ｂｐの診断用ＤＮＡ断片を増幅した。このようにして試験した２０個の形質転換体のうち、２個のＤｃｔｓ１ Ｋ．ラクチス系統を特定し、さらに分析した。 K. Disruption of the lactisquitinase gene (KlCTS1) A PCR-based method was used to construct a linear DNA disruption fragment consisting of an ADH2 promoter-G418 resistance gene cassette with 80-82 bp of KlCTS1 DNA at one end. When this fragment is integrated into the KlCTS1 site, it replaces the DNA encoding the first 168 amino acids of KlCts1p with a G418 resistance cassette. A primer comprising DNA that hybridizes to the ADH-G418 sequence (not underlined) and having ends (underlined) consisting of the KlCTS1 DNA sequence;

Was used to amplify the disrupted DNA fragment from the ADH2-G418-containing vector pGBN2 using Taq DNA polymerase. Using the amplification product, K. Lactis GG799 cells were transformed and colonies were selected on YPD agar containing 200 mg / ml G418. Whole cells using KlCTS1-specific forward primer 5'-GGGCCACAACAATGGCAGGG-3 '(SEQ ID NO: 3) (for upstream of the integration site) and G418-specific reverse primer 5'-GCCTCTCCACCCAAGCGGC-3' (SEQ ID NO: 4) By PCR, an approximately 600 bp diagnostic DNA fragment was amplified from a cell in which the disrupted DNA fragment was correctly integrated into the KlCTS1 site. Of the 20 transformants tested in this way, 2 Dcts1 K. Lactis lines were identified and further analyzed.

を用いて遺伝子をＰＣＲ増幅し、ｐＭＷ２０（３１）のＢａｍＨ Ｉ−Ｓｐｈ Ｉ部位にクローン化して、ＫｌＣＴＳ１の発現をガラクトース誘導性／グルコース抑制性Ｓ．セレビシエＧＡＬ１０プロモーターの制御下に置いた。この発現構築体をＳ．セレビシエΔｃｔｓ１系統ＲＧ６９４７に形質転換によって導入した。ＫｌＣｔｓｐ１の産生を誘導するために、スターターカルチャー２ｍｌを、２０ｍｇ／ｍｌウラシルを含むＳＤ培地中で３０℃で終夜増殖させ、その後、各培養物１ｍｌを用いてＹＰＤ培養物２０ｍｌ及びＹＰＧａｌ培養物２０ｍｌを接種した。各培養物を振とうしながら３０℃で終夜増殖させた後、使用培地のキチナーゼ産生及び顕微鏡法による細胞形態学の分析を行った。 S. Heterologous expression of KlCTS1 in S. cerevisiae In order to express KlCTS1 in cerevisiae,

The gene was PCR amplified using and cloned into the BamH I-Sph I site of pMW20 (31), and the expression of KlCTS1 was galactose-inducible / glucose-inhibitory S. cerevisiae. It was placed under the control of the cerevisiae GAL10 promoter. This expression construct is designated S. cerevisiae. It was introduced into S. cerevisiae Δcts1 strain RG6947 by transformation. To induce the production of KlCtsp1, 2 ml of starter culture was grown overnight at 30 ° C. in SD medium containing 20 mg / ml uracil, after which 20 ml of YPD culture and 20 ml of YPGal culture were used with 1 ml of each culture. Vaccinated. Each culture was grown overnight at 30 ° C. with shaking and then analyzed for chitinase production in the media used and cell morphology by microscopy.

Chitinase activity measurement 4-methylumbelliferyl N-acetyl-β-D-chitotrioside (4MU-GlcNAc) derivatized with a chitin oligosaccharide of 1-4GlcNAc residue and 4-methylumbelliferone (4-MU), 4-methylumbelliferyl N, N′-diacetyl-β-D-chitotetraoside (4MU-GlcNAc ₂ ), 4-methylumbelliferyl N, N ′, N ″ -triacetyl-β-D-chitotrioside (4MU- GlcNAc ₃ ) or 4-methylumbelliferyl N, N ′, N ″, N ′ ″-tetraacetyl-β-D-chitotetraoside (4MU-GlcNAc ₄ ) (Sigma-Aldrich Corp., St. Louis, MO and EMD) Biosciences, San Diego, CA) as a substrate Chitinase activity was determined by measuring 4-MU release at 37 ° C. using a Genios fluorescent microtiter plate reader (Tecan, San Jose, Calif.) And a 340 nm / 465 nm excitation / emission filter. The reaction mixture in each well of the titer plate was 100 ml and contained 50 mM substrate, 1 × McIlvaine buffer (pH 4-7 in different experiments) and sample 5-10 ml.Record initial release rate and release enzyme units. Calculated as 4-MU pmol / min A calibration curve of 4-MU (Sigma-Aldrich Corp., St. Louis, MO) was prepared under reaction conditions to convert from fluorescence units.

Microscopy Approximately 1-2 OD ₆₀₀ units of cells were collected and fixed on ice for 1 hour with 1 ml of 2.5% (v / v) glutaraldehyde. The cells were washed twice with water and resuspended in about 100 ml of mounting medium (20 mM Tris-HCl pH 8.0, 0.5% N-propyl gallate, 80% glycerin). In septal staining experiments, Calcofluor white (Sigma-Aldrich Corp., St. Louis, MO) was added to the mounting medium at a final concentration of 100 mg / ml. Cells were examined with a Zeiss Axiovert 200M microscope using light phase Normaski imaging or fluorescent DAPI filter settings.

Cell Chitin Measurement Cells were extracted with KOH, and chitin in an alkali-insoluble material was hydrolyzed to GlcNAc by chitinase (Bulik, DA, et al. Eukaryot. Cell 2: 886-900 (2003)). Quantified by the Micro Morgan-Elson assay.

K. B. Identification and biochemical characterization of lactisquitinase (KlCts1p) Polyclonal antibodies raised against the Circulance Chi1A chitin binding domain (α-CBD) are Native secreted K. containing a cross-reactive chitin binding domain used for Western blot analysis of Lactis GG799 working medium. Lactis protein was identified (Figure 1).

  Non-concentrated K.I. The secreted protein in 10 ml of Lactis GG799 working medium (96 hours growth) was separated using 4-20% polyacrylamide Tris-glycine SDS gel; The presence of the chitin binding domain was screened by Western blotting using a polyclonal antibody raised against the circulan skitinase A1 chitin binding domain (lane 1). Secreted proteins were bound to chitin beads and eluted directly in SDS-PAGE loading buffer by boiling for 2 minutes and separated using SDS-PAGE (lane 2).

  To test whether any of these proteins could bind to chitin, the working medium was mixed with chitin beads and rotated for 1 hour at room temperature. The bound protein was eluted directly into SDS-PAGE loading buffer by washing the chitin beads with water and boiling. According to Western blotting, only 85 kDa a-CBD cross-reactive protein was able to bind to chitin (FIG. 1, lane 2). The protein thus purified directly from chitin beads was subjected to N-terminal protein sequencing. As a result, the first 20 amino-terminal amino acids (FDINAKDNVAVYWGQASAT) (SEQ ID NO: 7) of the mature protein were confirmed. A translation sequence that exactly matches the query sequence by tBLASTn search using this amino acid sequence as a query to probe the sequence database, Partially sequenced K. cerevisiae containing a translated sequence with significant homology to S. cerevisiae extracellular chitinase Cts1p (ScCts1p). The lactis gene was identified.

Cloning and sequence analysis of KlCTS1 At the start of this study, The sequence of the lactis genome has not yet been reported. Therefore, using the combination of Southern blotting and anchor PCR, the remainder of the partial KlCTS1 sequence initially identified (above) by database search using the tBLASTn algorithm was cloned. The KlCts1p sequence has been reported recently in K.C. K. in the lactis genome sequence. It was identical to the translation product of Lactis ORF KLLA0C04730g (Dujon B., et al. Nature 430: 35-44 (2004)).

KlCTS1 encodes a protein containing 551 amino acids with a molecular weight of 85 kDa as determined by SDS-PAGE. KlCTS1p is S.I. It is 53% identical and 82% similar to S. cerevisiae Cts1p chitinase and has a similar modular domain structure consisting of a signal peptide, catalytic domain, Ser / Thr rich domain and chitin binding domain (FIG. 2A). Signal P software (Nielson et al Protein Eng 10: .. 1-6 (1997)) by ^the presence of a signal peptide that is cleaved after ^{A 19} is predicted (Fig. 2A). It ^begins with ^{F 20,} coincides with the amino-terminal protein sequence determined from the purified secreted KlCts1p. According to the predicted amino acid homology between the catalytic domain of KlCts1p and other chitinases, KlCts1p belongs to the chitinase family 18 (FIG. 2B). Furthermore, by SMART domain prediction software (Letonic, I., et al. Nucl. Acids Res. 30: 242-244 (2002) and Schultz, J., et al. PNAS 95: 5857-5864 (1998)), The presence of a C-terminal type 2 chitin binding domain containing 6 conserved cysteine residues was shown (FIG. 2C).

KlCts1p and S.C. The catalytic properties of cerevisiae chitinase (ScCts1p) were compared. S. Since S. cerevisiae Cts1p has an optimal acidic pH (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)), pH 4 was first used to define the substrate preference of KlCts1p. .5 was selected. As shown in FIG. 3B, chitinase from both yeasts hydrolyzed both 4MU-GlcNAc ₃ and 4MU-GlcNAc ₄ substrates. However, K. Lactiskininase differs from ScCts1p in that 4MU-GlcNAc ₄ is preferred over 4MU-GlcNAc ₃ . K. Similar results to those of lactis strain GG799 were obtained for chitinases secreted from strains CBS2359 and CBS683. Furthermore, KlCts1p showed maximum activity at pH 4.5 and was more alkaline by about 0.5 pH units than ScCts1p (FIG. 3C).

KlCts1p CBD-chitin affinity analysis The association of KlCts1p with chitin beads was examined under a range of conditions. According to FIG. 4A, the association of KlCts1p with chitin is stable in 5M NaCl and in pH 3, pH 5 and pH 10 buffers. In 8M urea, which is a condition that usually denatures proteins, only about 60% of KlCts1p bound to chitin was dissociated from chitin beads. However, it completely dissociated in 20 mM NaOH at pH 12.3. According to the elution profile of KlCts1p bound to chitin, an equal amount of protein is eluted in the first two fractions (including void volume), and KlCts1p-chitin association becomes immediately unstable in 20 mM NaOH. Was suggested (FIG. 4B). Surprisingly, KlCts1p eluted in this way retained chitinolytic activity (FIG. 4C). In fact, chitinase activity recovered almost 100% according to the measurement of chitinase activity before chitin binding and after elution with 20 mM NaOH.

  To determine whether KlCts1p CBD can function i) as an affinity tag for heterologously expressed proteins, independent of the KlCts1p catalytic domain, CBD from KlCts1p amino acids 470-551 (KlCBD) Human serum albumin (HSA) containing a C-terminal fusion with K. Secreted from lactis. For comparison, HSA was similarly used in B.I. It is also fused to a circulan skitinase A1 type 3 CBD (BcCBD). Secreted from lactis. The CBD fusion protein was bound to chitin beads as described in Example 1 and its chitin affinity was determined in the presence of 20 mM NaOH. According to FIG. 4D, the HSA-KlCBD fusion protein completely dissociated from the chitin beads in 20 mM NaOH, whereas the HSA-BcCBD fusion protein remained bound to chitin after extensive washing with 20 mM NaOH. there were. These results indicate that KlCBD functions independently of the KlCts1p catalytic domain and dissociation from chitin in 20 mM NaOH is an intrinsic property. These data also suggest the possibility that KlCBD can be used as an eluting affinity tag for the purification of alkaline or alkali-resistant proteins or for reversible chitin immobilization.

Destruction of KlCTS1 To investigate the in vivo function of KlCts1p in lactis, the KlCTS1 allele was split into haploid cells. A DNA disruption fragment containing a kanamycin selectable marker cassette was constructed by the method using PCR as described in “Materials and Methods”. Using this fragment, Lactis cells were transformed to G418 resistance. Transformants were screened by whole cell PCR for transformants incorporating the disrupted DNA fragment at the KlCTS1 site. Of the 20 colonies tested, 2 had correctly integrated disrupted DNA fragments (data not shown) and KlCTS1 was K. coli. It has been shown that it is not essential for the survival of lactis. Furthermore, as shown by the absence of KlCts1p (FIG. 5A) and chitinolytic activity (FIG. 3A) in the working medium, Lactis Dcts1 cells do not secrete chitinase.

  K. The growth and cell morphology of lactis wild type (GG799) and Δcts1 cells were examined. In YPD medium, the Δcts1 line grew as a small cluster of slowly aggregated cells and was easily dispersed into single cells by brief sonication. According to fluorescence microscopy of cells stained with the chitin-binding dye Calcofluor white, Δcts1 cells are linked via a septum (FIG. 5B, right panel), and Δcts1 cells cannot degrade chitin in the septum during cytokinesis It suggests. A similar phenotype is S. It was also observed in S. cerevisiae Δcts1 cells (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)). Therefore, KlCTS1 does not separate normal cells. It was tested whether cerevisiae Δcts1 cells could be restored. KlCTS1 was changed to S.C. It was placed under the control of a GAL10 galactose inducible promoter in a cerevisiae expression vector. S. cerevisiae expressing KlCTS1 S. cerevisiae Dcts1 cells secrete KlCts1p in galactose-containing medium (FIG. 6A) and do not form cell aggregates (FIG. 6B). In summary, these data suggest that KlCTS1 and ScCts1 encode functionally equivalent proteins involved in cell separation, presumably by promoting the degradation of septum chitin.

K. Characterization of lactisquitinase deletion mutants It was investigated whether Δcts1 cells could grow to high culture densities. K. The aggregation phenotype associated with lactis Δcts1 cells resulted in cell density measurements by absorbance at 600 nm (OD ₆₀₀ ) of less than 65% of wild type cells. However, cultures of wild-type and Δcts1 cells grown for 48 hours produced approximately the same dry weight of cells. Furthermore, the total chitin of the cells was not significantly different between the two lines. When cell chitin is extracted with KOH and hydrolyzed by chitinase, line GG799 produces GlcNAc 21.8 ± 1.9 nmol / mg dry cells, and Δcts1 line produces GlcNAc 20.8 ± 1.0 nmol / mg dry cells. Generated. Thus, despite its mild growth phenotype, Δcts1 cells can still obtain the same cell density as wild-type cells by culture, and this strain (strain background) is suitable for commercial production of CBD-labeled proteins. It has been suggested.

を使用して、ＫｌＣｔｓ１ｐのＣ末端の８１個のアミノ酸をコードするＤＮＡ断片をＤｅｅｐ Ｖｅｎｔ（商標）ＤＮＡポリメラーゼ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ，Ｉｎｃ．、Ｉｐｓｗｉｃｈ、ＭＡ）を用いて増幅した。ＫｌＣｔｓ１ｐ−ＣＢＤ断片をＫ．ラクチス組み込み発現プラスミドｐＧＢＮ２（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ，Ｉｎｃ．、Ｉｐｓｗｉｃｈ、ＭＡ）のＢｇｌ ＩＩ−Ｎｏｔ Ｉ部位にクローン化してｐＧＢＮ２−ＫｌＣＢＤを作製した。ＨＳＡを

を用いて増幅し、ｐＧＢＮ２−ＫｌＣＢＤのＸｈｏ Ｉ−Ｂｇｌ ＩＩ部位にクローン化した。生成した発現構築体は、Ｋ．ラクチスゲノムに組み込むと、（ｐＧＢＮ２中に存在する）Ｓ．セレビシエａ−接合因子プレプロ分泌リーダー、ＨＳＡ及びＫｌＣＢＤからなる単一のポリペプチドを産生する。 Preparation of pGBN2-HSA-KlCBD To create a fusion of KlCts1p CBD and human serum albumin (HSA)

Was used to amplify the C-terminal 81 amino acid DNA fragment of KlCts1p using Deep Vent ™ DNA polymerase (New England Biolabs, Inc., Ipswich, Mass.). The KlCts1p-CBD fragment was PGBN2-KlCBD was created by cloning into the Bgl II-Not I site of the lactis integration expression plasmid pGBN2 (New England Biolabs, Inc., Ipswich, Mass.). HSA

Was cloned into the Xho I-Bgl II site of pGBN2-KlCBD. The resulting expression construct was When integrated into the lactis genome, S. cerevisiae (present in pGBN2). A single polypeptide consisting of the cerevisiae a-mating factor preprosecretory leader, HSA and KlCBD is produced.

を使用して、ｐＴＹＢｌ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ，Ｉｎｃ．、Ｉｐｓｗｉｃｈ、ＭＡ）から得られるＢｃＣＢＤをＰＣＲ増幅した。増幅産物をｐＧＢＮ２のＢｇｌ ＩＩ−Ｎｏｔ Ｉ部位にクローン化してｐＧＢＮ２−ＢｃＣＢＤを作製した。ＨＳＡを、上述したように、増幅し、ｐＧＢＮ２−ＢｃＣＢＤのＸｈｏ Ｉ−Ｂｇｌ ＩＩ部位にクローン化した。 B. A control construct producing HSA containing a carboxy-terminal type 3 CBD (BcCBD) derived from B. circulans chitinase A1 was similarly assembled.

Was used to PCR amplify BcCBD obtained from pTYBl (New England Biolabs, Inc., Ipswich, Mass.). The amplified product was cloned into the Bgl II-Not I site of pGBN2 to generate pGBN2-BcCBD. HSA was amplified and cloned into the Xho I-Bgl II site of pGBN2-BcCBD as described above.

K. Production and secretion of HSA-CBD in lactis Δcts1 cells The vector pGBN2-HSA-KlCBD (5 μg) was linearized with SacII and Lactis Δcts1 cells were used for transformation by electroporation. Transformants were selected by growth for 4 days at 30 ° C. on yeast carbon-based agar medium (Difco ™, Becton Dickinson, Franklin Lakes, NJ) containing 5 mM acetamide. Individual transformants were used to initiate 2 ml culture of YPD (1% yeast extract, 2% peptone, 2% glucose) and grown overnight at 30 ° C. A 1: 100 dilution of the overnight culture was used to inoculate 2 ml of YPGaal (1% yeast extract, 2% peptone, 2% galactose) culture. The culture was incubated for 48 hours at 30 ° C. with shaking. The working medium was prepared by microcentrifuged 1 ml of the culture at 15,800 × g for 2 minutes to remove the cells. A 20 ml aliquot of working medium with the cells removed was transferred to a new tube, mixed with 10 ml of 3 × protein loading buffer and heated at 95 ° C. for 10 minutes. A 20 ml aliquot was separated on a 10-20% Tris-glycine polyacrylamide gel and secreted HSA-KlCBD fusion protein was detected by Coomassie staining.

Purification of HSA-KlCBD using chitin beads To isolate secreted HSA-KlCBD by immobilizing the fusion protein to chitin, the K. Lactis Dcts1 cells (above) were grown for 96 hours in 20 ml of YPD medium. Cells were removed from the culture by centrifugation and the working medium was transferred to a new tube containing 1 ml of washed chitin beads (New England Biolabs, Inc., Ipswich, Mass.) And incubated for 1 hour at room temperature with gentle rotation. . Chitin beads were collected by centrifugation, washed with 10 ml of water and resuspended in 1 ml of water. The immobilized HSA-KlCBD was eluted by boiling about 50 ml of protein-bound chitin beads in SDS sample buffer for 2 minutes, followed by microcentrifugation for 2 minutes to remove the chitin beads. Eluted HSA-KlCBD in the supernatant was visualized by SDS-PAGE and Coomassie staining or by Western analysis using a-CBD or a-HSA antibodies. Furthermore, HSA-KlCBD can be eluted from the chitin beads by passing 5 ml of 20 mM NaOH through the entire column. The HSA produced in this way binds to chitin by chance or endogenous K. which degrades chitin. Contains no lactis protein.

Concentration of HSA-KlCBD using magnetized chitin beads CBD-labeled human serum albumin (HSA-CBD) was used to demonstrate the association of CBD-labeled proteins and magnetic chitin beads at various stages of culture growth (see Figure 8). . K. 25 ml of 4 YPGal (1% yeast extract, 2% peptone and 2% galactose) cultures of the lactis line GG799 Δcts1PCK13 were inoculated with 100 ml of 2 ml starter culture grown at 30 ° C. for 24 hours.

  Culture 1: Before inoculation, 1 ml of settled chitin magnetic beads sterilized by autoclaving for 20 minutes was added into the medium. The culture is then about 300 r. p. m. And incubated at 30 ° C. for 72 hours with shaking.

  Culture 2: At 24 hours of growth, 1 ml of sterile magnetic chitin beads was added to the medium. The culture is about 300 r. p. m. And incubated at 30 ° C. for an additional 48 hours (72 hours total).

  Culture 3: After 72 hours, 1 ml of chitin magnetic beads was added to the third culture, followed by gentle shaking for 1 hour at room temperature.

  Culture 4: 5000 r. p. m. The cells were removed by centrifugation at 5 minutes, and 1 ml of magnetic chitin beads was added to the working medium from which the cells were removed, followed by gentle shaking for 1 hour at room temperature.

  Each culture was decanted into a standard 50 ml capped tube. Magnetic beads were collected by inserting the test tube into a 50 ml magnetic apparatus (FIG. 9) for 30 seconds, followed by decanting the supernatant. The test tube was then removed from the magnetic field and the magnetic chitin particle pellet was washed with 40 ml of water and isolated again in the magnetic field. After this washing process was repeated a total of 3 times, the beads were transferred to four screw capped microcentrifuge tubes. To elute the bound HSA-CBD, each centrifuge tube bead was suspended in 250 ml of 3 × protein loading buffer (New England Biolabs, Inc., Ipswich, Mass.) Containing dithiothreitol and 5 ° C. at 98 ° C. Heated for minutes. The eluted protein in 5 ml of each sample was separated by 10-20% SDS-PAGE gel and visualized by Coomassie staining (FIG. 8). Eluted HSA-CBD was observed in each sample, indicating that the magnetic chitin beads successfully captured the CBD-labeled protein during or after growth of the culture. The yield of captured HSA-CBD in each culture was estimated to be 4 mg / L.

Use of Magnetized Chitin Beads to Concentrate Secreted GluC-CBD Protein To test whether magnetized beads can concentrate CBD fusion protein from the culture medium, a fusion protein (GluC-CBD. GluC is a staphylococcus aureus). 20 ml of an overnight culture of Bacillus circulans transformed with DNA encoding the endoprotein from Aureus) was grown in a 125 ml flask, and the DNA encoding GluC was transferred to plasmid pGNB5 containing B. circulans CBD. Competent cell transformation was performed using standard techniques (Harwood and Cutting, Molecular Biological Methods, ed. John Wiley & Sons Lt. d., New York, NY, pp. 33-35, 67, 1990. For comparison, four cultures were grown, one containing a plasmid expressing the GluC endoprotease, Three contain plasmids for the fusion protein (GluC-CBD) In all experiments, 5% bead volume was added to the culture, as a control, culture containing the GluC construct to examine non-specific binding. Were grown overnight in the presence of magnetic beads (FIG. 12, lane 2) Magnetic beads were added to three GluC-CBD cultures at different times to see if incubation time resulted in binding capacity. Culture 1 was grown overnight at 37 ° C. with shaking in the presence of beads throughout the growth cycle (FIG. 12, lane 3). After 16 hours of growth at 37 ° C., magnetic beads were added and incubated for 1 hour at 37 ° C. with shaking (FIG. 12, lane 4). The magnetic beads were added to the supernatant and incubated for 1 hour at room temperature with shaking (Figure 12, lane 5). Collected using a separation rack (New England Biolabs, Inc., Ipswich, Mass.) And washed 3 times with 10 ml of LB broth and then twice with 1 M NaCl.The beads were suspended in 1 ml of 1 M NaCl and 1.5 ml Eppendorf. Transfer to a tube and centrifuge at 10,000 rpm for 1 minute to remove the liquid. The beads were suspended in 100 ml of 3 × SDS sample buffer containing DTT and boiled for 5 minutes to remove proteins. The beads were separated from the buffer by centrifuging at 10,000 rpm for 2 minutes. Samples were analyzed on 10-20% tricine gel, transferred to PVDF membrane by Western blot and stained with Coomassie blue. The identity of the eluted protein was confirmed by N-terminal sequencing. As a result, it was shown that the secreted GluC-CBD fusion protein is a main protein bound to magnetic chitin beads and can be efficiently eluted by boiling in SDS sample buffer. It has also been shown that beads can be added at any time during the experiment without changing their efficiency.

Production of luciferase and its elution from chitin beads. The gene encoding lactis CBD was cloned into the NotI / StuI restriction enzyme cleavage site of the vector pKLAC1 to prepare the vector pKLAC1-K1CBD. The gene encoding Gaussia luciferase (GLuc) is then cloned into the vector pKLAC1-KlCBD at the XhoI / NotI restriction site and the N-terminal fusion with the vector-derived secretion signal and the C-terminal fusion with the KlCBD gene. Produced. This construct is linearized and K.K. Lactis competent cells were transformed. K. secretes GLuc-KlCDB. 1 liter of the working medium obtained from lactis cells was mixed with chitin beads having a bed volume of 20 ml at room temperature for 1 hour. Chitin beads were poured onto the column and subsequently washed with 10 column volumes (200 ml) of water. The bound protein was eluted with 20 mM NaOH. 4 ml of the elution fraction was collected in a tube containing 1 ml of 1M Tris-Cl pH 7.5 to neutralize the eluent flowing out of the column. The luciferase activity of 25 microliters of a 1/40 dilution of each elution fraction was evaluated and expressed in RLU (relative light units). According to FIG. 13, active GLuc eluted in fractions 2 to 10, with the highest activity being fractions 3, 4 and 5.

It is a figure which shows a Western blot. K. Three proteins secreted by lactis into the working medium, with approximate masses> 200, 85 and 50 kDa It is cross-reacting with a polyclonal antibody raised against the circus Chi1A chitin binding domain (α-CBD) (lane 1). The 85 kDa protein binds to chitin beads; Corresponds to lactisquitinase (lane 2). FIG. 2 (A) shows a multi-domain KlCts1p chitinase having a signal peptide (stripe), a catalytic domain (grey), a Ser / Thr rich domain (white) and a chitin binding domain (black). Cleavage of the signal peptide occurs after A ^19. FIG. 2B shows that KlCts1p belongs to glycosyl hydrolase family 18. The predicted catalytic site of KlCts1p is between amino acids 150-158. Alternative amino acids for each of the nine positions are shown in parentheses. ("X" is any amino acid.) FIG. 2 (C) shows that KlCts1p contains a type 2 chitin binding domain. KlCts1p CBD was converted from SMART (Simple Modular Architecture Research Tool) software (Letonic, I, et al. Nucl. Acids Res. 30: 242-244 (2002); Schultz, J. 58: 57 S. 58. (1998)) in parallel with the type 2 CBD consensus sequence predicted by (SEQ ID NO: 14). KlCts1p CBD (SEQ ID NO: 15), fungi (Cladosporium fluvum (SEQ ID NO: 16); species-specific inducer A4 precursor), bacteria (Ralstonia solanacearum (SEQ ID NO: 17); Q8XZL0), C. elegans (Caenorhabditis elegans (SEQ ID NO: 18); putative endochitinase), mammal (Homo sapiens (SEQ ID NO: 19); chitinase) and insect (Drosophila melanogaster (SEQ ID NO: 20); putative chitinase 3) The alignment with the example protein containing the origin prediction type 2CBD is shown. Conserved cysteine residues are shown in bold. K. Lactis deletion mutants show no KlCts1p secretion as measured by chitinase activity. FIG. 3 (A) shows the chitinase activity measured after 22, 44, and 68 hours of cell growth at 30 ° C. in YPD medium. The activity was evaluated as the release rate 4-MU / min (relative fluorescence units, RFU / min) from 50 mM 4MU-GlcNAc ₃ at pH 4.5 and 37 ° C. K. Lactis deletion mutants show no KlCts1p secretion as measured by chitinase activity. FIG. 3 (B) shows that secretory chitinase could not be detected from the sample corresponding to the deletion mutant by Western blot (lane 1), whereas chitinase was easily detected by wild type (lane 2). FIG. FIG. 4 (A) shows K. on Western blot using α-CBD antibody. It is a figure which shows presence of lactis origin secreted chitinase (KLCts1p). This assay required the secreted chitinase to be bound to a chitin column and then eluted with various pH buffers or boiled for 2 minutes. FIG. 4B shows that elution of chitinase (KLCts1p) from chitin occurs almost immediately using 20 mM NaOH. KlCts1p bound to chitin was eluted as 5 consecutive 20 mM NaOH 1 ml fractions (E ₀ -E ₄ , where E ₀ is the column void volume), separated by SDS-PAGE, and α-CBD Western Detection was by blotting. FIG. 4 (c) is a graph showing that KlCts1p eluted with 20 mM NaOH retains chitinolytic activity. KlCts1p bound to chitin was eluted from the chitin minicolumn using various concentrations of NaOH. The chitinase activity of the eluate was evaluated by measuring the release rate 4-MU / min from 50 mM 4MU-GlcNAc ₃ at pH 4.5 and 37 ° C. FIG. 4 (D) shows that native KlCBD can function as an elution affinity tag alone or as part of a fusion protein. CBD is K.K. The fusion protein (human serum albumin (HSA) -KlCBD) obtained from lactis is chitinase-deficient K. It is expressed in lactis mutants (K. lactis Δcts1 cells) and elution conditions from chitin beads are 20 mM NaOH. In contrast, B.I. CBD fusion protein from B. circulans (HSA-BcCBD) cannot be eluted from chitin beads with 20 mM NaOH as well. Control (B-PB) is a fusion protein eluted by boiling chitin beads. It is a figure which shows a pGBN16 (pKLAC1) expression vector. The desired gene is cloned into the same translational reading frame as the mating factor alpha prepro secretion leader sequence present in the vector. A polylinker containing a unique restriction enzyme cleavage site is present to allow cloning of the desired gene. K. FIG. 2 shows the secretion of recombinant protein from lactis Δcts1 cells. FIG. 6A shows Δcts1 K.I. It is a figure which shows secretion of the maltose binding protein (MBP) derived from a lactis cell. The yield is equivalent to or better than that derived from wild type cells. K. FIG. 2 shows the secretion of recombinant protein from lactis Δcts1 cells. FIG. 6B shows Δcts1 K.I. It is a figure which shows secretion of the HSA-KlCBD fusion protein derived from a lactis cell, and elution of the fusion protein from chitin by 20 mM NaOH. K. 2 is a flowchart outlining the secretion of CBD-labeled protein from lactis cells. FIG. 2 shows SDS-PAGE separation of CBD-labeled human serum albumin (HSA-CBD) isolated from culture using magnetic chitin beads added to growth medium at various times of culture growth. Lane 1 is a molecular weight marker. Lane 2 shows K. as a medium component. HSA-KlCBD obtained from autoclaved chitin magnetic beads added to lactis culture for 72 hours. Lane 3 is K.I. HSA-KlCBD obtained from autoclaved chitin magnetic beads added to lactis culture for 48 hours. Lane 4 shows K.I. 1 hour prior to collection. HSA-KlCBD obtained from chitin magnetic beads added to lactis culture. Lane 5 is HSA-KlCBD obtained from magnetic chitin beads added to the cell culture supernatant. 9A and 9B are diagrams showing a method for purifying a protein from a dilute solution using chitin magnetic beads. FIG. 9A Stage 1: Sterilize magnetic chitin beads (eg, high pressure steam sterilization, ultraviolet light, irradiation, chemical treatment, etc.). Step 2: After adding sterile chitin beads to the medium, inoculate the growth medium with cells. During cell culture growth, cells secrete proteins (open circles) labeled with a chitin binding domain (black circles). Step 3: The secreted CBD labeled protein is immobilized on magnetic chitin beads in growth medium. Step 4: At some point during culture growth, magnetic chitin beads containing bound CBD-labeled protein are separated from the cells and growth medium by fixing in a magnetic field. Step 5: Wash the beads with the desired buffer or medium. Step 6: Release the chitin bead-protein complex from the magnetic field. Step 7a: If CBD that can be dissociated from chitin is used in the construction of the fusion protein, the purified CBD fusion protein is eluted from the magnetic chitin beads. Step 7b: Depending on the desired application, the collected protein remains immobilized on the chitin magnetic beads indefinitely. 9A and 9B are diagrams showing a method for purifying a protein from a dilute solution using chitin magnetic beads. FIG. 9B Stage 1: Inoculate cells in medium without magnetic chitin beads. Step 2: Proliferating cells secrete a protein (white circle) labeled with a chitin binding domain (black circle). Step 3: Cells can be removed from the culture (eg, centrifugation, filtration, aggregation, gravity sedimentation, etc.). Step 4a: At any point during the growth of the culture, sterile magnetic chitin beads can be added directly to the culture. Step 4b: Magnetic chitin beads added to the working medium with the cells removed. Steps 5a and 5b: CBD labeled protein is separated from the cells and / or growth medium by immobilizing the beads by applying a magnetic field. Step 6: Wash the beads with the desired buffer or medium. Step 7: Release the chitin bead-protein complex from the magnetic field. Step 8a: If a CBD that can be dissociated from chitin is used to construct the fusion protein, the purified protein is eluted from the magnetic chitin beads. Step 8b: Depending on the desired application, the collected protein remains fixed on the chitin magnetic beads indefinitely. FIG. 10 (a) shows a magnetic rack suitable for separating magnetic beads from a preparation in a microtiter dish. FIG. 10 (b) shows a magnetic rack suitable for separating magnetic beads from a preparation in a microcentrifuge tube. FIG. 10 (c) shows a magnetic rack suitable for separating magnetic beads from a preparation in a standard 50 ml test tube. FIG. 10 (d) shows a magnetic rack suitable for separating magnetic beads from a preparation in a standard 250 ml centrifuge bottle. FIG. 11 (a) shows an electromagnetic probe that can be put into water, suitable for separating magnetic beads from a preparation in a growth vessel or fermentor. Steps 1 and 2: Electromagnetic probes (dark grey) are submerged in a growth vessel or fermentor containing protein immobilized on magnetic beads (grey). Step 3: When the electromagnet is turned on, the magnetic beads are fixed to the surface. Step 4: Remove the electromagnet (on) from the growth vessel or fermentor, thereby isolating the magnetic beads. FIG. 11 (b) shows a magnetic device suitable for isolating magnetic beads from the effluent of a fermentor or growth vessel. Step 1: The effluent from the fermentor or vessel containing protein bound to the medium, cells and magnetic beads (gray) flows out of the fermentor into or through the magnetic isolator. Step 2: Separate the magnetic beads from the residual effluent by a magnetic isolator consisting of an electromagnet or a removable permanent magnet. Stage 3: The remaining effluent passes through a magnetic separator. FIG. 4 shows that a secreted GluC-CBD fusion protein derived from Bacillus circulans can be obtained regardless of whether magnetized chitin beads are added initially, during cell culture, or after collecting the culture supernatant. . The gel shows the amount of GluC-CBD obtained after elution from magnetized chitin beads by boiling in SDS sample buffer. Lane 1: Control-unstained standard (Mark 12-Invitrogen, Carlsbad, CA); Lane 2: Control-GluC protein; Lane 3: GluC-CBD transformed B. pylori. Incubate Circulation cells overnight. Magnetized chitin beads were added to the medium at the start of incubation; Lane 4: B. transformed with GluC-CBD. Incubate Circulation cells overnight. Magnetized chitin beads were added to the medium 1 hour prior to medium collection; Lane 5: B. transformed with GluC-CBD. Incubate Circulation cells overnight. After the medium was collected and centrifuged, magnetized chitin beads were added to the supernatant. Depending on the amount of luciferase obtained in a series of fractions from the chitin column, luciferase-CBD elutes as fractions 2 to 10 under non-denaturing conditions and is most active in fractions 3, 4 and 5 It is the histogram which showed.

Claims (28)

(A) transforming host-expressing cells with a vector comprising DNA encoding a fusion protein comprising a chitin binding domain (CBD) and a target protein;
(B) expressing the fusion protein in the host-expressing cell and secreting it from the host-expressing cell; and (c) as desired under non-denaturing conditions so as to obtain a concentrated preparation of the secreted recombinant protein. Binding the secreted fusion protein, eluting into a buffer volume, to the chitin preparation via the CBD;
To obtain a concentrated preparation of secreted recombinant protein.   The method of claim 1, wherein the vector of step (a) is a shuttle vector.   The DNA in the vector encoding the target protein is E. coli. 3. The method of claim 2, wherein the method is transcriptionally dormant in E. coli and is transcriptionally active in the host-expressing cell.   The method of claim 2, wherein the shuttle vector has a modified LAC4 promoter.   The method of claim 4, wherein the shuttle vector is pK1AC1.   The method according to claim 1, wherein the host-expressing cell is a chitinase-deficient cell.   4. The method of claim 3, wherein the host expression cell is a yeast cell.   6. The method of claim 5, wherein the yeast cell is a single species selected from Kluyveromyces, Yarrowia, Pichia, Hansenula, and Saccharomyces species.   8. The method of claim 7, wherein the host-expressing cell is Kluyveromyces.   10. The method according to claim 9, wherein the Kluyveromyces is Kluyveromyces marxianus variant fragilis or lactis.   The method of claim 1, wherein the host-expressing cell is in culture such that the fusion protein is secreted into the medium.   The method according to claim 11, further comprising adding chitin to the medium during culturing.   The method according to claim 12, wherein the chitin is sterile.   The method according to claim 1 or 11, wherein chitin is added to the mixture after culturing.   The method according to claim 1 or 13, wherein the chitin is in a form selected from coatings, colloids, beads, columns, matrices, sheets and membranes.   The method according to claim 1 or 13, wherein the chitin is a chitin bead and the bead is porous or non-porous.   The method of claim 16, wherein the chitin beads are magnetized.   The method of claim 17, wherein the recovery of the fusion protein bound to chitin in step (d) further comprises applying a magnetic force.   2. The method of claim 1, wherein binding of the fusion protein to chitin is reversible so that the fusion protein can be released from chitin under non-denaturing conditions different from the binding conditions. (A) providing a shuttle vector (the shuttle vector can be (i) integrated into the genome of a yeast host, and (ii) a DNA encoding a fusion protein comprising a chitin binding domain and a target protein) including.),
(B) transforming a chitinase-deficient host-expressing cell with the shuttle vector that expresses the fusion protein in a yeast host-expressing cell, and secreting the fusion protein from the chitinase-deficient host-expressing cell; and (c) secreted A method for obtaining a concentrated preparation of a secreted recombinant protein comprising linking a secreted fusion protein to a chitin preparation by CBD so that a concentrated preparation of the purified protein is obtained.   Kluyveromyces cell preparation characterized by a chitinase negative phenotype that is the result of a mutation in a chitinase gene that expresses a secreted chitinase and capable of growing to a cell density similar to wild type Kluyveromyces cells object.   22. The Kluyveromyces cell preparation of claim 21, further capable of expressing and secreting the recombinant protein.   23. The Kluyveromyces cell preparation of claim 22, wherein expression is regulated by the LAC4 promoter or a variant thereof.   Although the protein is expressed in Kluyveromyces, 22. The Kluyveromyces cell preparation of claim 21, comprising a shuttle vector having a modified Lac4 promoter that does not substantially express the protein in E. coli.   23. The Kluyveromyces cell preparation of claim 22, wherein the shuttle vector is pKLACI.   22. The Kluyveromyces cell preparation of claim 21, further comprising a medium capable of at least one of growth and maintenance of Kluyveromyces and further comprising sterile chitin.   27. The Kluyveromyces cell preparation of claim 26, wherein the sterile chitin is in the form of magnetic beads.   22. The Kluyveromyces cell preparation of claim 21, wherein the preparation is contained in a container that may contain a magnet that reversibly binds chitin beads or a container that may be in contact with the magnet.

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