KR20070085295A - Methods and compositions for concentrating secreted recombinant protein - Google Patents

Abstract

Methods and compositions are described that relate to obtaining concentrated preparations of secreted recombinant proteins. These proteins are expressed in the form of fusion proteins with a chitin-binding domain (CBD). The fusion proteins are capable of being concentrated in the presence of chitin. Also described is: a shuttle vector that includes a modified LAC4 promoter; a chitinase-negative host cell; a CBD capable of eluting from chitin under non-denaturing conditions; and sterilized chitin, which can be optionally magnetized for facilitating recovery of recombinant protein.

Description

Methods and Compositions for Concentrating Secreted Recombinant Protein

Chitin (β-1,4-linked unbranched polymer of N-acetylglucosamine (GlcNAc)) constitutes the second most abundant polymer after cellulose on earth. Chitin is known as the insect exoskeleton (Merzendorfer, H., et al., J. Exptl. Biol. 206: 4393-4412 (2003)), the shell and fungal cell walls of invertebrate crustaceans (Riccardo, A., et al. and fossil chitins ", in Chitin and Chitinases, ed. P. Jolles and RAA Muzzarelli, pub. Birkhauser Verlag: Basel, Switzerland (1999)). Chitinases hydrolyze the β-1,4-glycosidic bonds of chitin and are found in prokaryotic, eukaryotic and viral organisms. In yeast Saccharomyces cerevisiae, chitinases play a morphological role in efficient cell isolation (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991) ). In addition, plants express chitinases in defense against chitin-containing pathogens. Indeed, heterologous expression of the chitinase gene 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); Kim, J. et al. Trans. Res. 12: 475-484 (2003)). Chitinases belong to family 18 or family 19 of glycosylhydrolase based on their amino acid sequence similarity (Henrissat, B., et al. Biochem. J. 293: 781-788 (1993)). Family differences in the chitinase catalytic domain sequence reflect different mechanisms of their chitin hydrolysis causing maintenance (family 18) or reversal (family 19) of the anomeric arrangement of the product (Robertus, JD, et al. " The structure and action of chitinases, "in Chitin and Chitinases, ed. P. Jolles and RAA Muzzarelli, pub. Birkhauser Verlag: Basel, Switzerland (1999)).

Most chitinases have a modular domain configuration with distinct catalytic and non-catalytic domains that function independently of each other. O-glycosylated Ser / Thr-rich regions often can serve to separate the two domains, prevent proteolysis or help secrete chitinase (Arakane, Y., Q. et al. Insect Biochem.Mol Biol. 33: 631-48 (2003). Non-catalyst chitin binding domains (CBD is also called ChBD) belong to one of three structural classes (type 1, 2 or 3) based on protein sequence similarity (Henrissat, B. "Classification of chitinases modules," in Chitin and Chitinases, ed.P. Jolles and RAA Muzzarelli, pub.Birkhauser Verlag: Basel, Switzerland (1999)). Depending on the individual chitinases, the presence of CBD can enhance or inhibit chitin hydrolysis by the catalytic domain (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)). Hashimoto, M., et al. J. Bacteriol. 182: 3045-3054 (2000)).

The small size (~ 5-7 kDa) of CBD for chitin, substrate binding specificity and high avidity can be used as an affinity tag for immobilizing proteins on chitin surfaces (Bernard, MP, et al. Anal.Biochem.327: 278-283 (2004); Ferrandon, S., et al. Biochim. Biophys.Acta. 1621: 31-40 (2003)). For example, rain. The B. circulans chitinase A1 type 3 CBD provides a platform for intein-mediated protein splicing of fusion proteins expressed in bacteria (Ferrandon, 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) )). Since eukaryotic protein expression systems can perform biological processes that are not possible in bacterial systems (eg, protein glycosylation, chaperone-mediated protein folding, etc.), secreting CBD-tagged proteins from eukaryotic cells It is also desired. However, many eukaryotic cells, especially fungi, compete with CBD-tagged proteins for chitin-binding sites, co-purify with CBD-tagged proteins during chitin fixation applications, and destroy the target chitin-coated surface. Secretes endogenous chitinases that complicate the immobilization of tagged proteins to chitin.

Proteins secreted from the host cell into the surrounding medium must be substantially diluted, resulting in expensive and cumbersome purification from large volumes. It is desirable to reduce costs and increase the ease of protein separation from the medium in which they are secreted.

Various methods exist for purifying proteins from large volumes of medium. These methods differ in cost, efficiency, and length of time required to achieve the purification. For example, proteins in secreted culture can be harvested by precipitation. The solution requires the addition of a large amount of precipitant, for example ammonium sulfate, acetone or trichloroacetic acid, followed by centrifugation or filtration. Many of these agents are toxic or volatile and all add significant cost to protein collection. In addition, precipitation can cause significant loss of protein function.

Another approach is chromatography using various resins such as anion / cation exchange resins, hydrophobic interaction resins, or size exclusion gels. Protein collection by chromatography requires that all spent culture medium pass through the resin at a slow flow rate (usually 1-10 ml min ⁻¹ ). This can be very time consuming if a large volume of media has to be treated. For example, 100 liter of spent culture medium passed through the resin at 5 ml min ⁻¹ flow rate would take 333 hours to process. In addition, these types of chromatography resins do not selectively purify only the target protein, but often have to be used in conjunction with other methods in multistage purification processes.

Affinity chromatography resins that specifically bind to peptide sequences contained within the structure of a protein are often used because of their ability to selectively purify the target protein. In typical methods, peptide sequences (eg, peptide antibody epitopes or hexahistidine sequences) are engineered into the sequence of the protein of interest. Proteins expressed to have one of these tags will specifically interact with the corresponding resin (eg, a resin with immobilized antibodies or a nickel resin for hexahistidine bonds). These methods often produce highly purified proteins from small volumes, but because of their cost and performance they are limited in practicality in handling large volumes. For example, antibody affinity resins are very expensive and nickel resins can cause simultaneous purification of undesired proteins that inadvertently have a stretch of histidine residues.

Magnetic techniques using magnetic carriers containing beads have been used to purify proteins from cultures (Safarik et al. Biomagnetic Research and Technology 2: 7 (2004)). The problem with this solution is the need to set up each magnetic bead reagent to bind to the individual secreted protein. This may include complex chemistry to attach the affinity ligands to the beads. It also presents an obstacle in efficiency and cost.

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

Summary of the Invention

In one embodiment of the invention, (a) the host expressing cells are transformed with a vector comprising DNA (the DNA encodes a fusion protein comprising a CBD and a target protein); (b) expressing the fusion protein in a host expressing cell and secreting the fusion protein therefrom; (c) binding the secreted fusion protein to the formulation of chitin by CBD (the fusion protein can elute into the desired buffer volume under non-denaturing conditions) to obtain a concentrated formulation of secreted recombinant protein. The present invention provides a method for obtaining a concentrated preparation of secreted recombinant protein.

In another embodiment of the invention, (a) a shuttle vector is provided and said shuttle vector is (i) integrated into the genome of yeast expressing cells. Plasmids in E. coli, and (ii) DNA encoding a fusion protein comprising a CBD and a target protein}; (b) transforming the chitinase-deficient host expressing cells with a shuttle vector to express the fusion protein in yeast expressing cells and secrete the fusion protein therefrom; (c) binding the secreted fusion protein to the formulation of chitin by CBD to obtain a concentrated formulation of the secreted protein, thereby obtaining a concentrated formulation of the secreted recombinant protein.

Two embodiments are specific to certain embodiments. Can be cloned in E. coli but Illustrated is the use of shuttle vectors that are not expressed in E. coli and can be expressed in host expressing cells. An example of this type of shuttle vector is one containing a modified LAC4 promoter, further illustrated by pKLAC1.

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

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

Fusion proteins can be harvested when bound to magnetizing chitin by applying magnetic force. The binding of the fusion protein to the chitin is optionally reversible so that the fusion protein can be released from the chitin under non-denaturing conditions that differ from the conditions for binding.

In one embodiment of the invention, the preparation of the Kluyveromyces cells is characterized by a chitinase-negative phenotype, said phenotype being the result of a mutation in the chitinase gene expressing the secreted chitinase, said agent being wild type It can grow to a cell density similar to Veromyces cells. Cell density refers to the dry weight of cells in 48 h culture (Colussi et al. Applied and Environmental Microbiology 71: 2862-2869 (2005)).

Preferably, the preparation of Kluyveromyces cells can express and secrete recombinant fusion proteins. Expression is accomplished by the LAC4 promoter or a variant thereof, eg, E. coli. Can be controlled using a shuttle vector with a modified LAC4 promoter for expressing the protein in Kluyveromyces without substantially expressing the protein in E. coli. One example of a shuttle vector is pKLAC1.

The formulations of the Kluyveromyces cells described above may comprise a culture medium in which Kluyveromyces can do at least one of growth and maintenance. The culture medium may also include sterile chitin. Sterile chitin can be in the form of magnetic beads capable of binding to a magnet placed in the culture medium or in contact with a container containing the culture medium.

1 shows k with estimated masses> 200, 85 and 50 kDa. Three proteins secreted into the culture medium consumed by K. lactis were secreted. Western blots that cross-react with polyclonal antibodies raised against the Circula Chi1A chitin-binding domain (α-CBD) are shown (lane 1). 85 kDa protein binds to chitin beads and K. Corresponds to lactis chitinase (lane 2).

2 (A) shows multi-domain KlCts1p chitinase with signal peptide (stripes), catalytic domain (grey), Ser / Thr rich domain (white) and chitin-binding domain (black). Signal peptide cleavage occurs after A ¹⁹ .

2 (B) shows that KlCts1p belongs to family 18 of glycosylhydrolase. The predicted catalytic site of KlCts1p lies between amino acids 150-158. Alternative amino acids for each of the 9 positions are provided in parentheses. (“X” represents any amino acid.)

2 (C) shows that KlCts1p contains a type 2 chitin-binding domain. KlCts1p CBD was aligned with the Type 2 CBD consensus sequence (SEQ ID NO: 14) predicted by Simple Modular Architecture Research Tool (SMART) software (Letunic, I., et al. Nucl. Acids Res. 30: 242-244 (2002) Schultz, J., et al. PNAS 95: 5857-5864 (1998). KlCts1p CBD (SEQ ID NO: 15), fungus (Cladosporium fulvum (SEQ ID NO: 16); species-specific elicitor A4 precursor), bacteria (Ralsonia solanacearum) ( SEQ ID NO: 17); Q8XZL0), nematode (Caenorhabditis elegans (SEQ ID NO: 18); promising endocytase), mammal (Homo sapiens; human) (SEQ ID NO: 19); chitinase) And alignment with exemplary proteins containing predicted type 2 CBD from insects (Drosophila melanogaster; yellow fruit flies) (SEQ ID NO: 20); promising chitinase 3). Conserved cysteine residues are indicated in bold.

3 K. Lacked mutants of lactis do not secrete KlCts1p as measured by chitinase activity.

3 (A) shows the chitinase activity measured after cell growth for 22, 44 and 68 hours in YPD medium at 30 ° C. FIG. Activity was analyzed as the release rate of 4-MU min ⁻¹ (relative fluorescence unit, RFU min ⁻¹ ) from 50 mM 4MU-GlcNAc ₃ at pH 4.5 and 37 ° C.

FIG. 3 (B) shows that chitinase secreted on Western blotting could not be detected from the sample corresponding to the missing mutant (lane 1), while chitinase was easily detected from the wild type (lane 2).

Figure 4 (A) shows K. on Western blotting using α-CBD antibody. The presence of chitinase (KLCts1p) secreted from Lactis is shown. The assay required that the secreted chitinase be bound to the chitin column and then eluted by boiling in a buffer of varying pH or for 2 minutes.

4 (B) shows that elution of chitinase (KLCts1p) from chitin occurs almost immediately using 20 mM NaOH. Chitin-bound KlCts1p is eluted in five consecutive 1 ml fractions of 20 mM NaOH (E ₀ -E ₄ , where E ₀ represents column pore volume), separated by SDS-PAGE and α-CBD Western blotting Was detected by.

4 (C) shows that KlCts1p eluted at 20 mM NaOH retains chitinase activity. Chitin-bound KlCts1p is eluted from chitin minicolumns using various concentrations of NaOH, and the eluate is determined by measuring the release rate of 4-MU min ⁻¹ from 50 mM 4MU-GlcNAc ₃ at pH 4.5 and 37 ° C. The activity was analyzed.

4 (D) shows that native KlCBD can function as an elutable affinity tag alone or as part of a fusion protein, where CBD is K. Obtained from Lactis, the fusion protein (human serum albumin (HSA) -KlCBD) was deficient in chitinase K. Expressed in lactis mutants (K. lactis Δcts1 cells) and the conditions for elution from chitin beads are 20 mM NaOH. On the contrary, rain. The fusion protein of CBD from Circus (HSA-BcCBD) is similarly not elutable from chitin beads in 20 mM NaOH. Control (B-PB) is a fusion protein eluted by boiling chitin beads.

5 depicts a pGBN16 (pKLAC1) expression vector. The gene of interest is cloned in the same translation read frame as the mating factor alpha pre-pro secretion leader sequence present in the vector. Polylinkers exist that contain unique restriction sites to allow cloning of the gene of interest.

6 K. Secretion of recombinant protein from Lactis Δcts1 cells is shown.

6 (A) is Δcts1 k. Secretion of maltose-binding protein (MBP) from lactis cells is shown, yielding as good as or better than wild-type cells.

6 (B) is Δcts1 k. Secretion of HSA-KlCBD fusion protein from Lactis cells and elution of the fusion protein from chitin in 20 mM NaOH are shown.

7 K. Flow chart of the secretion of CBD-tagged proteins from lactis cells.

8 shows SDS-PAGE isolation of CBD-tagged human serum albumin (HSA-CBD) isolated from culture using magnetic chitin beads added to growth medium at various culture growth time points.

Lane 1 shows the molecular weight markers.

Lane 2 is K for 72 hours. HSA-KlCBD obtained from autoclave sterilized chitin magnetic beads added to the lactis culture as a media component.

Lane 3 is K for 48 hours. HSA-KlCBD obtained from autoclave sterilized chitin magnetic beads added to the lactis culture.

Lane 4 is K 1 hour before collection. HSA-KlCBD from chitin magnetic beads added to the lactis culture is shown.

Lane 5 shows HSA-KlCBD obtained from magnetic chitin beads added to the supernatant from cell culture.

9A and 9B show pictures of the manner in which chitin magnetic beads can be used to purify protein from dilute solutions.

Figure 9A

Step 1: Sterilize the magnetic chitin beads (eg autoclave, ultraviolet light, irradiation, chemical treatment, etc.).

Step 2: Sterile chitin beads are added to the growth medium before inoculating the cells into the medium. During growth of the cell culture, the cells secrete chitin-binding domains (black circles) and tagged proteins (open circles).

Step 3: The secreted CBD-tagged protein is fixed to magnetic chitin beads in growth medium.

Step 4: At some point during the growth of the culture, magnetic chitin beads comprising bound CBD-tagged proteins are separated from the cells and growth medium by exposure to magnetic fields to fix the beads.

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 capable of dissociating from chitin is used in the construction of the fusion protein, the purified CBD fusion protein is eluted from magnetic chitin beads.

Step 7b: Depending on the desired use, the harvested protein remains immobilized indefinitely on chitin magnetic beads.

Figure 9B

Step 1: Inoculate cells in culture medium without magnetic chitin beads.

Step 2: Growing cells secrete chitin-binding domains (black circles) and tagged proteins (open circles).

Step 3: The cells can be removed from the culture (eg centrifugation, filtration, aggregation, settling the cells by gravity, 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 clarified spent culture medium.

Steps 5a and 5b: CBD-tagged proteins are exposed to magnetic fields to immobilize the beads and separated from the cells and / or growth medium.

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 CBD capable of dissociating from chitin is used in the construction of the fusion protein, the purified protein is eluted from magnetic chitin beads.

Step 8b: Depending on the intended use, the harvested protein remains fixed indefinitely on chitin magnetic beads.

10 (a) shows a magnetic rack suitable for separating magnetic beads from a formulation in a microtiter dish.

Figure 10 (b) shows a magnetic rack suitable for separating magnetic beads from a formulation in a microcentrifuge tube.

10 (c) shows a magnetic rack suitable for separating magnetic beads from the formulation in a standard 50 ml laboratory tube.

10 (d) shows a magnetic rack suitable for separating magnetic beads from the formulation in a standard 250 ml centrifuge bottle.

11 (a) shows a picture of a submersible electromagnet probe suitable for separating magnetic beads from a formulation in a growth vessel or fermenter.

Steps 1 and 2: Electromagnet probes (dark gray) are immersed into growth vessels or fermenters containing proteins immobilized on magnetic beads (grey).

Step 3: Turn on the electromagnet, the magnetic bead is fixed on its surface.

Step 4: The electromagnet (turned on) is removed from the growth vessel or fermenter to isolate the magnetic beads.

11 (b) shows an illustration of a magnetic device suitable for isolating magnetic beads from the effluent of a fermenter or growth vessel.

Step 1: The effluent from the fermentor or vessel comprising the protein bound to the medium, cells and magnetic gray flows from or into the magnetic isolation device from the fermentor.

Step 2: A magnetic isolation device consisting of an electromagnet or removable permanent magnet separates the magnetic beads from the remaining effluent.

Step 3: The clarified effluent flows past the magnetic separation device.

12 shows that GluC-CBD fusion proteins secreted from Bacillus circulas can be obtained regardless of whether magnetized chitin beads are added at the beginning of the culture or during the culturing or after the culture supernatant is collected. The gel shows the amount of GluC-CBD obtained after elution from magnetized chitin beads by boiling in SDS sample buffer.

Lane 1: control group-unstained standard (Mark 12-Invitrogen, Carlsbad, Calif.);

Lane 2: control-GluC protein;

Lane 3: GluC-CBD transformed ratio. Overnight incubation of circular cells. Magnetized chitin beads were added to the culture medium at the start of incubation;

Lane 4: GluC-CBD transformed ratio. Overnight incubation of circular cells. Magnetized chitin beads were added to the culture medium 1 hr before collection of the medium;

Lane 5: GluC-CBD transformed ratio. Overnight incubation of circular cells. Magnetized chitin beads were added to the supernatant after harvesting and centrifugation of the culture medium.

FIG. 13 shows histograms showing the amount of luciferase obtained in a series of fractions from the chitin column, in which Luciferase-CBD eluted in fractions 2 to 10 under non-denaturing conditions and the highest activity was found in fractions 3, 4 and 5. Illustrated.

Described herein are methods for concentrating a protein after secretion from the host cell into which the protein is prepared into the culture medium. The method can be improved by using the binding affinity of CBD for chitin and using cells that do not secrete chitinase. Chitinase-negative cells can be produced as a result of genetic modifications or can occur naturally. It is desired that these chitinase-deficient modified cells can be grown in equal yield at a similar density as wild type cells. Host cells can be transformed using a vector encoding a target gene fused to DNA expressing CBD under a suitable promoter so that a relatively large amount of target protein can be secreted into the production medium by the host cell. Chitin substrates may be present in the production medium or in separate reaction vessels to extract the target protein from the mixture. Binding of the CBD fusion protein concentrates the secreted recombinant protein onto the surface of the chitin. Proteins can be further concentrated using any number of means. For example, in one embodiment, chitin is magnetized and a magnetic field is applied to the production medium to concentrate the chitin beads adjacent to the magnetic surface. Another embodiment includes precipitation of chitin beads by centrifugation. The target protein can then be harvested from the concentrated chitin substrate.

The term "concentrated" refers to a greater weight to volume ratio after running the procedure than before the procedure.

Chitinase  Modification of Host Cells to Knock Out Expression

Preferred host cell backgrounds for the secretion of recombinant CBD-tagged proteins do not (i) produce chitin-binding proteins or chitinase activity that will contaminate the preparation of secreted fusion proteins containing CBD; (ii) achieve high cell density in culture; (iii) can efficiently secrete recombinant proteins. Advantages of host cells that do not secrete chitinases include (i) elimination of competition for chitin-binding sites between CBD-tagged proteins and endogenous chitinases; (ii) elimination of the risk of contamination of chitin-fixed fusion proteins by endogenous chitinases; And (iii) elimination of degradation of the target chitin matrix by endogenous chitinase.

Suitable host cells include production lines of various insect cell cultures and mammalian cell lines and yeast producing strains and bacterial cells.

Examples of cells from which proteins are secreted therefrom for manufacturing purposes are E. coli. E. coli, Salmonella spp., Bacillus spp., Streptomyces spp., Etc., plant cells (eg, Arabidopsis spp., Taxus spp., Catharanthus spp. , Nicotiana species, Oryza species, soybeans, alfalfa, tomatoes, etc.), fungal cells (eg, Kluyveromyces spp., Saccharomyces spp., Peachia spp., Hanshenula spp., Yarrowia spp., Neurospora spp., Aspergillus spp., Penicillium spp., Candida spp., Schizosaccharomyces spp., Cryptococcus spp. , Coprinus spp., Ustilago spp., Magnaporth spp., Trichoderma spp., Etc., insect cells (e.g., Sf9 cells, Sf12 cells, Tricoplusia age ( Trichoplusia ni, Cabbage Silver Pattern Chestnut Moth), Drosophila And a bell, etc.), or mammalian cells (e.g., primary cell lines, HeLa cells, NSO cells, BHK cells, HEK-293 cells, PER-C6 cells, etc.). These cells can grow in culture ranging from microliter volumes to multiliter volumes.

Kluyveromyces species are used herein to describe how secreted proteins fused to CBD can be quickly and easily separated from the mixture. Yeast in the genus Kluyveromyces according to an embodiment of the invention is described in van der Walt in The Yeasts, ed. N. J. W. Kregervan Rij: Elsevier, New York, NY, p. 224 (1987), and yeast. Martian strain Lactis (K. lactis), K. Marcianus variant Marcianus (K. pragilis), K. Marcianus strains drosophilarum (K. drosophilarum) and K. K. waltii and other strains classified as Kluyveromyces in the art.

In host cells that naturally secrete one or more chitinases, chitinase deletion mutants can be produced by genetic modification. Genetic modification means any inhibition, substitution, deletion or addition of one or more bases in a target gene. The modification can be carried out in vitro (on isolated DNA) or in situ, for example by genetic engineering techniques, or alternatively, by transferring the host cell to a mutagen such as radiation (X-ray, gamma-ray, Ultraviolet light, etc.), or chemical agents capable of reacting with various functional groups of the base of DNA, for example alkylating agents: ethyl methanesulfone (EMS), N-methyl-N'-nitro-N-nitrosoguanidine, N-nitro Obtainable by exposure to quinoline 1-oxide (NQO), bialkylating agents, intercalating agents and the like. In addition, expression of the target gene can be inhibited by modifying all or part of the portion of the region encoding chitinase and / or the transcriptional promoter region.

Genetic modifications can also be obtained by gene disruption. One example of gene disruption of chitinase is provided in Example 1 for Kluyveromyces lactis. The method described in the examples is broadly applicable to any species of Kluyveromyces species.

In Example 1, the chitinase gene encoding KlCts1p is preferably K. Industrial Kay lacking the Lactis killer plasmid. Destroyed in lactis strain (GG799). Destruction is part of the chitinase gene, for example, natural k. This occurred by replacing the first 168 amino acids of the lactis chitinase gene with a selectable marker gene, eg, a G418 resistance cassette.

K. Lactis GG799 Δcts1 cells were able to achieve the same high cell density as wild-type cells in culture (Example 2) and did not produce proteins with detectable chitin binding or chitinase activity (FIG. 3A) and enriched with recombinant protein Could be secreted (Fig. 6). The strain proved to be very suitable as a host for the production of recombinant CBD-tagged proteins.

For manufacturing purposes, chitinase-negative mutant host cells will achieve high cell densities similar to wild-type cells in culture despite the lack of secreted proteins with detectable chitin binding or chitinase activity, and these cells are recombinant It is desirable to be able to secrete abundant proteins. Thus, chitinase-negative host cells can be used for fermentation to efficiently prepare purified recombinant proteins linked to CBD, either directly or via linker peptides or linkage chemistry with industrial utility.

In yeast High standard  Design and use of vectors to express and secrete proteins

Examples of expression vectors for various yeasts are described in Muller et al. Yeast 14: 1267-1283 (1998) and US 4,859,596, US 5,217,891, US 5,876,988, US 6,051,431, US 6,265,186, US 6,548,285, US 5,679,544 and US patent application 11 / 102,475.

Expression vectors can be exogenous. For example, YEp24 is an episomal shuttle vector used for gene overexpression in Saccharomyces cerevisiae (New England Biolabs, Inc., Ipswich, Mass., USA). . Other examples of episomal shuttle vectors for the organism are pRS413, pRS414, pRS415 and pRS416. The autologous vectors in Kluyveromyces are 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 full pKD1 vector, smaller vectors containing the pKD1 origin and cis-acting stability locus locus (CSL) are K. It was constructed and used for heterologous protein expression in Lactis (Hsieh, et al. Appl. Microbiol. Biotechnol. 4: 411-416 (1998)). Other episome vectors are also K. Replicate within Lactis. Plasmids having both cen and autoreplicating sequences (ars) are K. It was used for cloning the expression of fungal cDNA in lactis (van der Vlug-Bergmans, et al. Biotechnology Techniques 13: 87-92 (1999)). In addition, K. Vectors comprising a lactis ARS sequence (KARS) can be used for 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 be integrated into the host genome. See, for example, US Pat. No. 6,602,682, US Pat. No. 6,265,186 and US patent application Ser. No. 11 / 102,475 for plasmids that integrate into the genome of Kluyveromyces.

Vectors include (i) a strong yeast promoter; (ii) DNA encoding the secretory leader sequence (if secretion of the protein into the medium is required); (iii) a gene encoding a protein to be expressed; (iv) transcription terminator sequences; And (v) at least one of yeast-selectable marker genes. These sequence components are usually E. coli. After assembly in plasmid vectors in E. coli, it is delivered to yeast cells to achieve protein production. Vectors of this kind are called shuttle vectors.

Before transforming the host cell. Shuttle vectors are preferred because they can be prepared in E. coli, but embodiments of the present invention are not limited to shuttle vectors.

For example, DNA fragments that can be integrated into the yeast genome can be prepared by PCR or helicase dependent amplification (HDA) or introduced directly into yeast cells. Alternatively, the expression vector is E. coli. It can be assembled by a cloning step in bacteria other than E. coli or directly in yeast cells.

Overexpression of proteins in Kluyveromyces and more generally in yeast involves the construction of shuttle vectors containing DNA fragments having sequences suitable for directing high level transcription of the gene of interest upon introduction into the yeast host. For example, P _LAC4 can function as a strong promoter for protein expression in yeast when present on integrating plasmids or episomal plasmids such as pKD1-based vectors, 2 micron-containing vectors, and isotopic vectors. have. Secretion leader sequence (if secretion of the protein into the medium is required). Cerebizia may comprise an α-MF pre-pro secretory leader peptide. Other prokaryotic or eukaryotic secretion signal peptides (eg, Kluyveromyces α-Mating Factor Pre-Pro secretion signal peptides, Kluyveromyces killer toxin signal peptides) or synthetic secretory signal peptides may also be used. Alternatively, the secretory leader can be omitted entirely from the vector to achieve cellular expression of the protein of interest.

Shuttle vectors allow the cloned genes to proliferate in bacteria before they are introduced into yeast cells for expression. However, yeast expression systems using wild type P _LAC4 may be used in bacterial host cells such as _E. coli. It can be adversely affected by accidental expression of proteins from genes under the control of P _LAC4 in E. coli. The promoter activity may inhibit the cloning efficiency of genes whose translation products are harmful to bacteria.

P _LAC4 variants with mutated Pribnow box-like sequences were K. a. It can be generated by site-directed mutagenesis that substantially retains their ability to function as strong promoters in Cluyveromyces sp. Illustrated by but not limited to lactis. These mutant promoters function substantially as well as or better than the non-mutated _Pripno box-like sequence in wild type P _LAC4 .

The term “mutation” is intended herein to include any of the substitutions, deletions or additions of one or more nucleotides in a 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. E. coli and a series of P _LAC4 variants have the following characteristics: (a) the regions -198 to -212 of the promoter, eg, positions -201, -203, -204, -207, -209 and -210 . Does not substantially impair the ability of the promoter to function as a strong promoter in lactis; (b) the -133 to -146 regions of the promoter, such as positions -139, -140, -141, -142 and -144, do not substantially inhibit strong promoter activity; (c) zones -198 to -212 and -133 to -146; (d) K. _S. of 283 bp (-1 to -283) replacing the -1 to -283 region of Lactis P _LAC4 . In the cerevisiae a hybrid promoter consisting of the (Sc) PGK promoter was generated. Such 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 complement strain nutritional components (eg, ura3, trp1, his3). , lys2, etc.) or acetamidase (amdS) gene. Expression of acetamidase in transformed yeast cells lacks a simple nitrogen source but allows growth on medium containing acetamide. Acetamidases decompose acetamide into ammonia, which can be used as a nitrogen source by cells. The advantage of this selection method is that it enriches the population of transformants for cells that contain multiple tandem integration of pKLAC1-based expression vectors and produce more recombinant protein than a single integration (FIG. 5).

The vector described above comprising a mutant of P _LAC4 is _{E. coli} . E. coli / Kluyveromyces integration shuttle vectors, for example pGBN1 and pKLAC1 (US Patent Application 11 / 102,475), respectively, can be inserted into the Kluyveromyces genome after transformation of the omnipotent host cell and subsequently Specify protein expression.

US patent application 11 / 102,475 describes yeast, more particularly K., which provides improvements to vectors described in US 4,859,596, US 5,217,891, US 5,876,988, US 6,051,431, US 6,265,186, US 6,548,285, US 5,679,544. Shuttle vectors containing the mutant P _LAC4 for use in Kluyveromyces exemplified by _lactis are described. The improvement is useful for expression in yeast of modified LAC4 and E. coli. It is due to the lack of the ability to express proteins in E. coli, thereby preventing problems due to toxicity in bacterial cloning host cells.

Introduction of DNA Vectors into Host Cells

Methods of 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 gene crossover, protoplast fusion, lithium based transformation, electroporation, conjugation or, for example, 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: 147-153 (2000). Established techniques for the transformation of Kluyveromyces yeast 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.

Including elution characteristics CBD Characteristics of

CBD as a component of chitinase can be obtained from many different sources, for example fungi, bacteria, plants and insects. Any CBD derived from chitinase can be used herein, but CBD isolated from chitinase enzyme activity is preferred. Also preferred are CBDs that can dissociate from chitin under non-denaturing conditions that differ from those that permit binding. Not all CBDs can dissociate from chitin under non-denaturing conditions. For example, rain. Circula CBD binds tightly to chitin, which is described in U.S. 6,897,285, U.S. It is not reversible unless mutations are introduced into the protein as described in application publications 2005-0196804 and 2005-0196841. In contrast, Kluyveromyces species produce CBD which binds tightly to chitin but can be reversibly dissociated under altered conditions, eg NaOH (see Examples 5 and 6).

Kluyveromyces produces abundantly expressed secreted endo-kinase (KCBD), which is shown herein as an example of an affinity tag that is effective to allow reversible fixation or purification of alkaline or alkali resistant proteins. KCBD binds to chitin in the absence of a catalytic domain (see, eg, FIG. 4D), and functions as an affinity tag for heterologously expressed proteins in Kluyveromyces as illustrated in FIGS. 4D and 6B. Can be dissociated from chitin in NaOH from 5 mM to 500 mM, while B. CBD from Circus (BcCBD) cannot.

CBD Of chitin for binding to

Synthetic or natural chitin can be used to bind CBD fusion proteins. Examples of synthetic chitin are acetylated chitosan, polymerized N-acetylglucosamine monosaccharides, polysaccharides or oligosaccharides, or polymerized glucosamine monosaccharides, oligosaccharides or polysaccharides where glucosamine is subsequently chemically acetylated.

Examples of natural chitin are chitins derived from crab shells, insect exoskeleton or fungal cell walls, or from any source known in the art.

Chitin can optionally be immobilized on a substrate as described in FIG. Examples of substrates are polymers such as plastics. Alternatively, chitin can aggregate to form, for example, suspensions, colloids, beads, columns, matrices, sheets or membranes. Chitin may be sterilized for addition to the fermentation medium during fermentation or may be used in unsterile form to bind to the fusion protein at the end of the fermentation process.

Chitin-coated magnetic Bead  using CBD Generally applicable methods for concentrating any secreted protein fused to a

Chitin substrates for binding to secreted CBD fusion proteins can be optionally magnetized to facilitate removal of the target protein from the culture medium. Magnetized chitin is made by combining chitin with a magnetic material. The magnetic material may be dispersed debris, for example iron filings. In a preferred embodiment, the magnetized chitin is in bead form, but the magnetized chitin can be used as a coating on additional materials that may optionally be inert. In a preferred embodiment, the magnetized material is a magnetized chitin, while (a) can bind to or be expressed as a fusion with the target protein; (b) Other magnetic materials can be used that have the property of binding to a magnetizable material.

In one embodiment, magnetized chitin beads (New England Biolabs, Inc.) are used to bind to secreted CBD fusion proteins. Although the size of the beads is not critical, there is an advantage of forming colloids in the medium until beads formed from sizes less than 200 nm in diameter pass through the sterile filter and magnetic force is applied (see, eg, 5,160,726). Larger beads may also be used. Chitin beads can be solid (eg New England Biolabs, Inc.) or porous (eg JP 62151430). The beads may also be magnetized by various means, for example by dispersing iron filings throughout the beads or by forming beads with iron cores (coating iron with chitin) (see eg 5,262,176). Magnetized chitin, as used herein, is in bead form in preferred embodiments, but is not intended to exclude other shapes and sizes of chitin surfaces.

Magnetic chitin beads may also be added to the growth medium during or after growth of the cell culture, or after removing the grown cells from the culture (FIG. 9B). Thus, it was shown herein that the magnetized chitin beads can be sterilized without significantly altering their binding properties. When chitin beads are added to the culture medium during cell growth, the beads are preferably sterilized.

In general, the maximum binding of CBD-tagged proteins to chitin beads (FIG. 9B, steps 4a and 4b) will occur within 1 hour at 4 ° C., although other temperature and time structures are also possible. Proteins immobilized on magnetic chitin beads are harvested in the magnetic field (FIGS. 9B, steps 5A and 5B) and cells contaminating the protein and growth medium are washed away from the beads (FIG. 9B, step 6). The chitin bead-protein complex is then released from the magnetic field (FIG. 1B, step 7) and the harvested proteins can remain fixed indefinitely on the chitin magnetic beads (FIG. 9B, step 8b) or the elutable CBD is affinity When used as a tag it can be dissociated from chitin (FIG. 9B, step 8a).

By adding magnetized chitin beads to the medium in a fermentation vessel, a very efficient one step process for harvesting the target protein can be achieved. Cells are grown in a medium containing chitin beads magnetized so that the secreted CBD-tagged protein binds to the beads during incubation, and by a simple step of applying a magnetic force from a magnet outside the fermentation vessel or, if desired, inside the fermentation vessel. The harvest can be taken directly from the medium (see Figures 9-11). The solution requires that the magnetized chitin beads be sterilized. The yield of sterile chitin beads of size 50-70 μm added at the beginning of fermentation or during fermentation was found to be similar to the yield of protein obtained when beads were added at the completion of fermentation (FIG. 8).

A notable feature of the inventive solution for concentrating secreted proteins from large volumes is their universality. The method takes advantage of the ability of the chitin-binding domain to bind chitin when fused to the additional protein if the function of the additional protein is not impaired by the presence of CBD.

By ensuring that cells secreting a fusion protein do not naturally produce a protein that binds to chitin, competitive binding and contamination are prevented. CBD binds to chitin with significant binding activity. Allowing the desired protein CBD fusion protein to be harvested from chitin beads can be achieved by mutating the CBD (US 6,897,286) or alternatively releasing the protein from the CBD-chitin complex so that the binding can be reversed to release the fusion protein under controlled conditions. By using an intein cleavage system or protease cleavage (WO 2004/053460, US 5,643,758). In addition, if a proteolytic cleavage site is present between the CBD and the protein of interest, the CBD tag is released from the protein of interest by digestion with a protease (eg enterokinase, genenase, purine, factor X, etc.). Can be.

Due to the strong nature of the CBD-chitin interaction, CBD-tagged proteins are rapidly fixed to any form of magnetized chitin, for example to beads.

If the magnetized chitin is in bead form, the chitin beads containing the bound CBD-tagged protein can be collected within seconds in the magnetic field. If desired, CBD-tagged proteins can be dissociated from the substrate magnetized by incubation in the elution buffer (if elutable CBD is used). Advantages of the method include improved speed, cost effectiveness and simplicity.

Typical laboratory tubes (eg, 96 well microtiter plates, microcentrifuge tubes, 15 ml Falcon tubes, 50 ml Falcon tubes) for harvesting CBD-tagged proteins from several microliters to several liters of culture medium , Magnetic separation device with 250 ml Nalgene bottle) is used. The process can be easily scaled up to harvest CBD-tagged proteins from larger volumes of media. In a preferred embodiment, the magnet can be made from rare earth metals (eg, neodymium, samarium, cobalt, etc.) but other kinds of magnets can also be used (eg ferrites, ceramics, electromagnets, etc.).

Expression system  Usage

Production and isolation of target proteins from mixtures is necessary for proteins used in pharmaceuticals, food or industrial use. The list of proteins for which fermentation based production is present or desired is very large. Examples include superoxide dismutase, catalase, amylase, lipase, amidase, glycosidase, xylanase, laccase, ligninase, chymosin and the like or any fragment or derivative thereof, blood derivatives (e.g. For example, serum albumin, alpha- or beta-globin, coagulation factors such as factor VIII, factor IX, von Willebrand factor, fibronectin, alpha-1 antitrypsin, or any fragment or derivative thereof), Insulin and variants thereof, lymphokines such as interleukin, interferon, colony stimulating factors (G-CSF, GM-CSF, M-CSF, etc.), TNF and the like or any fragment or derivative thereof, growth factors (eg, Growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, or any fragment or derivative thereof, apolipoproteins and molecular variants thereof, antigenic polypeptides for vaccine preparation (hepatitis, cytomegalovirus, Epstein-Bar (Epstein-Bar) Barr) virus, herpes virus, etc.), single chain antibody (ScFv) or alternatively polypeptide fusions, eg, fusions containing biologically active moieties fused to stabilizing moieties.

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

Example  1: follow up Example  Materials and Methods

Yeast strains, culture conditions and conditions for transformation

K. Lactis and S. Strains of cerevisiae (Table 1) were conventional at 3O <0> C in YPD medium (1% yeast extract, 2% peptone and 2% glucose) or YPGal medium (1% yeast extract, 2% peptone and 2% galactose). Cultured in a phosphorous manner.

K. Lactis and S. Transformation into Serevisia was achieved using electroporation. K. Transformants of lactis were selected by growth on YPD agar containing 200 mg G418 ml ⁻¹ , whereas S. Transformants in cerevisiae are either SD medium (0.67% yeast nitrogen base, 2% glucose) or SGal medium (0.67% yeast nitrogen base, 2% galactose) containing the appropriate supplements needed to supplement strain nutritional components. Obtained by growth on the phase.

Secreted K . Lactis  Detection and Isolation of Chitin-binding Proteins

Western blotting is used to cross-react with polyclonal anti-chitin-binding domain antibodies (α-CBD) generated against chitin-binding domains derived from Bacillus circus chitinase A1 (New England Biolabs, Inc.). Secreted K. Lactis protein was detected.

(a) K after 48-96 hours growth. The spent culture medium was isolated from the Lactis GG799 culture by centrifugation at 4000 × g for 10 minutes.

After centrifugation, the protein in spent media was separated by SDS-PAGE on 4-20% Tris-glycine polyacrylamide gel (Daiichi Pharmaceutical Corp., Montvale, NJ), Protran nitrocellulose membrane (Schleicher & Schuell Bioscience, Key, New Hampshire, USA). Membranes were blocked overnight at 4 ° C. in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) and 5% skim milk (w / v), and α-CBD polyclonal antibody (containing 5% skim milk) 1: 2000 in PBS-T) followed by horseradish peroxidase conjugated anti-rabbit secondary antibody (Kirkegaard & Perry Laboratories, Gettersburg, MD); 1: 2000 in PBS-T containing 5% skim milk). Protein-antibody complexes were visualized using LumiGlo ™ detection reagent (Cell Signaling Technologies, Beverly, Mass.).

(b) K. To isolate secreted protein that binds to chitin. Lactis GG799 cells were grown 96 hours in 20 ml YPD medium. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a new tube containing chitin beads (New England Biolabs, Inc.) washed with 1 ml of water and incubated with gentle rotation for 1 hour at room temperature. . Chitin beads were collected by centrifugation and washed with 10 ml of water. Approximately 50 μl volume of protein-bound chitin beads are removed and the bound protein is eluted by boiling in a protein loading buffer for 2 minutes, after which the eluted protein is separated by SDS-PAGE and α-CBD polyclonal Western analysis using antibodies or amino-terminal protein sequencing was applied.

K . Lactis Chitinase  Derived chitin-binding domain ( KlCts1p CBD Analysis of chitin-binding properties

20 ml K. KlCts1p from Lactis GG799 spent culture medium was bound to 1 ml volume of chitin beads as described above. KlCts1p-bound beads were washed with 10 ml of water and resuspended in 1.5 ml of water. Minicolumns were prepared by dispensing 100 μl aliquots of KClTs1p-bound beads into individual disposable columns (Bio-Rad Laboratories, Hercules, Calif.). The following buffers in 1 ml volume (˜10 bed volumes) were passed onto individual minicolumns: 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, 5 M NaCl and 8 M urea. Each column was then washed with 2 ml of water, and the beads were resuspended in 200 μl of water, transferred to a microcentrifuge tube and collected by centrifugation for a short time. The remaining protein bound to chitin was eluted by boiling the beads for 5 minutes in 50 ml of 3 × SDS-PAGE loading buffer (New England Biolabs, Inc.). Eluted proteins were separated by SDS-PAGE as described above and detected by Western analysis.

KlCts1p elution profile using varying concentrations of NaOH (0-40 mM) was performed in a similar manner. An aliquot of chitin-bound KlCts1p (100 μl) was prepared as described above and inserted into a 1.5 ml microcentrifuge tube to create a spin column (Millipore, Billerica, Mass.) ) Into). Flow-through was collected by microcentrifugation at 15,800 x 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. Chitinase activity in each eluate was measured as described below.

K . Lactis Chitinase  gene ( KlCTS1 Destruction of)

PCR-based methods were used to construct linear DNA disruption fragments consisting of the ADH2 promoter-G418 resistant gene cassette with 80-82 bp of f KlCTS1 DNA on both ends. When integrated at the KlCTS1 locus, the fragment replaces the DNA encoding the first 168 amino acids of KlCts1p with a G418-resistant cassette. Primer with tail, comprising DNA hybridizing to ADH-G418 sequence (not underlined) and consisting of KlCTS1 DNA sequence (underlined), 5'- CCAGTAATG CAACTATCAATCATTGTGTTAAACTGGTCACCAGAAATACAAGATATCAAAAATTACTAATACTACCATAA GCCATCATCATATCGAAG-3 '(SEQ ID NO: 5) Destructive DNA fragments were amplified from ADH2-G418-containing vector pGBN2 using Taq DNA polymerase using CCAAACTAGCGTATCCGGTTGGATTATTGTTTTCGATATCGAAATCGAAACCATCGACGACAGCAGTGTC GAATGGTCTTTC CCCGGGGTGGGCGAAGAACTCC-3 '(SEQ ID NO: 2). The amplified product was k. They were selected on YPD agar containing ^1-lock tooth GG799 transformed cells and colonies 200 mg ml G418. Pre-cells using KlCTS1-specific forward primer 5'-GGGCACAACAATGGCAGG-3 '(SEQ ID NO: 3) (designed upstream of the integration site) and G418-specific reverse primer 5'-GCCTCTCCACCCAAGCGGC-3' (SEQ ID NO: 4) PCR was used to amplify ˜600 bp diagnostic DNA fragments from cells that correctly incorporated the disruption DNA fragments at the KlCTS1 locus. Of the 20 transformants tested in this manner, two Dcts1 K. Lactis strains were identified and further characterized.

s . In Serbia KlCTS1 Heterologous expression of

s. Three Levy Jia to express the KlCTS1 within, 5'-GGC GGATCC gene primers GCCACCATGTTTCACCCTCGTTTACTT-3 '(BamHI site is also underlined) (SEQ ID NO: 5) and 5'-ACAT GCATGC CTAGAAGACGACGTCGGGTTTCAA-3' (SphI site Are underlined) (SEQ ID NO: 6) and cloned into the BamHI-SphI site of pMW20 (31) to express KlCTS1 expression by galactose-inducible / glucose-inhibitable S. a. Serevidia was placed under the control of the GAL10 promoter. The expression construct was transformed by S. a. Cerebizia was introduced into Δcts1 strain RG6947. In order to induce the production of KlCts1p, 2 ml from the culture medium 20 mg of uracil ml ^- After overnight growth at 3O ℃ in SD medium containing ^1, 20 ml using each of the culture medium of 1 ml YPD, and 20 ml YPGal broth Was inoculated. After each culture was grown overnight with shaking at 30 ° C., spent culture medium was analyzed for cell morphology and chitinase production by microscope.

Chitinase  Active measurement

Chitin oligosaccharides of 1-4 GlcNAc residues each induced with 4-methyl umbelliferone (4-MU) were used as substrates: 4-methylumbelliferyl N-acetyl-β-D-chitotrioxide (4MU-GlcNAc). ), 4-methylumbeliferil N, N'-diacetyl-β-D-chitotetraoxide (4MU-GlcNAc ₂ ), 4-methylumbeliferil N, N ', N "-triacetyl-β- D-chitotrioxide (4MU-GlcNAc ₃ ) or 4-methylumbeliferyl N, N ', N ", N'"-tetraacetyl-β-D-chitotetraoxide (4MU-GlcNAc ₄ ) {(sigma Aldrich Corporation (Sigma-Aldrich Corp., St. Louis, MO) and EMD Biosciences (San Diego, CA, USA) .Genios fluorescence microtiter reader at 37 ° C (Tecan, CA, USA) Chitinase activity was determined by measuring the release of 4-MU using a primary acid jose)) and a 340 nm / 465 nm excitation / emission filter.Reaction horn in each well of a 96 well black microtiter plate The mixture was 100 ml and contained 50 mM substrate, 1 × McIlvaine buffer (pH range was 4-7 in different experiments) and 5-10 ml of sample, the initial release rate was recorded and the enzyme unit was 4-MU released. Calculated as pmol of min ⁻¹ A standard curve of 4-MU (Sigma-Aldrich Corporation) was prepared under the conditions used for the reaction for conversion from fluorescence units.

microscope

About 1-2 OD ₆₀₀ units of cells were harvested and fixed for 1 hour in 1 ml of 2.5% (v / v) glutaraldehyde on ice. 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-propylgallate, 80% glycerol). In septum staining experiments, Calcofluor white (Sigma-Aldrich Corp.) was added to the mounting medium at a final concentration of 100 mg ml ⁻¹ . Cells were observed with a Zeiss Axiovert 200M microscope using optical phase Normaski imaging or fluorescent DAPI filter settings.

Cellular chitin measurement

The cells are extracted with KOH and the chitin in the alkali insoluble material is quantified by Micro Morgan-Elson assay as described in Boulk, DA, et al. Eukaryot Cell 2: 886-900 (2003). Hydrolyzed to GlcNAc by chitinase for this purpose.

Example  2: K . Lactis Chitinase  ( KlCts1p Identification and Biochemical Characterization

Naturally secreted K comprising a cross reactive chitin-binding domain. K to check lactis protein. B. in Western blotting assay of culture medium spent lactis GG799. Polyclonal antibodies generated against the circular Culran Chi1A chitin-binding domain (α-CBD) were used (FIG. 1).

10 ml uncondensed K. Secreted proteins in lactis GG799 spent culture medium (96 hours growth) were separated on 4-20% polyacrylamide Tris-glycine SDS gel and b. Screening for the presence of chitin-binding domains by Western blotting using polyclonal antibodies generated against the circular Cultinase A1 chitin-binding domain (lane 1). Secreted protein was bound to chitin beads and boiled directly into SDS-PAGE loading buffer for 2 minutes before separation by SDS-PAGE (lane 2).

To test if any of these proteins can bind to chitin, the spent culture medium was mixed with chitin beads and spun at room temperature for 1 hour. Chitin beads were washed with water, bound proteins were boiled and eluted directly into SDS-PAGE loading buffer. Western blotting showed that only 85 kDa a-CBD cross-reactive protein could bind chitin (FIG. 1, lane 2). Protein purified directly from chitin beads in this manner was N-terminal protein sequencing to identify the first 20 amino-terminal amino acids of the mature protein (FDINAKDNVAVYWGQASAAT) (SEQ ID NO: 7). The tBLASTn search using the amino acid sequence as a question for probing the sequence database is exactly identical to the question sequence. Partially sequenced K with translation having significant homology to the extracellular chitinase Cts1p (ScCts1p) in cerevisiae. The lactis gene was identified.

KlCTS1 Cloning  And sequence analysis

K at the beginning of this work. The sequence of the lactis genome has not been reported yet. Thus, a combination of Southern hybridization and anchored PCR was used to clone the remainder of the partial KlCTS1 sequence (see above) originally identified by database search using the tBLASTn algorithm. KlCts1p sequence was reported recently. K in the lactis genomic sequence. Same as the translation product of Lactis ORF KLLA0C04730g (Dujon B., et al. Nature 430: 35-44 (2004)).

KlCTS1 encodes a protein with 551 amino acids having a molecular weight of 85 kDa as measured by SDS-PAGE. KlCTS1p S. Cerebizia is 53% identical to 82% similar to Cts1p chitinase and has a similar modular domain configuration consisting of 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)) predicted the presence of signal peptide cleaved after A ¹⁹ (FIG. 2A). This is consistent with the amino-terminal protein sequence determined from purified secreted KlCts1p starting at F ²⁰ . Amino acid homology between the predicted KlCts1p catalytic domain and the domains of other chitinases indicates that KlCts1p belongs to chitinase family 18 (FIG. 2B). In addition, SMART domain prediction software (Letunic, I., et al. Nucl.Acids Res. 30: 242-244 (2002) and Schultz, J., et al. PNAS 95: 5857-5864 (1998)) was 6 The presence of a C-terminal type 2 chitin-binding domain comprising two conserved cysteine residues is shown (FIG. 2C).

KlCts1p and S. The catalytic properties of chilease (ScCts1p) in cerevisiae were compared. s. Since Cts1p has an optimal acidic pH in Serevisiae (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)), pH 4.5 was initially set to determine substrate preference of KlCts1p. Selected. As shown in FIG. 3B, chitinases from both yeasts hydrolyze both 4MU-GlcNAc ₃ and 4MU-GlcNAc ₄ substrates. However, K. Lactis chitinase differs from ScCts1p to the extent that 4MU-GlcNAc ₄ is preferred over 4MU-GlcNAc ₃ . K. Similar results were observed for lactis strain GG799 against chitinases secreted from strains CBS2359 and CBS683. In addition, KlCts1p showed maximal activity at pH 4.5, which is about 0.5 pH units more alkaline than ScCts1p (FIG. 3C).

KlCts1p CBD -Chitin Affinity Analysis

Association of KlCts1p and chitin beads was investigated under various conditions. 4A shows that the chitin association of KlCts1p is stable in 5M NaCl and in buffers of pH 3, pH 5 and pH 10. Of the 8 M ureas, which are conditions that normally denature proteins, only about 60% of chitin-bound KlCts1p was dissociated from chitin beads. However, complete dissociation was observed in 20 mM NaOH at pH 12.3. The elution profile of chitin-bound KlCts1p revealed that an equivalent amount of protein was eluted in the first two fractions (including the pore volume), suggesting that destabilization of the KlCts1p-chitin association occurred immediately in 20 mM NaOH (FIG. 4B). Surprisingly, KlCts1p eluted in this manner retained chitinase activity (FIG. 4C). Indeed, measurement of chitinase activity after elution with 20 mM NaOH prior to chitin binding showed that almost 100% of the chitinase activity was restored.

KlCts1p CBD is i) independent of the KlCts1p catalytic domain; And ii) human serum albumin (HSA) containing a C-terminal fusion to CBD (KlCBD) derived from amino acids 470-551 of KlCts1p to determine if it can function as an affinity tag for a heterologously expressed protein. K. Secreted from lactis. For comparison, also HSA rain. K. in the same manner by fusion to Cercurans chitinase 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. 4D shows that HSA-KlCBD fusion protein completely dissociates from chitin beads in 20 mM NaOH, while HSA-BcCBD fusion protein remained bound to chitin even after extensive washing with 20 mM NaOH. These results indicate that KlCBD functions independently of the KlCts1p catalytic domain and its dissociation from chitin in 20 mM NaOH is a unique property. The data also raise the possibility that KlCBD can be used as an elutable affinity tag for the purification or reversible chitin-fixation of alkaline or alkali resistant proteins.

KlCTS1 Destruction of

K. To investigate the in vivo function of KlCts1p in Lactis, KlCTS1 allele was disrupted in haploid cells. PCR based methods were used to assemble DNA disruption fragments comprising the kanamycin selectable marker cassette as described in Materials and Methods. K using the fragment. Lactis cells were transformed with G418 resistance. Transformants were screened by whole-cell PCR for incorporation of disruption DNA fragments at the KlCTS1 locus. Of the 20 colonies tested, two colonies correctly integrated the destructive DNA fragments (data not shown), which resulted in KlCTS1 k. Not essential for the viability of lactis. In addition, K. Lactis Dcts1 cells do not secrete chitinase in the spent culture medium, as evidenced by the absence of KlCts1p (FIG. 5A) and chitinase activity (FIG. 3A).

K. The growth and cell morphology of Lactis wild type (GG799) and Δcts1 cells were investigated. In YPD medium, the Δcts1 strain grew as a small cluster of loosely aggregated cells that readily disperse into single cells upon short sonication. Fluorescence microscopy of cells stained with the chitin-binding dye Calcofluor white showed that Δcts1 cells were linked through their septum (FIG. 5B, right panel), suggesting that these cells were unable to degrade septal chitin during cytoplasmic division. . s. Similar phenotypes were observed in Δcts1 cells in Serevisia (Kuranda, M., et al. J. Biol. Chem. 266: 19758-19767 (1991)). Thus, S. The ability of KlCTS1 to restore normal cell segregation to Δcts1 cells in Serevisiae was tested. KlCTS1 S. Serevisiae were placed under the control of the GAL10 galactose-inducible promoter of the expression vector. S. expressing KlCTS1. Dcts1 cells in cerevisiae secrete KlCts1p in galactose containing medium (FIG. 6A) and do not form cell aggregates (FIG. 6B). Taken together, the data suggest that KlCTS1 and ScCTS1 encode functionally equivalent proteins that are believed to participate in cell separation by facilitating the degradation of septum chitin.

K . Lactis Chitinase Characterization of Deletion Mutants

The ability to grow to high culture density of Δcts1 cells was investigated. K. Aggregation phenotypes associated with lactis Δcts1 cells distorted measurements of cell density by absorbance at 600 nm (OD ₆₀₀ ) to less than 65% of wild type cells. However, cultures of wild-type and Δcts1 cells grown for 48 hours produced cells of approximately the same dry weight. In addition, total cellular chitin was not significantly different between the two strains. Strain GG799 produced 21.8 ± 1.9 nmole GlcNAc / mg dry cells upon KOH extraction of cellular chitin and hydrolysis by chitinase, and the Δcts1 strain produced 20.8 ± 1.0 nmole GlcNAc / mg dry cells. Thus, despite its moderate growth phenotype, Δcts1 cells can still achieve cell density in the same culture as wild type cells, suggesting that the strain background is suitable for commercial production of CBD-tagged proteins.

Example  3: pGBN2 - HSA - KlCBD Formulation

To generate a fusion between CBD of KlCts1p and human serum albumin (HSA), primers 5'-GGA AGATCT GACTCCTGGGCTGTTACAAGA-3 '(BglII sites are underlined) (SEQ ID NO: 8) and 5'-ATAAGAAT GCGGCCGC CTAGAAGACGACGTCGGGTTTCAAATA-3 (NotI sites are underlined) (SEQ ID NO: 9) to amplify DNA fragments encoding the C-terminal 81 amino acids of KlCts1p using Deep Vent ™ DNA polymerase (New England Biolabs, Inc.). I was. KlCts1p-CBD fragment. PGBN2-KlCBD was produced by cloning into the BglII-NotI site of the lactis integrated expression plasmid pGBN2 (New England Biolabs, Inc.). HSA uses primers 5'-CCG CTCGAG AAAAGAGATGCACACAAGAGTGAGGTTGCT-3 '(SEQ ID NO: underlined) (SEQ ID NO: 10) and 5'-CGC GGATCC TAAGCCTAAGGCAGCTTGACTTGC-3' (SEQ ID NO: 11) (SEQ ID NO: 11) And amplified and cloned into the XhoI-BglII site of pGBN2-KlCBD. K. When integrated into the lactis genome, the resulting expression construct is S. aureus. Cerebizia produces a single polypeptide consisting of the a-mate factor pre-pro secretion leader (present in pGBN2), HSA and KlCBD.

ratio. A control construct that produced HSA containing carboxy-terminal type 3 CBD (BcCBD) derived from circular curinase A1 was assembled in a similar manner. PTYB1 using primers 5'-GGA AGATCT ACGACAAATCCTGGTGTATCCGCT-3 '(SEQ ID NO: 12) (SEQ ID NO: 12) and 5'-ATAAGAAT GCGGCCGC TTATTGAAGCTGCCACAAGGCAGGAAC-3' (SEQ ID NO :) (SEQ ID NO: 13) BcCBD from (New England Biolabs, Inc.) was PCR amplified. The amplification product was cloned into the BglII-NotI site of pGBN2 to generate pGBN2-BcCBD. HSA was amplified and cloned into the XhoI-BglII site of pGBN2-BcCBD as described above.

Example  4: K . Lactis  Δ cts1  In the cell HSA - CBD Production and secretion

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

Example  5: chitin Bead  Used HSA - KlCBD Tablets

K. carrying an integrated HSA-KlCBD expression fragment (see above) to isolate HSA-KlCBD secreted by immobilization to chitin of the fusion protein. Lactis Dcts1 cells were grown 96 hours in 20 ml of YPD medium. The cells were removed from the culture by centrifugation and the spent medium was transferred to a new tube containing chitin beads (New England Biolabs, Inc.) washed with 1 ml of water 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. Immobilized HSA-KlCBD was eluted by boiling ˜50 ml volume of protein-bound chitin beads for 2 minutes in SDS sample buffer, followed by microcentrifugation for 2 minutes to remove 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 antibody. In addition, HSA-KlCBD can be eluted from chitin beads by passing 5 ml of 20 mM NaOH on the column. HSA produced in this manner includes endogenous K. which inadvertently binds to or degrades chitin. There is no lactis protein.

Example  6: magnetized chitin Bead  using HSA - KlCBD Enrichment of

CBD-tagged human serum albumin (HSA-CBD) was used to demonstrate the association of CBD-tagged proteins with magnetic chitin beads during various stages of culture growth (see FIG. 8). K. Four 25 ml YPGal (1% yeast extract, 2% peptone and 2% galactose) cultures of Lactis strain GG799 Δcts1PCKl3 were inoculated with 100 ml of 2 ml starting culture grown at 30 ° C. for 24 hours.

Culture 1: Prior to inoculation, 1 ml of precipitated chitin magnetic beads sterilized for 20 minutes by autoclaving was added to the culture medium. The cultures were then incubated at 30 ° C. for 72 hours with shaking at ˜300 r.p.m.

Culture 2: At 24 h growth, 1 ml of sterile magnetic chitin beads were added to the culture medium. Cultures were further incubated at 30 ° C. for 48 hours (72 hours total) with shaking at ˜300 r.p.m.

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

Culture 4: After removing cells from the fourth culture by centrifugation for 5 minutes at 5000 r.p.m., 1 ml of magnetic chitin beads were added to the clarified spent culture medium, followed by gentle shaking at room temperature for 1 hour.

Each culture was poured into a standard laboratory tube with 50 ml stopper. Magnetic beads were collected by draining the supernatant after inserting the tube into a 50 ml magnetic device (FIG. 9) for 30 seconds. The tube was then removed from the magnetic field and the pellets of magnetic chitin particles were washed with 40 ml of water and cut off in the magnetic field. After the washing process was repeated three times in total, the beads were transferred to a microcentrifuge tube with four screw caps. To elute bound HSA-CBD, the beads in each tube were suspended in 250 ml 3X protein loading buffer (New England Biolabs, Inc.) containing dithiothritol and heated at 98 ° C. for 5 minutes. It was. Eluted protein in 5 ml of each sample was separated on a 10-20% SDS-PAGE gel and visualized by Coomassie staining (FIG. 8). Eluted HSA-CBD was observed in each sample, indicating that magnetic chitin beads successfully captured CBD-tagged proteins during or after growth of the culture. The yield of HSA-CBD captured in each culture was estimated to be 4 mg L-1.

Example  7: secreted GluC - CBD  Magnetized Chitin for Concentrating Proteins Bead  use

To test the ability of magnetized beads to concentrate CBD fusion proteins from the culture medium, coding for fusion proteins (GluC-CBD, where GluC is an endoprotein from Staphylococcus aureus) 20 ml overnight cultures of Bacillus Circus transformed with the DNA to grow were grown in 125 ml flasks. Rain the DNA encoding GluC. Insertion was made into plasmid pGNB5 containing the circular CBD. Transformation of pluripotent cells was achieved using standard techniques (Harwood and Cutting, Molecular Biological Methods, ed. John Wiley & Sons Ltd., New York, NY, pp. 33-35, 67, 1990). For comparison, four cultures were grown, one containing plasmids expressing GluC endoprotease and three containing plasmids for fusion protein (GluC-CBD). For all experiments, 5% bead volume was added to the culture. As a control, cultures containing GluC constructs were grown overnight in the presence of magnetic beads to investigate nonspecific binding (FIG. 12, lane 2). To investigate if incubation time affects binding capacity, magnetic beads were added to three GluC-CBD cultures at different time points. Culture 1 was grown overnight at 37 ° C. with shaking in the presence of beads during the entire growth cycle (FIG. 12, lane 3). For culture 2, magnetic beads were grown after 16 hours at 37 ° C. and added and incubated for 1 hour at 37 ° C. with shaking (FIG. 12, lane 4). Culture 3 was grown overnight at 37 ° C. with shaking, and cells were removed by centrifugation (10,000 rpm, for 10 minutes). Magnetic beads were added to the supernatant and incubated with shaking for 1 hour at room temperature (FIG. 12, lane 5). In all cases, the beads were collected using a magnetic separation rack (New England Biolabs, Inc.) and washed three times with 10 ml LB broth and then twice with 10 ml of 1 M NaCl. The beads were suspended in 1 ml of 1M NaCl, transferred to a 1.5 ml Eppendorf tube and centrifuged at 10,000 rpm for 1 minute to remove the liquid. Beads were suspended in 100 ml 3X SDS sample buffer with DTT and boiled for 5 minutes to remove protein. Beads were separated from the buffer by centrifugation at 10,000 rpm for 2 minutes. Samples were analyzed on 10-20% Tricine gels, transferred onto PVDF membrane by western blot and stained with Coomassie Blue. The identity of the eluted protein was confirmed by N-terminal sequencing. The results indicate that the secreted GluC-CBD fusion protein is the major protein bound by magnetic chitin beads and can be eluted efficiently by boiling in SDS sample buffer. The results also show that the beads can be added at any point during the experiment without changing their efficiency.

Example  8: Luciferase  Production and chitin From beads  His dissolution

Wild type K. The gene encoding Lactis CBD was cloned into the NotI / StuI restriction site of vector pKLAC1 to generate vector pKLAC1-KlCBD. The gene encoding Gaussia luciferase (GLuc) was then cloned into the XhoI / NotI restriction site of the vector pKLAC1-KlCBD to produce an N-terminal fusion with the vector-derived secretion signal and a C-terminal fusion with the KlCBD gene. Linearize the constructs; Transformed into lactis pluripotent cells. K secreting GLuc-KlCDB. One liter of spent culture medium obtained from lactis cells was mixed with 20 ml bed volume of chitin beads for 1 hour at room temperature. Chitin beads were poured into the column and subsequently washed with 10 column volumes (200 ml) of water. The bound protein was eluted with 20 mM NaOH. A 4 ml elution fraction was collected in a tube containing 1 ml 1M Tris-Cl pH 7.5 to neutralize the eluent when introduced from the column. A 40-fold dilution of each eluted fraction of 25 microliters was analyzed for luciferase activity (expressed in RLU (relative light units)). 13 shows that active GLuc eluted in fractions 2 to 10 and the highest activity was found in fractions 3, 4 and 5.

                          <110> New England Biolabs, Inc.        Taron, Christopher H.        Colussi, Paul H.   <120> Methods and Compositions for Concentrating Secreted Recombinant        Protein <130> NEB-247-PKR <150> 60 / 690,470 <151> 2005-06-14 <150> PCT / US2005 / 035697 <151> 2005-10-03 <150> 60 / 616,420 <151> 2004-10-06 <160> 20 <170> PatentIn version 3.2 <210> 1 <211> 98 <212> DNA <213> artificial <220> <223> primer <400> 1 ccagtaatgc aactatcaat cattgtgtta aactggtcac cagaaataca agatatcaaa 60 aattactaat actaccataa gccatcatca tatcgaag 98 <210> 2 <211> 104 <212> DNA <213> artificial <220> <223> primer <400> 2 ccaaactagc gtatccggtt ggattattgt tttcgatatc gaaatcgaaa ccatcgacga 60 cagcagtgtc gaatggtctt tccccggggt gggcgaagaa ctcc 104 <210> 3 <211> 18 <212> DNA <213> artificial <220> <223> primer <400> 3 gggcacaaca atggcagg 18 <210> 4 <211> 19 <212> DNA <213> artificial <220> <223> primer <400> 4 gcctctccac ccaagcggc 19 <210> 5 <211> 36 <212> DNA <213> artificial <220> <223> primer <400> 5 ggcggatccg ccaccatgtt tcaccctcgt ttactt 36 <210> 6 <211> 34 <212> DNA <213> artificial <220> <223> primer <400> 6 acatgcatgc ctagaagacg acgtcgggtt tcaa 34 <210> 7 <211> 20 <212> PRT <213> Kluyveromyces lactis <400> 7 Phe Asp Ile Asn Ala Lys Asp Asn Val Ala Val Tyr Trp Gly Gln Ala 1 5 10 15 Ser Ala Ala Thr             20 <210> 8 <211> 30 <212> DNA <213> artificial <220> <223> priimer <400> 8 ggaagatctg actcctgggc tgttacaaga 30 <210> 9 <211> 43 <212> DNA <213> artificial <220> <223> primer <400> 9 ataagaatgc ggccgcctag aagacgacgt cgggtttcaa ata 43 <210> 10 <211> 39 <212> DNA <213> artificial <220> <223> primer <400> 10 ccgctcgaga aaagagatgc acacaagagt gaggttgct 39 <210> 11 <211> 33 <212> DNA <213> artificial <220> <223> primer <400> 11 cgcggatcct aagcctaagg cagcttgact tgc 33 <210> 12 <211> 33 <212> DNA <213> artificial <220> <223> primer <400> 12 ggaagatcta cgacaaatcc tggtgtatcc gct 33 <210> 13 <211> 43 <212> DNA <213> artificial <220> <223> primer <400> 13 ataagaatgc ggccgcttat tgaagctgcc acaaggcagg aac 43 <210> 14 <211> 57 <212> PRT <213> artificial <220> <223> Type 2 chitin-binding domain consensus sequence <400> 14 Gln Asp Cys Thr Asn Ala Leu Asp Gly Leu Tyr Ala Leu Gly Glu Cys 1 5 10 15 Glu Pro Gln Phe Leu Thr Cys Ser Gly Gly Ile Ala Arg Ile Met Asp             20 25 30 Cys Pro Ala Asp Leu Ile Tyr Asn Glu Pro Leu Leu Ile Cys Asp Trp         35 40 45 Arg His Asn Val Ile Gly Cys Glu Gly     50 55 <210> 15 <211> 48 <212> PRT <213> Kluyveromyces lactis <400> 15 Cys Ser Asp Gly Glu Ile Ser Cys Thr Ala Asp Gly Lys Ile Ala Ile 1 5 10 15 Cys Asn Tyr Gly Ala Trp Val Tyr Thr Glu Cys Ala Ala Gly Thr Thr             20 25 30 Cys Phe Ala Tyr Asp Ser Gly Asp Ser Val Tyr Thr Ser Cys Asn Phe         35 40 45 <210> 16 <211> 64 <212> PRT <213> Cladosporium fulvum <400> 16 Thr Lys Cys Met Gly Pro Lys Asp Cys Leu Tyr Pro Asn Pro Asp Ser 1 5 10 15 Cys Thr Thr Tyr Ile Gln Cys Val Pro Leu Asp Glu Val Gly Asn Ala             20 25 30 Lys Pro Val Val Lys Pro Cys Pro Lys Gly Leu Gln Trp Asn Asp Asn         35 40 45 Val Gly Lys Lys Trp Cys Asp Tyr Pro Asn Leu Ser Thr Cys Pro Val     50 55 60 <210> 17 <211> 68 <212> PRT <213> Ralstonia solanacearum <400> 17 Phe Lys Cys Pro Ala Pro Ser Gly Arg Tyr Leu Val Asp Asp Gly Thr 1 5 10 15 Asn Asn Arg Gly Pro Asn Gln Val Pro Arg Thr Asn Cys Thr Arg Ala             20 25 30 Tyr Ala Val Cys Asp Ala Gln Ser His Ala Thr Leu Asp His Cys Pro         35 40 45 Ser Gly Gln Val Phe Asp Lys Arg Phe Ser Thr Cys Val Val Lys Asp     50 55 60 Ala Cys Asp Glu 65 <210> 18 <211> 54 <212> PRT <213> Caenorhabditis elegans <400> 18 Phe Lys Cys Thr Lys Asp Gly Phe Phe Gly Val Pro Ser Asp Cys Leu 1 5 10 15 Lys Phe Ile Arg Cys Val Asn Gly Ile Ser Tyr Asn Phe Glu Cys Pro             20 25 30 Asn Gly Leu Ser Phe His Ala Asp Thr Met Met Cys Asp Arg Pro Asp         35 40 45 Pro Ser Lys Cys Ala Lys     50 <210> 19 <211> 48 <212> PRT <213> Homo sapiens <400> 19 Cys Ala Gly Arg Ala Asn Gly Leu Tyr Pro Val Ala Asn Asn Arg Asn 1 5 10 15 Ala Phe Trp His His Cys Val Asn Gly Val Thr Tyr Gln Gln Asn Cys             20 25 30 Gln Ala Gly Leu Val Phe Asp Thr Ser Cys Asp Cys Cys Asn Trp Ala         35 40 45 <210> 20 <211> 52 <212> PRT <213> Drosophila melanogaster <400> 20 Leu Glu Cys Thr Glu Gly Asp Tyr Tyr Pro His Arg Asn Cys Arg Lys 1 5 10 15 Tyr Tyr Ile Cys Asn Lys Ala Leu Val Pro Ser Glu Cys Gly Gly Asp             20 25 30 Leu His Trp Asp Gly Ile Lys Lys Leu Cys Asp Trp Pro Glu Asn Val         35 40 45 Gln Cys Val Thr     50

Claims (28)

(a) transforming host expressing cells with a vector comprising a 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 the fusion protein from the host expressing cell; (c) binding the secreted fusion protein to the chitin preparation by CBD, which can be eluted into the desired buffer volume under non-denaturing conditions, to obtain a concentrated formulation of secreted recombinant protein. Method of obtaining a concentrated formulation. The method of claim 1 wherein the vector in step (a) is a shuttle vector. The method of claim 2, wherein the DNA encoding the target protein in the vector is E. coli. Transcriptionally inactive in E. coli and transcriptional activity in host expressing cells. The method of claim 2, wherein the shuttle vector has a modified LAC4 promoter. The method of claim 4, wherein the shuttle vector is pKlAC1. The method of claim 1, wherein the host expressing cells are chitinase-deficient cells. The method of claim 3, wherein the host expressing cells are yeast cells. 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 of claim 9, wherein the Cluyveromyces is Cluyveromyces marxianus variety fragilis or lactis. The method of claim 1, wherein the host expressing cells are present in the culture such that the fusion protein is secreted into the culture medium. The method of claim 11, further comprising adding chitin to the culture medium during the culture. The method of claim 12, wherein the chitin is sterile. The method of claim 1 or 11, wherein the chitin is added to the mixture after incubation. The method of claim 1 or 13, wherein the chitin is in the form selected from coatings, colloids, beads, columns, matrices, sheets and membranes. The method of claim 1 or 13, wherein the chitin is chitin beads and the beads are porous or non-porous. The method of claim 16, wherein the chitin beads are magnetized. 18. The method of claim 17, wherein recovering the fusion protein bound to the chitin in step (d) further comprises applying a magnetic force. The method of claim 1, wherein the binding of the fusion protein to chitin is reversible such that the fusion protein can be released from the chitin under non-denaturing conditions that differ from the conditions for binding. providing a shuttle vector comprising (a) (i) can be integrated into the genome of a yeast host cell and (ii) a DNA encoding a fusion protein comprising a chitin binding domain and a target protein; (b) transforming the chitinase deficient host expressing cells with a shuttle vector to express the fusion protein in yeast host expressing cells and secreting the fusion protein from the host expressing cells; (c) binding the secreted fusion protein to the chitin preparation by CBD to obtain a concentrated formulation of secreted protein A method of obtaining a concentrated formulation of secreted recombinant protein comprising a. A preparation of Cluiveromyces cells characterized by a chitinase-negative phenotype, wherein the phenotype is the result of mutation of a chitinase gene expressing secreted chitinase, the preparation having a cell density similar to that of wild type Cluiberomyces cells Formulation that can grow to furnace. 22. The preparation of cluyveromyces cells according to claim 21, which is further capable of expressing and secreting recombinant proteins. 23. The preparation of Kluyveromyces cells according to claim 22, wherein the expression is regulated by a LAC4 promoter or a variant thereof. The method of claim 21, wherein: A preparation of a Kluyveromyces cell comprising a shuttle vector with a modified Lac4 promoter for expressing the protein in Kluyveromyces without substantially expressing the protein in E. coli. The preparation of Kluyveromyces cells according to claim 22, wherein the shuttle vector is pKLACI. 22. The preparation of Cluyveromyces cells according to claim 21, further comprising a culture medium capable of growing and / or maintaining Cluyveromyces, wherein the culture medium further comprises sterile chitin. 27. The preparation of Cluyveromyces cells according to claim 26, wherein the sterile chitin is in the form of magnetic beads. 22. The preparation of Cluyveromyces cells according to claim 21, wherein the preparation is contained in a container, the container being able to contact the magnet to include a magnet or to reversibly bind to chitin beads.

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