WO2004090549A1

WO2004090549A1 - Method of screening for secreted glycosylated polypeptides

Info

Publication number: WO2004090549A1
Application number: PCT/DK2004/000246
Authority: WO
Inventors: Kirk Matthew Schnorr
Original assignee: Novozymes A/S
Priority date: 2003-04-11
Filing date: 2004-04-05
Publication date: 2004-10-21

Abstract

The present invention relates to a method of screening for a secreted polypeptide of interest comprising the steps of a) providing a host cell expressing and secreting a glycosylated polypeptide of interest and culturing the said host cell under conditions promoting the said expression; b) contacting in solution and in a compartment a liquid sample from (a) comprising the glycosylated polypeptide of interest with an immobilized lectin compound capable of binding to the glycosylated polypeptide of interest, under conditions where binding capacity of the immobilized lectin compound per compartment volume is at least 10 ng polypeptide/300 microl compartment volume; c) isolating the immobilized lectin compound retaining the polypeptide of interest; d) releasing the polypeptide of interest from the immobilized lectin compound.

Description

TITLE: METHOD OF SCREENING FOR SECRETED GLYCOSYLATED POLYPEPTIDES

FIELD OF INVENTION

The present invention relates to a method of screening for secretion of a polypeptide of interest.

BACKGROUND OF THE INVENTION

Screening procedures, in particular high throughput procedures, applied by biotech industry often involve testing of thousands or even millions of small volume test fermentations for new and/or improved biological compounds expressed by cells and/or micro organisms. Separation and/or isolation of the biological compound of interest may frequently offer an advantage in determining if the expressed biological compound is indeed novel or does indeed offer any improvements such as improved yields, improved activity, improved stability etc. Assessment of such properties of a biological compound of interest will often be impeded if various interfering components from the fermentation culture broth are present in a sample. Such interfering components often generate false positive results.

It is thus desirable for many industries to have available a large spectra of relatively pure secreted proteins for use in there own high throughput screening assays.

Cloning and over-expression of candidate gene/proteins is one way to overcome many of the purity problems relating to separation of proteins from a wild type candidate. PCT/DK02/00717 discloses a general method for high throughput purification of tag-free polypeptides in small volume samples. In case of a specific class of enzymes or proteins such as secreted proteins a fast and easy screening system for the recovery of secreted proteins would be desirable thereby allowing detection and recovery of recombinant lines expressing secreted proteins in an assay independent manor. This way one could maximize the number of secreted protein candidates per recombinant DNA library.

Various attempts have been made to find a fast and simple way of purifying secreted proteins from eukaryotic host cells, especially from filamentous fungi such as Aspergillus. Screening in a filamentous o rganism i ntroduces a n umber of p roblems that a re a ssociated with this type of screening host (see e.g. EP1230348). Firstly, fermentation and recovery of culture fluids in High Throughput Screening (HTS) formats, such as 96 well microtitre plates, is difficult as the mycelia interfere with sample collection. Secondly, most filamentous fungi chosen for screening or production of secreted proteins themselves secrete major amounts of native secreted proteins such as proteases, amylases and hydrophobins. This becomes a problem when one wants to detect a unique recombinant product in a sea of natively secreted protein. Thirdly, cell lysis and cell leakage which normally occurs in standard fermentation procedures as well as HTS systems further increase the background from which a recombinant secreted protein must be identified.

In e ukaryotes secreted p roteins are often glycosylated. Protein secretion plays an important role in all fungi. The majority of proteins secreted by fungi are thought to be glycosylated (see review by Peberdy: Protein and secretion in filamentous fungi-trying to understand a highly productive black box., J.F. Peberdy, TIBTECH, 12:50-57, 1994).

Lectins are secreted proteins capable of binding carbohydrate moieties. Lectins have been separated into various classes based on what type of carbohydrate they bind to. Lectin based affinity matrixes have been in use for many years and are used to capture and purify glycosylated proteins. Physical Biochemistry, by D.M. Freifelder, copyright 1982 p.260. Lectin affinity chromatography has become a standard and widely used technique for the isolation of soluble glycoproteins, hormones, antigens and polysaccharides, as well as detergent- solubilized membrane-bound glycoconjugates and cell surface receptors (see review by Lotan and Nicolson, Biochim. Biophys. Acta, 559, 329-376, 1979).

Lectin affinity chromatography is known in large scale purification of glycosylated proteins, and concanavalin A is one of the favored glycoprotein affinity lectins because it binds to a commonly occurring sugar structure in glycoproteins; alpha linked mannose.

However, a need exists for a fast and simple method for screening e.g. a library of recombinant clones for secreted proteins.

SUMMARY OF THE INVENTION Surprisingly we have now found that lectins can be applied in a fast and convenient screening method for screening samples from a recombinant library of secreted polypeptides of interest in a high throughput format. By use of lectin affinity based matrixes in a multi-well, automation enabled high throughput format, it is possible to recover many of the secreted proteins from a library, e.g. a cDNA library, expressed in a recombinant host.

In a first aspect the present invention relates to a method of screening for secretion of a polypeptide of interest comprising:

a) providing a host cell expressing and secreting a glycosylated polypeptide of interest and culturing the said host cell under conditions promoting the said expression; b) contacting in solution and in a compartment a liquid sample from (a) comprising the glycosylated polypeptide of interest with an immobilized lectin compound capable of binding to the glycosylated polypeptide of interest, under conditions where binding capacity of the immobilized lectin compound per compartment volume is at least 10 ng polypeptide/300 microl compartment volume,

c) isolating the immobilized lectin compound retaining the polypeptide of interest;

d) releasing the polypeptide of interest from the immobilized lectin compound.

DEFINITIONS

Prior to a discussion of the detailed embodiments of the invention, a definition of specific terms related to the main aspects of the invention is provided.

Lectins are multivalent carbohydrate-binding proteins or glycoproteins except for enzymes and antibodies. The lectins of interest in the invention are proteins capable of binding sugar moieties in the glycosyl backbone, branchpoints or terminal sugars of eukaryotic secreted proteins. The category includes the general grouping "plant lectins".

Concanavalin A: The structure of ConA has been reported by Becker et al. (1976, Nature 259: 406). It is composed of identical subunits of 237 amino acid residues (M.W.: 26,000). ConA exists as a single dimer (M. W.: 53,000). Above pH 7 it is predominantly tetrameric (Wang et al., 1975, J. Biol. Chem. 250: 1490). Its optimal activity is near pH 7. ConA reacts with non- reducing alpha-D-glucose and alpha-D-mannose.

The term "library" as used herein is to be understood as a collection of different or diversified biological compounds, in particular present in separate discrete samples. A library usually consist of at least 10 different biological compounds, particularly at least 50, more particularly at least 100, even more particularly at least 500, more particularly at least 1000 b iological compounds. In a particular embodiment the library originates from fermenting a population of host cells, transformed or transfected with nucleotide sequences encoding gene(s) or cDNAs which have been recombinantly engineered for expression in the host cell so as to allow for expression in that host cell.

The term "population" as used herein is to be understood as a collection of similar entities. For example a population of host cells is a collection of cells of the same strain, while a population of samples or containers is a collection of samples or containers having the same volume. A population usually comprises more than 10 units of the entity, in particular more than 20 units, more particular more than 50, more particular more than 95, more particular more than 300, more particular more than 383, more particular more than 500, more particular more than 1000, more particular more than 5000 units of the entity.

Endogenous: The term "endogenous" in the context of the present invention means that e.g. the gene or protein originates from within the host organism.

The term "isolating" as used herein is to be understood as treating a first solution comprising a polypeptide of interest in a manner to yield a second solution comprising the polypeptide of interest, wherein either the concentration of polypeptide of interest of the second solution is higher than that of the first solution and/or the ratio of polypeptide of interest to other dissolved or suspended matter in the second solution is higher than that of the first solution. In the context of the present invention the term "purified" is intended to have the same meaning as the term "isolated".

DETAILED DESCRIPTION OF THE INVENTION

Glycosylation of secreted proteins. In eukaryotes secreted proteins are often glycosylated. Protein secretion plays an important role in all fungi. The majority of proteins secreted by fungi are thought to be glycosylated (see review by Peberdy: Protein and secretion in filamentous fungi-trying to understand a highly productive black box., J.F. Peberdy, TIBTECH, 12:50-57, 1994). In eukaryotic organisms, proteins destined for secretion are first synthesized on ribosomes that are associated with the rough endoplasmic reticulum (RER). The step that determines that the translation product is targeted for secretion is the processing of the N terminal signal sequence that must be present in order to enter the RER. Before proteins are secreted in fungi, they undergo several post translation modifications: Proteolytic cleavage of the signal peptide, folding of the protein usually through disulphide bond formation, and glycosylation. Glycosylation involves the attachment of sugar chains to Asn, Ser and Thr residues on the peptide. Oligosaccharides attached to an asparagine are said to be N-linked while those attached to serine or threonine are O-linked. N-linked glycans have a core region of two N- acetylglucosamine linking the rest of the chain; usually a number of mannose residues and can contain other sugars. Mannose side chain decorations have been observed to be over 200 residues. By contrast, O-glycan side chains are usually between 1 and 5 sugar residues which are exclusively mannose. Lectins.

Lectins are s ecreted p roteins capable of b inding carbohydrate m oieties. ectins h ave been separated into various classes based on what type of carbohydrate that they bind to. For example, Hevein binds chitin polysaccharide, and concanavalin A binds alpha linked mannose residues in, for example, glycosylated proteins. Probably the best single information source on plant lectins is Handbook of Plant Lectins: Properties and Biomedical Applications by Els J.M. Van Damme, Willy J. Peumans, Arpad Pusztai, Susan Bardocz, John Wiley & Sons, Chichester, New York etc. 1998 ISBN 0-471 -96445-X.

Legume lectins are one of the largest lectin families with more than 70 lectins reported. Leguminous plant lectins resemble each other in their p hysicochemical p roperties a Ithough they differ in their carbohydrate specificities. They consist of two or four subunits with relative molecular mass of 30 kDa and each subunit has one carbohydrate-binding site. The interaction with sugars requires tightly bound calcium and manganese ions. The primary structural analyses and X-ray crystallographic studies report the structural similarities of these lectins. X-ray studies have shown that the folding of the polypeptide chains in the region of the carbohydrate-binding sites is also similar, despite differences in the primary sequences. The carbohydrate-binding sites of these lectins consist of two conserved amino acids situated on the secondary structure of beta pleated sheets and two loops. One of these loops contains transition m etals, calcium a nd manganese, and keep the amino acid residues of the sugar binding site at the required positions. Amino acid sequences of these loops play an important role in the carbohydrate-binding specificities of these lectins.

Lectin based affinity matrixes have been in use for many years and are used to capture and purify glycosylated proteins. Lectin affinity chromatography has become a standard and widely used technique for the isolation of soluble glycoproteins, hormones, antigens and polysaccharides, as well as detergent-solubilized membrane-bound glycoconjugates and cell surface receptors. A review article on this topic has been published by Lotan and Nicolson (Biochim. Biophys. Acta, 559, 329-376, 1979). Lectin affinity chromatography combines simplicity with potentially high resolution. During the purification of glycoconjugates, the immobilized lectin is allowed to bind to the glycoconjugate, and the unbound residual material can b e readily removed by subsequent washing. The bound glycoconjugates are displaced from the immobilized lectins by the addition of a solution of a sugar known to inhibit binding of the particular lectin. Beads are commonly used in affinity chromatography. At least two basic types exist; porous and non porous. The two types are defined by whether the beads are permeable to solutes, large and small molecules. Agarose, sepharose, and sephacryl are all examples of porous bead matrixes. Covalent couplings between the lectin and the bead are then made through various activated groups on the bead and specific side chains and/or terminals of the lectin. Common activated groups include -NH2, -SH, -COOH, and -OH. To minimize steric hindrance a molecular spacer from a few to e.g. ten C-C bonds between the bead and the activated group is often employed. To name one such m anufacturer, Vector Laboratories supplies stable and high quality agarose-lectin bead products. Heat stable, cross- linked 4% agarose beads with a molecular weight exclusion limit of about 2x10⁷ are used as the solid-phase matrix to which the lectins are covalently bound. The attachment of the lectins to the solid phase is carefully controlled in order to preserve the activity of the lectins as well as to minimize conformational changes of the bound lectins which might result in nonspecific ionic or hydrophobic interactions. The technique developed to couple lectins to agarose provides a very hydrophilic spacer arm between the protein and the matrix. This ensures maximum expression of the carbohydrate binding activity of the lectin. The linkage is very stable over a range of p H values a nd, u nlike cyanogen b romide I inkages, p roteins a re n ot leached off the gel by Tris or other routinely used buffers. In addition, residual charges generated during cyanogen bromide conjugation which can produce nonspecific binding are not present on the gel following our coupling procedure.

Fluid impervious matrixes are covered with a layer of functional groups in their surface. These groups can be further modified so as to bind functional proteins such as lectins. Silica and chemically m odified d erivatives, p olymethacrylate, z irconia, p olystyrene (PS), copolymers of PS and modifications of PS such as PS and divinyl benzine (PS-DVB) are examples of non porous bead matrixes. Agarose can be made essentially non porous by shrinkage and cross linking in organic solvents, (Lee, W., Protein separation using non-porous sorbents. J. Chromatography, 699(1997) 29-45).

The lectin compounds of the invention should be capable of binding sugar moieties in the glycosyl backbone, branch point or terminal sugars of glycosylated polypeptides, e.g. alpha linked mannose.

The lectin compounds of the invention comprise plant lectins such as lectins selected from the list below: a) a Ipha-D-mannose-specific p lant I ectin ( monocot I ectin) s uch a s: a maryllis, Bluebell SCA- FET, Bluebell SCA-MAN, daffodil amaryllidaceae, garlic bulbs lectin, snowdrop lectin b) b-prism plant lectins such as: artocarpin, heltuba, jacalin, Madura pomifera MPA c) Agglutinin with hevein domain such as: Hevein, Urtica dioica UDA, Wheat germ WGA-1 , Wheat germ WGA-2, Wheat germ WGA-3 d) Legume lectin such as: canavalia brasiliens, concanavalin A, Dioclea grandiflora DGL, Dioclea guianensis lectin, Dolichos biflorus DB58, Dolichos biflorus DBL, Dolichos lablab FRIL, Erythrina corallodendron EcorL, Erythrina cristallogali ECL, favin, Griffonia simplicifolia GS-I, Griffonia simplicifolia GS-IV, Lathyrus ochrus LOL-1 , Lathyrus ochrus LOL-2, Lentil LCL, lima bean LBL, Maackia amurensis MAL, Pea PSL, Peanut PNA, Phaseolus vulgaris PHA-L, Pterocarpus angolensis, Robinia pseudoacacia bark lectin I, Soybean SBA, Ulex europaeus UEA-1 , Ulex europaeus UEA-2, Vicia villosa WL-B4, winged bean agglutinin I, winged bean agglutinin II.

Particularly, the lectin according to the invention comprises lectins capable of binding alpha mannose, alpha glucose or N-acetylglucosamine (GlcNAc) in secreted glycosylated proteins such as concanavalin A (Con A), Lens culinaris agglutinin (LCA), Pisium staivum agglutinin (PSA), Galanthus Nivalis Lectin (GNL), Hippeastrum Hybrid Lectin (HHL), Narcissus Pseudonarcissus Lectin (NP), Datura Stramonium Lectin (DSL), Lycopersicon Esculentum (Tomato) Lectin (LEL), Solanum Tuberosum ( Potato) Lectin ( STL), W heat G erm Agglutinin (WGA).

In a particular embodiment the lectin is concanvalin A.

Concanavalin A

Concanavalin A is one of the favored glycoprotein affinity lectins because it binds to commonly occurring sugar structure in glycoproteins; alpha linked mannose. Since a wide variety of serum and membrane glycoproteins have a "core oligosaccharide" structure which includes alpha-linked mannose residues, many glycoproteins can be examined or purified with Con A and its conjugates. For example ConA-sepharose, sephacryl, acryl and agarose have all been used for various protein purification methods. Other lectins that bind mannose residues and may b e u seful, but in a non limiting way, are Galanthus Nivalis Lectin (GNL), Hippeastrum Hybrid lectin (HHL), Lens Culinaris Agglutinin (LCA9, Narcissus Pseudonarcissus; Daffodil lectin (NPL) and Pisum Sativum Agglutinin (PSA).

Plastic coating technology has also made available ConA coated microtitre plates capable of binding glycosylated proteins (CALBIOCHEM®. Concanavalin A plate, clear polystyrene cat. no. 234590). ConA coated plates or those coated with other lectins have been used previously for purification of glycosylated proteins in the form of coated surfaces in e.g. a microtiter plate format. In these cases however, sensitive enzymatic or binding assays are typically employed. Such lectin coated plates are not useful in a high throughput format in which it is desirable to screen many samples at the same time in a small volume and where detection of the captured polypeptide is by way of a general protein detection assay.

We have established (see example 1 for details) that concanavalin A (conA) coated plates do not have the binding capacity needed to detect secreted proteins applying one of the most sensitive general protein assays (SYPRO Orange). The binding capacity of the affinity matrix (immobilized lectin compound bound to solid carrier material) is essential for the technique to work. In our experience, the matrix must bind at least the minimum amount of glycosylated, secreted protein needed for detection in the SYPRO orange or similar sensitive general proteins assay. At least 10 ng polypeptide, particularly at least 40 ng or even more particularly at least 100 ng polypeptide should be captured by the immobilized lectin compound in a compartment volume of 300 microl for a high throughput screening assay. By selecting to elute the bound glycosylated protein in a reduced volume, one can concentrate the protein and thus even further increase the detection potential in a general protein assay.

In order to solve the problem of binding capacity we have in the present invention developed means for immobilizing lectins in a way that will increase the surface area that will be in contact with the samples to be purified and screened. This will make possible the application of a lectin based purification of secreted proteins. Examples of such means include those described below, e.g. the use of conA beads instead of conA coated microtiter plates.

The proteins can be purified according to the method of the invention without the need for any artificial tags. The host cell expressing the protein of interest should be a host cell that will add carbohydrate moieties to the proteins known as glycosylation, and secrete the glycosylated protein.

In one aspect the present invention therefore relates to a method of screening for secretion of a polypeptide of interest comprising:

a) providing a host cell expressing and secreting a glycosylated polypeptide of interest and culturing the said host cell under conditions promoting the said expression;

b) contacting in solution and in a compartment a liquid sample from (a) comprising the glycosylated polypeptide of interest with an immobilized lectin compound capable of binding to the glycosylated polypeptide of interest, under conditions where binding capacity of the immobilized lectin compound per compartment volume is at least 10 ng polypeptide/300 microl compartment volume, c) isolating the immobilized lectin compound retaining the polypeptide of interest;

d) releasing the polypeptide of interest from the immobilized lectin compound.

Isolation of the immobilized lectin compound comprised on the solid carrier material (the affinity matrix) can be accomplished by any conventional way of isolating particulate material from a liquid solution, such as e.g. filtration or centrifugation.

The polypeptide of interest can subsequently be released from the immobilized lectin compound by elution with a solution containing soluble sugar or polysaccharide.

In a further embodiment the host cell expressing the polypeptide of interest expresses a library of polypeptides to be understood as a population of different or diversified biological compounds, in particular present in separate discrete samples.

Preparing a library of biological compounds

The library can be prepared as described in PCT/DK03/00106 (Expression Cloning Methods in Filamentous Fungi). The starting material for this method is double stranded cDNA adapted with suitable restriction sites for cloning into the vector system(s) used in PCT/DK03/00106. A specific example of a method for generating cDNA for a fungal cDNA library preparation is found in WO 01/12794 under the section: "Construction of EcoRI/Notl-directional cDNA library from Malbranchea cinnamomea".

A library of biological compounds may be prepared by any conventional method, such as genetic engineering. The preparation of a library of polypeptides of interest may for instance be achieved by:

(1) preparing a library of nucleic acid sequences (a gene library) encoding polypeptides, (2) inserting the gene library into a population of host cells and (3) expressing the gene library in the host cells so as to produce a library of polypeptides.

Procedures for transformation of a host cell by insertion of a plasmid comprising a DNA or cDNA fragment from a gene library is well known to the art, e.g. Sambrook et al., "Molecular cloning: A laboratory manual", Cold Spring Harbor lab., Cold Spring Harbor, NY. (1989), Ausubel et al. (eds.), Current protocols in Molecular Biology, John Wiley and Sons, 1995 and Harwood and Cutting (eds.), "Molecular Biological Methods for Bacillus", John Wiley and Sons, 1990. The plasmid to be inserted into a host cell may contain a nucleotide sequence encoding a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics (denoted as an antibiotic marker) like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, zeocin, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs. In one embodiment the pSJ1678 plasmid DNA of WO 94/19454 and Diderichsen et al., J. Bacteriol., 172, pp 4315-4321 , 1990., which enables resistance to chloramphenicol, may be used for transforming a SJ2 E. coli host cell. Alternatively the plasmid pZErO-2 (Invitrogen, CA, USA) may be used.

Bacterial DNA libraries might also be screened by this method of the invention if the secreted products are glycosylated. Some bacteria, for example Campylobacter jejuni are known to glycosylate membrane associated and secreted proteins.

Host cells

The h ost cell according to the definition may be a ny cell a ble of hosting and expressing a polypeptide of interest as well as glycosylating the protein. In one embodiment the host cell is a eukaryotic cell and in another embodiment the host cell is a prokaryotic cell.

As mentioned above, the host may also be a prokaryote such as an E. coli or Lactococcus modified to comprise genes from a bacterial glycosylation pathway, e.g. the glycosylation pathway from a Campylobacter sp, particularly Campylobacter jejuni. (Wacker, Michael, Linton, Dennis, Hitchen, Paul G., Nita-Lazar, Mihai, Haslam, Stuart M., North, Simon J., Panico, Maria, Morris, Howard R., Dell, Anne, Wren, Brendan W., Aebi, Markus, N-Linked Glycosylation in Campylobacter jejuni and Its Functional Transfer into E. coli: Science 2002 298: 1790-1793).

The host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

In a particular embodiment, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the O omycota (as cited i n Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

In another particular embodiment, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfect! (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980). In an even more particular embodiment, the yeast host cell is a Candida, Hansenula,

Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In a most particular embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most particular embodiment, the yeast host cell is a Kluyveromyces lactis cell, a Yarrowia lipolytica cell, a Scizosaccharomyces pombi cell, or a Pichia pastoris cell or a Lipomyces starkii cell.

In another more particular embodiment, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

In an even more particular embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.

In a most particular embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus o ryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, F usarium g raminearum, Fusarium g raminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In an even most particular embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another most particular embodiment, the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium p urpurogenum, Thielavia terrestris, Trichoderma harzianum,

Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1 984, P roceedings of the N ational Academy of Sciences USA 81 : 1470- 1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

In a further embodiment, the mammalian cells are immortalized mammalian cell lines.

Host background When applying the method of the invention for screening, a significant reduction in background, meaning a reduction in the expression and secretion of the major secreted proteins of the expression host, is highly desirable. As an example, our production host, Aspergillus oryzae, has been mutated and selected to have both the major amylase genes and the major proteases knocked out (for further details see WO 00/39322 as well as other suitable hosts as described in PCT/DK03/00198). This results in a significantly reduced background as observed by SDS gel analysis. It is desirable to remove this background in order to improve the detection limits of the recombinant secreted protein being screened for (WO 00/39322).

Remaining host background results from cell leakage and lysis. Such proteins a re largely not glycosylated and thus removed by the lectin affinity treatment.

In one embodiment of the present invention, the host cell expressing the p olypeptide(s) of interest therefore has a minimum number of endogenous genes encoding and expressing secreted proteins in the said host.

In a further embodiment, a number of endogenous genes encoding secreted proteins has been reduced or knocked out in the host cell.

The b ackground l evel of s ecreted p roteins in preferred host strains should not exceed 100 mg/liter particularly 10 mg/liter even more particularly 1 mg/liter. Elimination of major secreted products from the supernatant by mutation of the gene encoding said proteins is one way to achieve this. Another way to achieve this is by control of the fermentation conditions. Naturally the two techniques may be combined.

Methods of reducing expression of genes or inactivating genes are well known in the art.

Immobilization of lectin

The lectin compounds of the invention are immobilized on e.g. a solid particulate carrier material. Such carrier material can be porous or non-porous. In case of non-porous particles such as non-porous beads, reducing the diameter of the bead will result in an increase in the available surface area per volume occupied by the beads.

An essential feature of the present invention is to provide a large enough surface area for the immobilization of the lectin compound thus resulting in the maximum binding capacity of the affinity matrix.

In one embodiment of the invention the lectin compound is immobilized on beads. Other ways of immobilization could also be imagined such as fibres, amorphous materials, diatomaceous earth. Both porous and non-porous material may be used.

In one embodiment of the invention the lectin compound may be immobilized on solid particulate carrier material such as on porous or non-porous beads, having a diameter of between 500- 0.05 microm. Porous or non-porous beads typically has a diameter of less than 500 microm, particularly less than 200 microm, more particularly less than 100 microm, such as less than 50 microm or 10 microm.

When the beads are non-porous the diameter of the beads has to be smaller than in the case of a p orous b ead i n o rder to a chieve the s ame s urface a rea available for i mmobilizing t he lectin compound.

Typically the beads according to the invention have a diameter comprised in the range from 0.05-10 microm, particularly in the range from 0.08-5 microm.

The beads may in one embodiment be acryl beads (Sigma Aldrich, C6160-1G) coated with concanavalin A, in the present termed conA-acryl beads. Other suitable beads could be conA- sepharose beads, conA-agarose beads, conA sephacryl beads, see van Sommeren, A.P.G. et al. Comparison of three activated agaroses for use in affinity chromatography: effects on coupling performance and ligand leakage, J. Chromatogr. 639, 23 (1993). Other examples of relevant beads include but are not limited to: conA-silica beads, conA- polystyrene beads or conA-epoxy-activated silica beads or their derivatives. In another embodiment, epoxy-activated silica may be used as the solid matrix.

ConA beads can be prepared in a number of ways. A specific example for epoxy-activated silica beads is provided here: 8.0 g dry epoxy-silica (modified with glycidyloxypropyltri- methoxysilane by Daiso co./FeF Chemicals) and 32 ml 25 g/l ConA in 0.1 M KH2P04/NaOH, pH 8.15 is mixed and stirred gently for five days at room temperature. Residual epoxy-groups are blocked by adding 5ml 1 Tris/HCI, pH 8.0, and stirring for another day.

When applying a multi well format for screening secreted polypeptides according to the invention, a 96-well microtiter plate format is one appropriate embodiment. In this format the surface area available for the immobilized lectin compound should at least be 500 mm² per compartment/well (volume pr. well is approximately 300 microl in a 96 well plate). In another particular embodiment the surface area is at least 1000 mm², more particularly at least 5,000 mm² per compartment having a volume of 300 microl.

According to the method of the invention several samples may be screened at the same time by contacting each liquid sample with the immobilized lectin compound in separate compartments for each sample. In one embodiment at least 10 separate compartments are applied, such as at least 24, particularly at least 96, more particularly at least 384.

Each compartment is filled with the immobilized lectin on e.g. a particulate carrier material. The a mount of t he i mmobilized l ectin on the particulate carrier m aterial (the affinity matrix) may vary and will typically depend on the sample size to be screened.

Each separate compartment may in general comprise between 5-100% of the maximum number of particles constituting the particulate carrier material with immobilized lectin compounds that each compartment is capable of containing, particularly between 10-80%, more particularly between 25-50%.

A common p roblem when p urifying e .g. p roteins o btained from fermentation cultures is the need for mechanical separation of cells or cell debris by e.g. centrifugation or filtration in order to avoid clogging problems when loading samples on columns. The present invention makes possible the rapid purification of multiple samples at the same time without any need for an initial separation or purification step before bringing the sample in contact with the immobilized lectin compound. The discrete liquid samples can according to the invention be provided as crude cell culture samples. The crude culture samples can be grown by any suitable method promoting growth of the host cell of choice and the host cells can be grown with or without the immobilized lectin compound being present. If grown without the immobilized lectin compounds appropriate amounts of sample can subsequently be brought in contact with the lectin compound.

The library of polypeptides of interest to be retained, e.g. in the form of fermentation broths, is in a particular embodiment in the form of small volume discrete samples of less than 3.7 ml. However, when preparing a library of polypeptide samples, e.g. by fermenting a population of host cells comprising a library of nucleotide sequences encoding the library of secreted poplypeptides of interest, the sample volumes may be smaller, such as volumes sufficiently small to be contained in wells of modern micro plates. Small volumes may be applied to increase the sample capacity. Accordingly the sample volume may be comparable to the well volume of commercially available microtitre plates. A suitable volume is less than the well volume of a 24 well micro plate, particularly less than the volume of a well on a 96 or 384 well plate.

Retention of the biological compound on the solid chromatographic material

Retention of the biological compound may be achieved by contacting, in solution, the tag-free polypeptide of interest with the immobilized lectin compound comprising lectin immobilized to a suitable carrier as described above.

The immobilized lectin compound material may be in any suitable form, in particular a form, wherein a maximum of the immobilized lectin compound material is exposed to the biological compound. The immobilized lectin compound material may also be combined with a magnetic material so that the immobilized lectin compound material may be physically controlled by applying a magnetic field. In one embodiment the immobilized lectin compound material, particularly in the form of a solid ball or a bead or another solid structure comprising the immobilized lectin material, is simply added to a sample comprising the secreted polypeptide of interest at conditions wherein the polypeptide is capable of binding to the immobilized lectin compound material.

In one embodiment, the host cells are grown in the presence of the immobilized lectin compounds and in a further embodiment the growth and expression of the polypeptide of interest is conveniently performed in a multi-well format, such as e.g. a 96 well microtitre plate format. In one embodiment contacting the discrete samples comprising the library of tag-free polypeptides of interest with the solid immobilized lectin compound material may be performed in a population of wells in a micro-titer plate, in particular those fitted with a filter, particularly at the bottom of the wells. These are commonly called microtitre filter plates because they are able to filter the material in each well through a filter and orifice at the bottom.

In another embodiment, the vessels are a population of hollow vessels, such as small volume chromatography columns, in which the immobilized lectin material is packed. These hollow vessels allow samples comprising biological compounds to pass the immobilized lectin material whereby secreted polypeptides of interest are retained in the columns.

To ensure that only biological compounds with substantial affinity towards the immobilized lectin compound material are retained, the amount of solid immobilized lectin material may be limited so that the immobilized lectin material is substantially saturated with biological compounds of interest leaving little or no space available for retaining other constituents of the samples. In particular one may use less than 10.000 mg immobilized lectin compound material per mg biological compound of interest in a sample, in particular less than 5000 mg, more particularly less than 1000 mg immobilized lectin compound material per mg biological compound of interest in a sample. One advantage of using a limited amount of immobilized lectin compound material is that a constant amount of biological compound may be retained, so that a need for subsequent quantification (which is useful when using the isolated biological compound in tests for improved properties) may be eliminated. By applying a limiting amount of immobilized lectin material, only a fraction of the biological compound in a sample may be bound, thus differences in levels of biological compound in different samples may not affect the amount of isolated biological compound. Such an approach may make automation simpler as it eliminates the necessity for using variable sample volumes for each sample of isolated biological compound when testing for improved properties.

Isolating the immobilized lectin material retaining the secreted polypeptides of interest from the samples When the secreted polypeptides of interest have been retained on the lectin material the lectin material may be isolated from the unbound constituents of the samples.

When a vessel or container with a filter is used the lectin material retaining the biological compounds may be isolated by filtering the sample through the filter leaving the lectin material on the filter. When the vessel is a hollow vessel, such as a column wherein the lectin material is packed, the isolation of the lectin material retaining the polypeptides of interest may be achieved by flushing the samples through the hollow vessel allowing impurities to flow past the lectin material.

In a particular embodiment, microtitre plates (Multiscreen HV Plate (Millipore MAHVN4550) e.g. Whatman, Unifilter 800 microl, 25-30 microm MBPP) equipped with filters in the bottom of the wells may be used. In particular the method of the invention may be carried out by placing the filter plates on top of a standard microtitre vacuum unit (such as a Whatman Univac 3), which provides controlled sub-atmospheric pressure underneath the filters, while the top side of the microtitre plate is open to the ambient atmospheric pressure. To minimize differences in drainage from each well a special lid as that described in WO 2003037914 may be used. In short said lid divides the space above the filter plate into individual compartments isolated from ambient pressure. The number of compartments on the lid should correspond to the number of wells in the microtitre plate. The compartments may e.g. be provided by a suitable grid of a rubber material or similar material. The basic shape of the lid is the same as a microtitre plate turned upside down. A coating with rubber or similar material ensures sufficient contact between the bottom of the lid and the filter plate so that each individual compartment is air tight. This construction ensures that a vacuum is maintained in each filter-bottomed well until the liquid from the well is drained through the filter. Draining of one well has no influence on the other wells. Regarding the physical form of the lids, we have obtained successful results with 1 cm³ head space in each lid compartment. Obviously, increasing this volume only benefits the technique. We expect that the compartment size may be as low as 0.1 cm³ head space depending on the particular resistance towards liquid drain. An alternative method of separation is low speed centrifigation of a stacked microtitre filter plate, microtiter collection plate configuration. In such cases, it is helpful to have a microtitre plate "stacker" to stabilize the two stacked plates during centrifugation. The stacker is basically a sleeved precision plastic frame that allows the two plates to be stacked to be seated tightly to each other.

Washing the retained biological compound

When a polypeptide of interest has been retained on the immobilized lectin material it may optionally be further freed of impurities by washing the lectin material in one or more washing steps.

The continuous phase of the washing liquid may be aqueous or organic. However, it is important that the affinity matrix preserves its functional properties while in contact with the washing liquid. In the case, supra, the vessel holding the polypeptide of interest retained on the immobilized lectin material is a well in a micro plate fitted with a filter in the bottom of the well, the washing liquid in each washing cycle may be removed through the filter leaving the purified polypeptide of interest retained on the immobilized lectin material on the filter.

In the case, supra, the chromatographic material is packed in a hollow vessel, such as a column, the washing l iquid i n each washing cycle m ay s imply b e flushed through the hollow vessel allowing impurities to flow with the washing liquid, while retaining biological compound retained on the immobilized lectin material.

Releasing the biological compound from the solid chromatographic material

Polypeptides may be eluted from the solid particulate immobilized lectin compounds also referred to as the matrix, by contacting the matrix with a solution containing soluble sugar or polysaccharide. In this way the lectin binding sites may be competed out by the more abundant sugar solution provided resulting in the release of the bound glycosylated protein. In general, it is good practice that the sugar solution is buffered at the appropriate pH with a buffer solution. Optionally, a non-ionic or zwitterionic detergent may also be included as needed by the specific lectin-protein interaction to increase elution efficiency (Ochoa, J.L.,

Sierra, A., and Cordoba, F. "On the specificity and hydrophobicity of lectins" in: Lectins;

Biology-biochemistry-clinical biochemistry: proceedings of the third Lectin Meeting;

Copenhagen, June 1980-1981 ISBN3-11-008483-X pp.73-79. T.C. Bøg Hansen editor, copyright 1981). The choice of sugar solution depends on the lectin compound immobilized on the particulate carrier since the sugar should be capable of competing with the polypeptide for binding to the lectin. In particular the bound glycosylated protein may be eluted with methyl- alpha-D-mannopyranoside, alpha-methyl glucoside or a mixture of the two.

Collecting the polypeptide of interest Once the polypeptide of interest has been released it may be separated from the immobilized lectin material (which then may be reused) to produce a liquid comprising the isolated polypeptide of interest.

In the case, supra, the vessel holding the polypeptide of interest retained on the immobilized lectin material is a well in a microtitre plate fitted with a filter in the bottom of the well, collection of the released liquid comprising the polypeptide of interest may be achieved by filtering the released liquid through the filter leaving the chromatographic material on the filter.

General protein detection A sensitive general protein detection method is needed to determine if a glycosylated protein was eluted from the immobilized lectin material. The method should be able to detect protein at a concentration of between 10ng/100microl and 500ng/100microl. A detection method that does not have a strong bias based on protein structure would be desirable.

One detection method is the Nano-Orange protein quantification system optimized for detection and quantification of proteins in liquids and another detection system is the SYPRO Orange system used for SDS acrylamide gel analysis. Both SYPRO Orange S-6551 and the NanoOrange Protein Quantification kit were o btained from M olecular P robes I nc., ( PO Box 22010, Eugene OR, 97402-0469, USA; www.probes.com). Both the SYPRO Orange and NanoOrange detection kits utilize the same dye chemistry to stain SDS-protein interactions for highly sensitive quantification of proteins.

The NanoOrange quantization reagent, a component of the NanoOrange Protein Quantitation Kit (N-6666), binds to a hydrophobic SDS detergent coat around the proteins, which results in a fluorescent s ignal. Because p roteins bind similar amounts of detergent on a mass basis, there is very little protein-to-protein variation, allowing accurate quantification of protein mixtures of unknown composition. The NanoOrange protein assay detects as little as 10 ng of protein per ml, which is 1000 times more sensitive than UV absorbance (A280) measurements and 100 times more sensitive than the Lowry or Bradford assays. The assay takes only 30 minutes. The method was used as directed according to the manufacturer, Molecular Probes, and for further details see e.g. product information sheet MP 06666.

The method affords a reliable detection range of between 100ng-10microg protein/ml when used with fluorescence-based m icroplate readers. B riefly, 1 Omicrol of s ample is mixed with 290microl of diluted NanoOrange protein quantification reagent (500X diluted in NanoOrange protein diluent component B). A sample volume of no more than 4% of the total volume is desired for a ssay a ccuracy. T he s ample i s covered with a vapour tight seal and heated to 95°C, protected from light. The samples are then cooled to room temperature for 20 minutes protected from light. The signal is detected in a standard fluorescence-based microplate reader according to the NanoOrange protocol available from Molecular Probes.

Detection of glycosyl groups

A detection method that specifically detects glycosyl groups on the secreted proteins could also b e u sed. Two main m ethods exist, one is a MALDI (Matrix Assisted Laser Desorption lonization) method where one first adds a commercially available deglycosylating enzyme (such as endoA) then desalt the mix or dilute if necessary. The blend is then mixed with a suitable matrix such as de-hydroxy benzoic acid (2,5 DHB) and applied on a MALDI target plate. The spots are analyzed by MALDI-TOF MS (Matrix Assisted Laser Desorption lonization-Time Of Flight Mass Spectrometry) and the presence of free glycosyl groups are identified in the spectrum of the positive candidates.

Another method for detection of glycosyl groups is an ELISA (Enzyme linked immunosorbent assay) type sandwich assay with antibodies directed towards glycosyl side chains.

Test for monitoring the degree of purity of the isolated biological compound

When a polypeptide of interest e.g. from a crude broth of a cell culture is isolated and/or purified it may be useful to establish the purity of the polypeptide before carrying out any further tests for improved properties. This may be achieved, depending on the nature of the biological compound, by any conventional method. For example it is possible to do simple quantifications, such as UV (280 nm) absorbance and protein fluorescence to measure the purity and amount of biological compound isolated. Both techniques require no substrate and consume no sample; concentration is simply determined by an almost instant read in a spectrophotometer or spectrofluorometer.

In standard protein fluorescence, one typically runs excitation of sample at 280 nm and emission around 340 nm. Fluorescence is more sensitive than UV a bsorbance a nd i s I ess prone to giving a false signal if sample contaminated with e.g. DNA/RNA absorbance. However, m utations i nvolving a romatic a mino a cids ( especially T ryptophan) c an m ake b oth fluorescence and UV absorption determination inaccurate.

Other methods of quantifying the amount of isolated biological compound include use of commercially available kits from Bio-Rad to quantify protein contents of a sample.

Instead of using a sensitive general protein assay, MALDI (MALDI means Matrix Assisted Laser Desorption lonization) can be used for high throughput detection of conA based screening. Briefly, the eluate containing the secreted glycosylated protein may be processed with a protease such as Trypsin (cleavage after Arginine or Lysine residues) or otherwise degraded into smaller fragments suitable for detection with MALDI-TOF. The treated material may then be desalted with a HTS desalting column setup and then mixed with a matrix such as 4 hydroxy-alpha-cyano cinnapic acid (HCCA). A standard peptide map for the expression strain is produced by MALDI-TOF MS and subtracted from the sample peptide maps. In this way proteins only expressed by the sample and not the standard expression strain may be identified as peaks only present in the spectrum obtained from the sample and not the standard. Furthermore, the new peaks may be used for ID (identity) or homology searches in sequence databases providing a possible identification for the new expressed protein. Modification of the MALDI-TOF method is also a useful detection method for identifying secreted proteins directly in the conA-protein matrix (glycosylated protein bound to the immobilized conA on solid particulate carrier material) without e lution of the bound protein.

Such methods as described in: Nordhoff E, Krogsdam AM, Jorgensen HF, Kallipolitis BH, Clark BF, Roepstorff P, Kristiansen

K. (1999) Rapid identification of DNA-binding proteins by mass spectrometry. Nature

Biotechnology, 17(9) 884-888.

An interesting variation is called SELDI-TOF (Surface Enhanced Laser Desorption-Time of

Flight). This method is also adaptable to detection of secreted proteins directly in the conA- protein matrix.

Issaq HJ, Veenstra TD, Conrads TP, Felschow D. (2002) The SELDI-TOF MS approach to proteomics: protein profiling and biomarker identification. Biochem Biophys Res Commun

5;292(3):587-92

In one embodiment the screening for lectin trapped protein eluates may be detected by general protein quantification assays such as SYPRO Orange or Nano Orange or by mass spectrometry such as MALDI-TOF.

The screening according to the invention may in a further embodiment comprise a further screening step. Such an additional screening comprises screening for e.g. activity, functionality, stability, allergenicity, and anti-microbial effect of the secreted polypeptide.

The polypeptide of interest to be screened for may be any secreted polypeptide of interest. In one embodiment the biological compound is a protein, a polypeptide or a peptide, particularly an enzyme or a pharmaceutical such as a hormone or a receptor.

EXAMPLES

The following examples illustrate the applicability of immobilized lectin material for screening and capturing secreted proteins. One example relates to concanavalin A coated plates and one example relates to conA-acryl beads.

Materials used

Concanavalin A coated Microtitre Strips (Merck Eurolab, 90006386-0). Beads (Sigma Aldrich, C6160-1G) Methyl-alpha-D-mannopyranoside (Sigma Aldrich, 67770-25G). Buffers used

Equilibration and washing buffer: 10mM TRIS pH 7.5, 10mM CaCI2, MnCI2). Elution buffer (1 M Methyl-alpha D-mannopyranoside in 10mM Tris pH7.5).

Test enzyme used

The endo type cellulase EG1 of Thermoascus aurantiacus was used (Hong,J.; Tamaki.H.; Yamamoto.K.; Kumagai,H.; Endo-beta-1 ,4-glucanase genomic DNA from Thermoascus aurantiacus IF09748. Submitted (FEB-2002) to the EMBL/GenBank/DDBJ databases; SPTREMBL:Q8TG26). The peptide was expressed in the Aspergillus oryzae strain EXP0512 as d escribed i n patent application PCT/DK03/00039. Standard shake flask fermentations in YPM media were made of strain EXP0512 exactly as described in the above patent application. Fermentations of the expression strain Bech2 (WO 00/39322) were performed in parallel.

Example 1. Concanavalin A coated plates for purification: determination of binding capacity parameters of concanavalin A binding.

Experiments were performed in order to determine if microtitre plates coated with ConA could be used to trap enough glycosylated protein for detection by 1) a sensitive target enzyme assay, in the present case a beta glucanase assay, or by a general protein staining assay such as 2) a SYPRO orange stained SDS gel, or 3) a small volume liquid SYPRO orange protein stain.

Procedure: Strain EXP0512 was cultured allowing expression of the secreted polypeptide, which under these conditions acts as a beta-glucanase. The culture medium was used to test for detection of secreted proteins. The culture medium was used in undiluted form and in different dilutions as described below. Dilutions were made using corresponding Bech2 supematants since Bech2 is the screening host and thus dilutions in Bech2 provide realistic background levels throughout the dilutions series. This provides relevance in detection limits of proteins expressed in Bech2 background. The following dilutions of the culture medium were tested:

EXP0512 YPM grown- undiluted

1 :2 1:5 1 :10 1 :20 1:40 Bech2 undiluted

100 microl of Equilibration and washing buffer were added to well of the conA coated microtiter strip (from Merck) and allowed to equilibrate for 10 minutes. The buffer was removed and 50 microl of sample material (in all dilutions) was added to each well. The wells were incubated with gentle shaking for 2 hours, 37°C and washed 2X 30 minutes with

Equilibration and washing buffer (lOOmicrol).

50 microl Elution buffer was added and the wells were incubated with gentle shaking for 30 minutes.

The supernatant comprising the retained proteins was transferred to a normal microtitre plate. These samples were then tested by the different assays for enzyme activity, and general protein content.

1 ) Standard AZCL Beta glucan assay

Materials: AZCL-Barley beta glucan (Megazyme, Ireland) A suspension of 0.1% was made by adding a AZCL-beta glucan wetted in 70% ethanol, to

10mM Tris pH 7.5.

10 microl of treated samples from the conA experiment were added to 90 microl buffered

AZCL-beta glucan in a microtitre plate. The experiment was assayed with the following treatments and controls: All the samples described above, which were treated with conA were included. Furthermore some untreated samples were included in the assay, which includes: controls, EXP0512 undiluted, and Bech2 undiluted.

The assay was observed visually after 2 hours of incubation at 37°C and after an overnight incubation at the same temperature. Detection of the beta glucanase enzyme was possible down to 1 :5 dilution after two hours in the conA treated material and down to 1 :20 dilution after an overnight incubation. This result indicate that enzyme activity was captured by the conA-microtitre strips down to 1 :20 dilution based on the most sensitive assay available; the specific enzyme assay.

Small volume liquid SYPRO orange protein staining assay The same sample treatments were assayed with the Liquid SYPRO Orange assay for small sample volumes as described above and in e.g. product sheet MP06666 from Molecular Probes.

Positive protein detection was only possible on the undilutted EXP0512 material that had not been treated with conA. We conclude that the binding capacity of the conA-microtitre plates was insufficient.

SYPRO orange stained SDS gel

The same samples as above were run on a SDS gel with SYPRO Orange staining. Supernatants were run on NuPage 10% Bis-Tris SDS gels (Invitrogen) as recommended by the manufacturer (NuPage, Inc., USA) with the modifications suggested according to Molecular Probes (see e.g. product sheet MP06650).

Results: The results of t he e nzyme a ctivity staining experiment for beta glucanase activity indicated that the conA coated plates could trap enough enzyme to detect activity even after 40 fold dilution in supernatant from the expression host. This indicated not only that the method worked in principle but that dilution of the enzyme producing supernatant with culture fluid from the expression strain did not interfere with capture or detection of the activity even in high dilution. Next we wanted to see if the captured enzyme would be sufficient for detection in a very sensitive general protein detection assay. Results indicated that neither in the SYPRO Orange liquid assay nor in the SYPRO stained SDS gel could a band of any kind be seen. This indicates that the binding capacity of conA coated microtitre plates is not high enough for use in a general protein assay detecting enzyme activity in eluate.

Example 2. Use of conA-acryl beads

One way to increase the interaction of conA with the glycosylated protein is to increase the surface area displaying conA to the protein. This can be achieved by using conA coated beads. The surface area of beads is several folds higher than a coated vessel surface (i.e. microtitre wells).

The same procedures were used as in the above experiment with the coated microtitre strips with the following modifications: Normal 96 well polystyrene microtitre plates were used. Two grams conA-acryl beads were rehydrated overnight in 7 ml equilibration and washing buffer + 0.1% sodium azide. The bead suspension was aliquoted into a microtitre plate so that, after mild centrifugation, approximately 0.5 mm of beads was deposited at the bottom of the wells.

The beads were washed twice in 500 microl equilibration and washing buffer (as from previous experiment) and the excess buffer removed with a pipette. Elution was performed exactly as described in the previous experiment.

A SYPRO Orange SDS gel was run on samples recovered from the conA-acryl bead treatments. It was established that a single band corresponding to the correct molecular weight was seen on the gel. Interestingly, background bands observed in both the crude EXP0512 and the Bech2 fermentation broths were completely removed by the conA-acryl bead treatments. A band corresponding to EXP0512 beta glucanase was observed in the undiluted, 1 :2 and 1 :5 dilutions only.

Visualization method:

The SYPRO stained gels were placed directly on the UV transilluminator of a Stratagene Eagle Eye 2 image documentation system. The gel was exposed to 260nm wavelength UV light and the image captured as a JPEG file using Gel-Pro Analyzer software version 3 (Media Cybernetics, www.mediacy.com). The image taken captured by the Eagle Eye 2 was analyzed with the densiometric analysis software of the Gel-Pro Analyzer. After the gel lanes were assigned, the bands were marked and then the density value of the marked band calculated as total relative OD.

Table 1

Relative densiometric measurement of supernatant of EXP0512

maxOD conA

EXP0512

Undiluted 0.0214

1 :2 dilution 0.0362

1 :5 dilution 0.0035

1 :10 - no conA undiluted 2.3027

Background, in the form of a zone of the gel not containing a band, has been subtracted from the values shown.

This indicates that the binding capacity was not sufficient to trap enough EXP0512 protein in higher dilutions. A rough estimation, based on the beta glucanase, protein indicates that, for the proposed conA HTS system to be workable with expression levels normally observed in our expression hosts, we must be able to detect glycosylated protein down to at least 200mg/liter in crude culture broths. This roughly corresponds to a 1:20 dilution of EXP0512. In order to detect the protein in the dilution, it was decided to establish whether adding more conA-acryl bead matrix would capture more of the secreted beta glucanase from EXP0512 in the full dilution range especially 1 :20 and over.

An identical experiment to the one previously described was run with the exception of adding conA-acryl beads up to half of the void volume of the wells of a 96 well microtitre plate. The results this time confirmed that EXP0512 could be visualized by SYPRO-Orange stained SDS gels down to a 1 :40 dilution. As with the previous experiment, the elimination of background proteins from the fermentation broth was also observed.

Table 2.

Relative Densiotometric measurement of supernatant of EXP0512 conA acryl beads

maxOD conA

EXP0512

Undiluted 2.4065

1 :2 dilution 0.1204

1 :5 dilution 0.1579

1 :10 dilution 0.1065

1 :20 dilution 0.1863

1 :40 dilution 0.0794 no conA undiluted 2.4065

This experiment shows detection by SDS gel electrophoresis of the EXP0512 peptide down to a dilution of at least 1 :20. This is roughly equivalent to 250mg/liter in the culture broth and is a realistic lower limit for the expression levels of a plasmid based high throughput Aspergillus oryzae transformation and expression system.

Example 3. Co-Fermentation of Aspergillus beta glucanase producing strain EXP05 2 with conA acryl beads It could be considered an advantage from the standpoint of economy of plastic ware and liquid handling to co-ferment the recombinant samples in microtitre plates in which conA has been added. 500 microl of conA beads were added to each well of a millipore filter plate. Beads were washed 2X in equlibration and washing buffer. 3M clear adhesive microtitre tape was applied to the bottom of the filter plate to seal the holes. 400 microl of YPM media + ampicillin (50 microg/ml) was added to each of 5 wells. An additional 3 wells were also filled with media. The wells were inoculated with between 10 and 100 spores of EXP0512 or Bech2. Microtiter dishes were sealed with porous adhesive tape and incubated at 37°C with shaking (200 rpm). After 2.5 days of growth, it was clear that both the recombinant Aspergillus strain and Bech2 were able to grow quite well in the presence of conA-acryl beads.

The sealing tape from both the top and bottom of the plate was removed and the fluid removed from the plate with mild centrifugation. The filtrates were saved in a microtitre collection tray and frozen until later use. The microtitre filter plate was washed twice with equilibration and washing buffer. Material bound to the conA beads were harvested in the following manner: 100 microl of 1 M alpha mannopyranoside was applied to each well and allowed to incubate for 30 minutes at room temperature. The filtrate was then harvested by light centrifugation and collection into a microtitre plate. The plate was frozen until later use. A SDS gel was run of the treatments and stained with SYPRO orange as done previously. The results indicated that co-cultivation of the recombinant strain EXP0512 with conA-acryl beads: 1) did not lead to any perceived toxic effects, 2) demonstrated that conA- acryl beads could still specifically bind the EXP0512 beta glucanase, and 3) only minor high molecular weight background was seen in the experiment.

The experiments above shows that a lectin based affinity matrix is applicable for purification of secreted recombinant proteins in a 96 well microtiter plate format due to satisfactory recovery of the secreted glycosylated recombinant protein and the elimination of host background.

Example 4. Detection of conA trapped eluates in liquids using the NanoOrange detection method

In order for the screening for conA captured secreted proteins to function in high throughput systems, the detection method must work as a liquid detection system. The following experiment was performed to verify that the NanoOrange detection method could be used.

Methods: Standard shake flask fermentations in YPM media were made of strain EXP0512 exactly as described in experiment example 1. Fermentations of the expression strain Bech2 (WO 00/39322) were performed in parallel. The fermentations were purified in the microtiter filter plate based method described in example 2 of this patent application and a SYPRO orange stained SDS gel was run on the samples to confirm that the purification had worked. The remainder of the samples was frozen until the NanoOrange assay could be performed.

The NanoOrange Assay:

All measurements were performed on a 1420 Victor Multilabel Counter (EG&G Wallac, Turku Finland). The continuous wave quartz halogen reflector lamp was used for fluorescence detection with the light filters configured for 485 nm excitation and 572 nm emission and emission filter configured to "normal" mode as defined by the equipment manufacturer. Each sample was measured for 1 second.

Detection of BSA (bovine serine albumin standards)

Standard dilutions were made of the BSA supplied with the NanoOrange kit exactly as described by the manufacturer of the kit for microtitre based screening except the samples were placed in 0.2 ml PCR microtube trays (Thermo-fast 96 non skirted, AB Gene). Pretreatment of the samples was performed in an MJ Research DYAD thermal cycler under continuous heat for 10 minutes at 90°C and then cooling at room temperature protected from light. Samples were transferred to a black well, flat bottomed microtiter plate (Sero-Wel, Bibby Sterilin Ltd., UK) and measured directly in the 1420 Victor. The results are displayed below in table form:

Table 3.

Nano Orange dilution detection experiment for BSA

Final BSA cone. F Flluuoerescence units microg/ml

0 118

10 716

6 378

3 202

1 94

0,6 84

0,3 90

0,1 108 The results demonstrate that the detection limit of BSA with the assay and equipment used in the experiment was between 2 and 3 ng/ml protein standard. This detection limit is somewhat higher than reported in the NanoOrange instruction sheet. A possible explanation is the non optimal wavelength of the emission filter in our 1430 Victor detection instrument (572 nm) versus the optimal wavelength for the NanoOrange dye (590 nm). Detection of the BSA standard was however calculated to be sufficient so as to expect that conA bound proteins could also be detected in the concentration range of the conA eluate.

Detection of EXP512 protein in dilution using the NanoOrange assay

Dilutions of non conA treated EXP512 were made in 1X NanoOrange working solution for a total volume of 250microl according to the manufacturer's instructions. For the conA treated and the Bech2 control samples, lOmicrol was mixed directly with 1 X N anoOrange working solution. All samples were heat pre-treated and measured in exactly the same way as the BSA standard experiment above. The results are shown below.

Table 4.

Nano Orange dilution detection experiment for EXP512 and Bech2 control dilution. Fluorescence units

0 180 undiluted 880

1 2 dilution 884

1 5 dilution 690

1 10 dilution 572

1 20 dilution 308

1 40 dilution 206 conA eluate undiluted 476

B ech2 undiluted 830

The result clearly demonstrates that conA purified EXP512 eluate can be detected with the NanoOrange system with a detection range comfortably within the limits of the system. When summarized, the body of the results presented in examples 1 through 4 show that a typical glycosylated enzyme expressed in Aspergillus oryzae host Bech2 can be purified with conA and detected with SYPRO reagents in either SDS gels (SYPRO orange) or liquid format (NanoOrange). Example 5: Detection of previously identified Aspergillus recombinant lines expressing secreted enzymes from an automomously replicating plasmid with the NanoOrange assay

Five clones identified by an immunoscreening method and expressed in an Aspergillus host were tested with the NanoOrange assay. Each clone comprised the secreted protein identified below encoded on an autonomously replicating plasmid:

Culture fluid supernatants of the clones all produce, to varying degrees, a visible band on an SDS gel stained with Coomasie blue according to standard procedures.

Experimental setup: The six clones listed and the control host strain Bech2 were grown in 96 well microtiter plates in 200 microl YPM medium at 37°C for 3 days and 50 rpm shaking. 100 microl of the culture fluid was transferred manually to a filter microtiter plate in which each well had received conA acryl beads exactly as described in example 4. The purification, elution procedure and NanoOrange treatment and detection were also performed exactly as described in example 4. 10 microl of supernatant or the remaining culture fluid was used to run a standard SDS PAGE gel which was then stained with Coomasie blue to confirm the presence of secreted protein.

Results of the NanoOrange assay.

Treatment FU FU corrected

Clone 1 298 204

Clone 2 396 302

Clone 3 324 230

Clone 4 312 218 Clone 5 348 254

Clone 6 252 158

BECH2 216 122

The corrected are minus the background for the assay; 94 FU units. FU means Fluorescence units.

As can be seen from the results, the treated supernatants from the Aspergillus lines secreting enzyme can easily be detected above the control expression host Bech2. In one case, the signal is over 2X the signal of Bech2. The experiment shows that the method described can be used to detect Aspergillus expression clones encoding secreted enzymes that are encoded on autonomously replicating plasmids. Such autonomous plasmid systems are routinely used for recombinant library screening in Aspergillus.

Claims

1. A method of screening for secretion of a polypeptide of interest comprising:

b) contacting in solution and in a compartment a liquid sample from (a) comprising the glycosylated polypeptide of interest with an immobilized lectin compound capable of binding to the glycosylated polypeptide of interest, under conditions where binding capacity of the immobilized lectin compound per compartment volume is at least 10 ng polypeptide/300 microl compartment volume,

d) releasing the polypeptide of interest from the immobilized lectin compound.

2. The method according to claim 1 , wherein the polypeptide is provided from a host cell expressing and secreting a library of polypeptides.

3. The method according to any of the preceding claims, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

4. The method according to claim 3, wherein the eukaryotic cell comprises fungi, yeast, mammalian cells, and insect cells.

5. The m ethod a ccording to claim 4 , wherein the fungi comprises Aspergillus oryzae, Aspergillus niger, and Fusarium graminearium.

6. The method according to claim 4, wherein the yeast comprises Saccharomyces cerevisiae, Scizosaccharomyces pombi, Pichia pastoris, and Lipomyces starkii

7. The method according to claim 4, wherein the mammalian cells are immortalized mammalian cell lines.

8. The method according to claim 3, wherein the prokaryotic host cell has been modified to comprise genes from a bacterial glycosylation pathway.

9. The method according to claim 8, wherein the genes from a bacterial glycosylation pathway comprises genes involved in glycosylation in Campylobacter sp.

10. The method according to claim 9, wherein the Campylobacter sp. is Campylobacter jejuni.

11. The method according to any of the preceding claims, wherein the host cell expresses a minimum of secreted proteins endogenous to the host.

12. The method according to claim 11 , wherein expression of a number of endogenous genes encoding secreted proteins has been reduced or knocked out in the host cell.

13. The method according to any of the preceding claims, wherein the lectin compound comprises lectins capable of binding sugar moieties in the glycosyl backbone, branch point or terminal sugars of glycosylated polypeptides.

14. The method according to claim 13, wherein the lectins comprises plant lectins, particularly lectins capable of binding alpha mannose, alpha glucose or GlcNAc in secreted glycosylated proteins selected from the group comprising concanavalin A (Con A), Lens culinaris agglutinin (LCA), Pisium staivum agglutinin (PSA), Galanthus Nivalis Lectin (GNL), Hippeastrum Hybrid Lectin (HHL), Narcissus Pseudonarcissus Lectin (NP), Datura Stramonium Lectin (DSL), Lycopersicon Esculentum (Tomato) Lectin (LEL), Solanum Tuberosum (Potato) Lectin (STL), Wheat Germ Agglutinin (WGA).

15. The method according to claim 1, wherein the lectin compound is immobilized on solid particulate porous or non-porous carrier material having a diameter of less than 500 microm.

16. The method according to claim 15, wherein the diameter is less than 200 microm.

17. The method according to claim 16, wherein the diameter is less than 100 microm, particularly less than 50 microm, more particularly less than 10 microm.

18. The method according to claim 1-14, wherein the lectin compound is immobilized on a non-porous or porous carrier in the form of fibers, diatomaceous earth or beads.

19. The method according to claim 1, wherein the lectin compound is immobilized on beads such as conA-acryl beads, conA-sepharose beads, conA-agarose beads, conaA sephacryl beads, conA-silica beads, conA-polystyrene beads or conA-epoxy-activated silica beads or their derivatives.

20. The method according to any of the preceding claims, wherein the said contacting of a liquid sample with the immobilized lectin compound is performed in separate compartments for each culture sample.

21. The method according to claim 20, wherein at least ten separate compartments are applied, such as at least 24, particularly at least 96, more particularly at least 384.

22. The method according to claim 21, wherein each separate compartment comprises between 5-100% of the maximum number of particles constituting the particulate carrier material with immobilized lectin compounds, that each compartment can hold, particularly between 10-80%, more particularly between 25-50%.

23. The method according to any of the preceding claims, wherein the surface area of the particulate carrier material is at least 500 mm² per compartment volume of 300 microl, particularly at least 1000 mm², more particularly 5000 mm².

24. The method according to any of the preceding claims, wherein the screening is followed by an additional screening step, which screening step comprises screening for activity, functionality, stability, allergenicity, and anti-microbial effect of the secreted polypeptide of interest.

25. The method according to any of the preceding claims, wherein the screening for conA trapped protein eluates is detected by general protein quantification assays such as SYPRO orange or NanoOrange, or Mass spectrometry detection such as MALDI-TOF.

26. The method according to claim 1 where the recombinant hosts are fermented in a filter system such as 96 well microtitre filter plates.

27. The method according to claim 26 wherein the particulate carrier material with immobilized lectin compounds is co-fermented in the filter system.

28. The method according to any of the preceding claims, where the bound polypeptides on the immobilized lectin compounds are eluted with an inhibiting/eluting sugar which sugar is capable of competing with the polypeptide for lectin binding.

29. The method according to any of the preceding claims, where the bound polypeptides are eluted with alpha-methyl-mannoside, alpha-methyl glucoside or a mixture of the two.