WO2003050501A2 - Indirect fluctuation screening - Google Patents

Abstract

The present invention describes a general method of isolation genetic elements, encoding bioproducts that exert their biological activity from the outside the target cell. The method allows the effective identification of the desired gene products (and the factors induced by the activity of such products in target cells) that can act from the outside of the cells and, therefore, do not require additional delivery systems for their in vivo applications.

Description

Indirect Fluctuation Screening

Field of the Invention

The present invention relates to screening of expression libraries. More particularly, the present invention relates to isolation of genetic elements that encode biologically active products that exert their biological activity from the outside the target cell.

Background of the Invention Expression cloning involves screening cDNA expression libraries to identify clones that express proteins that interact with a given target protein. Gene expression libraries have been used to identify, investigate and produce the target molecules. This powerful technique allows genes to be identified and isolated based on gene function or phenotype in the absence of any prior knowledge of the protein or nucleic acid sequence or other physical properties of the protein.

A wide variety of gene types have been cloned from cDNA libraries by using functional assays designed to select or screen for a specific trait. Such genes include, for example, cell surface receptors, extracellular receptors, growth factors, oncogenes, cell cycle proteins, signaling proteins, apoptotic factors, metastates-inducing genes and the like.

In many instances, these genes encode proteins are potential disease targets, e.g. , receptors (Simonsen et al. 1994, Trends Pharmacol Sci 15:437-441; Nakayama et al. 1992, Curr Opin Biotechnol 3:497-505; Aruffo, 1991, Curr Opin Biotechnol, 2:735-741), and signal-transducing proteins (Margolis et al. , U.S. Pat. No. 5,434,064). See Seed et al. , 1987, Proc Natl Acad Sci 84:3365-3369; Yamasaki et al., 1988, Science 241:825-828; and Lin et al. , 1992, Cell 68:775-785, for examples of proteins identified by functional expression cloning in mammalian cells.

Once a disease target is identified, the protein target or engineered host cells that express the protein target have been used in biological assays to screen for lead compounds (Luyten et al. 1993, Trends Biotechnol 11:247-54).

Thus, within the scheme of drug discovery, the use of gene expression libraries has been largely limited to the identification and production of potential protein disease targets. Only in those instances where the drug is a protein or small peptide, e.g., antibodies, have expression libraries been prepared in order to generate and screen for molecules having the desirable biological activity (Huse et al. 1991, Ciba Foundation Symp 159:91-102).

Today, the vast majority of gene products that have been used as drugs or diagnostic tools in modern medical practice function from the outside of the cell. These include growth factors (i.e., insulin, erythropoietin, GMCSF, etc.), "death" ligands (TNF, TRAIL, etc.) and antibodies (Heceptin as an example of antibody-based drug, numerous diagnostic antibodies). However, isolation of such useful products from expression libraries by a direct screening is problematic.

In traditional functional cloning, a host cell is transiently transfected with the cDNA library and cultured. After a suitable period of time, cells are selected or screened for the desired phenotypes and colones or selected populations of cells are expanded or enriched. Subsequently, the genomic DNA is isolated and the cDNA inserts are retrieved using PCR and sequenced using conventional techniques. Since this conventional screening involves the use of expression gene libraries that are delivered to the test-cells by gene transfer and subsequent isolation of cells with altered phenotype, the direct practical use of this technique is hampered by the difficulties of targeted delivery of the cDNA into the cells of interest.

Moreover, the intracellular expression of the cDNA may not result in the desired phenotype which will allow recover of the products.

The progress in systematic isolation of proteins or peptides (or other cellular factors associated with their expression) would be greatly facilitated by the development of an effective screening of expression libraries for gene products with the desired properties (changing cell phenotype, capable of specific binding, etc.) that act from the outside of target cells. If developed, such methodology would streamline the development of new useful pharmaceuticals from the products of expression libraries. Summary of the Invention

The present invention described a method of screening of expression libraries for those gene products that are active if externally added to the cell and, therefore, generates bioactive factors that are much more suitable for conversion into in vivo active products. This method is applicable to a broad variety of libraries and can be used for isolation of both natural and artificial bioactive molecules with extracellular activity. The screening method of the present invention can be easily converted into a high throughput screening system using conventional equipment that is broadly used for screening of small molecules. It does not necessarily require creation of new libraries and can be applied for many existing expression libraries of different nature. The prospective products that could be generated by this approach are: proteins, peptides or, other factors that are generated by host cell after exposure to certain gene product. Their practical use involved the whole spectrum of application: clinical (factors correcting the disease by inducing/inhibiting cell growth, differentiation, death, secretion), nutritional (food supplements), agricultural (factors modulating growth and development), etc.

More specifically, the present invention describes a method of a method of isolating a polynucleotide encoding a biologically active product by culturing host cells comprising an expression library, wherein said expression library comprises a pool of expression constructs, said expression constructs each comprising a nucleic acid which encodes a candidate biologically active product operably linked to an expression control sequence, to effect the expression of said biologically active product in said host cell; contacting a target cell with the expression products obtained from said host cell under conditions which permit ^• candidate biologically active expression products to exert a biological effect on said target cell, wherein said target cell is different from the host cell comprising the expression library; and identifying a subpool of expression constructs from the pool of expression constructs of step (a) wherein said subpool comprises an expression construct comprising the polynucleotide which encodes the biologically active product that exerts a detectable biological effect on said target cell in step (b).

The host cells used in the present invention may be any host cell traditionally used for the expression of products from an expression library. In preferred embodiments, the host cell is Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Pseudomonas aeruginosa, Myxococcus xanthus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, Nicotiana tabacum, COS cells, 293 cells, VERO cells, NIH/3T3 cells, or CHO cells. The host cells are transformed with an expression vector comprising the expression constructs. Any expression vector capable of directing high levels of expression of the library in the host cells can be used for library preparation, including, but not limited to: plasmids for prokaryotic expression, bacteriophages for intracellular expression and for surface expression (phage display vectors), plasmids and viral vectors for expression in mammalian or other vertebrate cells (retroviral, adenoviral, vaccinia virus-based, etc.), vectors for expression in insect cells (Baculovirus system), yeast expression systems. In preferred embodiments, the expression vector is selected from the group consisting of a plasmid, a bacteriophage, a viral vector, a cosmid vector or an artificial chromosome.

In performing the screening assay the target cell is contacted with the expression products produced by the expression system. The contacting step may comprise contacting the target cells with supernatant from the media of said host cells. In certain embodiments, the supernatant of said host cells is collected after said host cells have been lysed. In other embodiments, the supernatant comprises candidate biologically active expression products that have been secreted from said host cells.

The target cells of the invention may be any cell that is responsive to the expression product. Such a cell may have a receptor for the expression product, an enzyme for which the expression product acts as a substrate, or may be otherwise responsive to the expression products. In preferred aspects the target cells are selected from the group consisting of bacterial, insect, plant or vertebrate origin. In preferred embodiments, the expression product is an agent that has an extracellular effect on the target cell. Preferably, the target cell comprises receptor for the candidate biologically active expression product, wherein the receptor is exposed to the extracellular environment of the cell. More particularly, the extracelluar effect is a receptor mediated effect and the target cell comprises a receptor for the expression product. In specific embodiments, the expression product is a cytokine, a, chemokine, a tumor suppressor, a growth factor, a hormone, a toxin, a survival factor and the like. In other particular embodiments, the biological effect is selected from the group consisting of cell death, cell survival, phosphorylation, cell proliferation, activation/suppression of a selectable marker or reporter.

In preferred aspects, the libraries are enriched for expression products of the invention. In preferred aspects, the invention further comprises isolating an expression construct that encodes an expression product that exerts a desired biological effect. Additional aspects of the invention involve identifying the expression product for example, by sequencing the expression product or sequencing the expression construct. Protein and nucleic acids sequencing techniques are well known to those of skill in the art and are contemplated for use in the present invention.

A further aspect of the invention describes a method of screening for a biologically active product comprising culturing an expression library in a host cell, wherein the expression library comprises a pool of expression constructs, each expression construct comprising a nucleic acid which encodes a candidate biologically active product operably linked to an expression control sequence, to effect the expression of the biologically active product in the host cell; contacting a target cell with the expression products obtained from the host cell under conditions which permit candidate biologically active expression products to exert a biological effect on the target cell, wherein the target cell is different from the host cell comprising the expression library; monitoring the biological effect of the expression products on the target cell to identify an expression product that exerts biological effect; and identifying a subpool of the pool of expression constructs which subpool comprises an expression construct which encodes the biologically active product that exerts a biological effect on the target cell. Also contemplated by the present invention is a method of producing a biologically active compound comprising isolating a polynucleotide encoding a biologically active product according to the methods described herein, preparing an expression construct comprising the polynucleotide operably linked to an expression control sequence; expressing the expression construct in an appropriate host cell to produce the biologically active compound. The host cell for the production of the biologically active compound may be the same host cell as used in the library screening. Such host cells may be cultured for bulk production of the biologically active compound. Once produced, the biologically active compound may be recovered from the host cell using techniques well known to those of skill in the art. Techniques for recombinant protein production are well known to those of skill in the art and are contemplated to be particularly useful in the present invention.

Brief Description of the Drawing

Figure 1. General scheme and principles of the methods of the present invention. Figure 2. Optimization of conditions of bacterial induction and lysis to generate cell-free inducible gene product (on the model of lacZ-encoded β- galactosidase). FIG. 2A -dynamics of distribution of β-gal activity between cells and medium in growing bacterial culture. FIG. 2B-the same after simultaneous addition of IPTG and ampicillin; c-the same as "b" ampicillin is added 1 hour after IPTG. Figure 3. Biological effect of the indicated bacterial lysates on

NIH3T3 cells, u/t-untreated cells (all cells were incubated in the media containing CHI and IPTG; TNF(l) and TNF(2) - treatment with mouse TNF protein, 2 and 20 ng/ml, respectively; bac.;ys. -bacterial lysate from non-transformed cells.

Figure 4. Biological effect of the indicated bacterial lysates on TF-1 cells. FIG. 4A- induction of recombinant IL-3 production by IPTG treatment. FIG.

4B-relative cell death after the indicated treatment.

Detailed Description of the Preferred Embodiments

While there are presently many expression library systems commercially available at present, these cDNA libraries are being under-utilized because of the difficulties of delivering cDNA into the target cells and ensuring that the DNA is expressed in such cells in such a way as to present a phenotype which will allow recover of useful expression products. The present invention addresses circumvents these problems by providing an indirect fluctuation screening method which avoids the necessity of expressing the genetic elements of a cDNA library within the target cell.

Currently available methodologies allow screening of expression libraries for those gene products that are synthesized and act inside target cells. Therefore, practical applications of such biologically active clones, even if they have the desired properties, are often jeopardized by the difficulties of specific and effective delivery of the identified gene products. Our method is specifically focused on selection of such factors that are active if externally added to the cell and, therefore, generates bioactive factors that are much more suitable for conversion into in vivo active products. This method is applicable to a broad variety of libraries and can be used for isolation of both natural and artificial bioactive molecules with extracellular activity. The screening method of the present invention can be easily converted into a high throughput screening system using conventional equipment that is broadly used for screening of small molecules. It does not necessarily require creation of new libraries and can be applied for many existing expression libraries of different nature. The prospective products that could be generated by this approach are: proteins, peptides or, less likely, other factors that are generated by host cell after exposure to certain gene product. Their practical use involved the whole spectrum of application: clinical (factors correcting the disease by inducing/inhibiting cell growth, differentiation, death, secretion), nutritional (food supplements), agricultural (factors modulating growth and development), etc. The present invention provides a method of isolating genetic elements, encoding bioproducts that exert their biological activity from the outside the target cell. This method will allow one of skill in the art to effectively identify desired gene products (and the factors induced by the activity of such products in target cells) that can act from the outside of the cells and, therefore, do not require additional delivery systems for their in vivo applications.

More particularly, the present invention provides a method in which clones from an expression library are expressed in host cells that are used exclusively for the generation of gene products but not for the screening of such products. The screening step is performed on separate target cells that do not express the library by themselves. Using this method, one of skill in the art will be able to select those expression products generated from the library that exert their biological effect by acting from the outside of the target cell. Given that many therapeutically useful agents act by exerting an extracellular effect, it is envisioned that the methods of the present invention will greatly increase the efficiency of identifying new agents that are useful in therapeutic and/or diagnostic applications. The methods of the invention are limited only by the availability of appropriate target cells. Such target cells may naturally be responsive to the external application of the candidate expression product being screened or alternatively have been engineered to be responsive to such agents. For example, the methods of the present invention may employ target cells that express native receptors for the type of molecule being identified from the expression library. For example, in the event that the methods are being employed to identify a novel chemokine, target cells expressing receptors for chemokines may be particularly useful. Target cells either naturally expressing such receptors, or engineered to express such receptors are well known to those of skill in the art. The methods of the present invention were used for the isolation of bioactive cytokines (FIG. 1). A mammalian cDNA library was constructed in prokaryotic expression vector suitable for high-level inducible expression in E. coli that is used as host cell system. Plasmid library growing as multiple bacterial clones on agar is split into batches containing approximately 100 clones each (step 1 in FIG. 1), the mixture of plasmid is isolated from each batch and is stored in the solution in

96-well plates. Plasmid mixtures from every batch are transformed into competent E. coli cells (step 2, FIG. 1) that are induced to express library-encoded products. Cells are then lysed by ampicillin treatment, the intracellular products, including the library- encoded products, are released into the media and are administered to target cells to monitor the biological effect (step 3). Batches that demonstrate the desired effect (cell death or rescue from death) are identified (step 6) and subjected to individual clonal analysis (steps 5-7) to isolate individual genetic elements responsible for the biological activity of the batch. As a readout for screening one can use target cells that are sensitive for biological activity of the cytokine of interest (i.e., die from cytokine-induced apoptosis or can be rescued from apoptosis by the cytokine in the media). Steps 2, 3, 6 and 7 are most critical for the success of the methodology. They have been tested in two screening systems: isolation of TNF and IL-3. All other steps are routinely used in a variety of protocols and do not require additional verification.

A more detailed description of the methods and compositions involved in the present invention is presented below.

A. Expression Libraries

Expression libraries are widely available for use in expression cloning. Such libraries may be prepared from human and mammalian tissues using techniques well known to those of skill in the art ( Simonsen et al. 1994, Trends Pharmacol Sci 15:437-441; Nakayama et al. 1992, Curr Opin Biotechnol 3:497-505; Aruffo, 1991,

Curr Opin Biotechnol, 2:735-741; Margolis et al, U.S. Pat. No. 5,434,064; Seed et al., 1987, Proc Natl Acad Sci 84:3365-3369; Yamasaki et al., 1988, Science 241:825-828; Lin et al., 1992, Cell 68:775-785; Huse et al. 1991, Ciba Foundation Symp 159:91-102). Additionally, many such libraries are commercially available. The expression libraries may be prepared from any organism that is likely to yield a candidate biologically active expression product. It is contemplated that the source of genetic material for the preparation of expression libraries may be any polynucleotide sequences capable of encoding polypeptides. Such a source may include mixtures of fragmented genomic DNA of viruses, procaryotic and eukaryotic organisms, mixtures of full length or fragmented cDNAs cloned in sense or anti-sense orientation, and mixtures of artificial sequences. Non-protein- encoding sequences expressed as RNA transcripts also may be used as sources of genetic material.

Cells from such organisms may be obtained from private or public laboratory cultures, culture depositories such as the American Type Culture

Collection, or from environmental samples either cultivable or uncultivable. In certain circumstances, it may be desirable to use human tissues as the source of genetic material for the preparation of the expression libraries. In certain circumstances, donor organisms or tissues chosen for the preparation of expression libraries may have been a traditional source of drug leads, such as terrestrial bacteria, fungi and plants. The donor organisms may be transgenic, genetically manipulated or genetically selected strains that have been useful in generating and/or producing drugs.

The cDNA or genomic DNA is isolated from the donor tissue and ligated to an appropriate vector for delivery and expression in expression host cells. Preferably, the vectors have the capacity to shuttle between two or more expression hosts.

The expression libraries may be prepared from any organism that is likely to yield a candidate biologically active expression product. Cells from such organisms may be obtained from private or public laboratory cultures, culture depositories such as the American Type Culture Collection, or from environmental samples either cultivable or uncultivable. In certain circumstances, it may be desirable to use human tissues as the source of genetic material for the preparation of the expression libraries. In certain circumstances, donor organisms or tissues chosen for the preparation of expression libraries may have been a traditional source of drug leads, such as terrestrial bacteria, fungi and plants. The donor organisms may be transgenic, genetically manipulated or genetically selected strains that have been useful in generating and/or producing drugs.

For preparing cDNAs from the donor organisms, specific growth conditions or the presence of certain chemicals in the culture may be required to induce or enhance the transcription of gene products encoding the activities of interest in the donor organisms. Standard growth conditions may be used to culture the organisms if only genomic DNA is required.

Donor organisms contemplated by the invention may include, but are not limited to viruses; bacteria; unicellular eukaryotes, such as yeasts and protozoans; algae; fungi; plants; tunicates; bryozoans; worms; echinoderms; insects; mollusks; fishes; amphibians; reptiles; birds; and mammals. The source of genetic material may involve any polynucleotide sequences capable of encoding polypeptides: mixtures of fragmented genomic DNA of viruses, procaryotic and eukaryotic organisms, mixtures of full length or fragmented cDNAs cloned in sense or anti-sense orientation, and mixtures of artificial sequences may be used. Non-protein-encoding sequences expressed as RNA transcripts can be also considered.

Nucleic acids may be isolated from donor organisms by a variety of methods depending on the type of organisms and the source of the sample. It is important to obtain high quality nucleic acids that are free of nicks, single stranded gaps, and partial denaturation, and are of high molecular weight (especially for genomic DNA cloning), in order to construct gene expression libraries that are fully representative of the genetic information of donor organisms. To prepare high quality nucleic acid, the methods of the invention provide gentle, rapid and complete lysis of donor organisms in the sample, and rapid and complete inactivation of nucleases and other degradative proteins from the organisms. Any nucleic acid isolation procedure requires efficient disruption of the donor organism to release the cellular milieu. A number of standard techniques may be used, including freezing in liquid nitrogen, grinding in the presence of glass or other disruptive agents, as well as simple mechanical shearing or enzymatic digestion. RNA isolated from donor organisms can be converted into complementary DNA (cDNA) using reverse transcriptase.

The term "host organism" as used herein broadly encompasses unicellular organisms, such as bacteria, and multicellular organisms, such as plants and animals. Any cell type may be used, including those that have been cultured in vitro or genetically engineered. Any host-vector systems known in the art may be used in the present invention. The use of shuttle vectors that can be replicated and maintained in more than one host organism is advantageous.

Host organisms from which host cells are generated or host cells may be obtained from private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers. Such host organisms or cells may be further modified by techniques known in the art for specific uses. According to the invention, it is preferable that the host organism or host cell has been used for expression of heterologous genes, and are reasonably well characterized biochemically, physiologically, and/or genetically. Such host organisms may have been used with traditional genetic strain improvement methods, breeding methods, fermentation processes, and/or recombinant DNA techniques. It is desirable to use host organisms which have been developed for large-scale production processes, and that conditions for growth and for production of secondary metabolites are known.

The host organisms may be cultured under standard conditions of temperature, incubation time, optical density, and media composition corresponding to the nutritional and physiological requirements of the expression host. However, conditions for maintenance and production of a library may be different from those for expression and screening of the library. Modified culture conditions and media may also be used to emulate some nutritional and physiological features of the donor organisms, and to facilitate production of interesting metabolites. For example, chemical precursors of interesting compounds may be provided in the nutritional media to facilitate modifications of those precursors. Any techniques known in the art may be applied to establish the optimal conditions.

Preferred prokaryotic host organisms may include but are not limited to Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor

Pseudomonas aeruginosa, Myxococcus xanthus. Yeast species such as Saccharomyces cerevisiae (baker's yeast), Schi∑osaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts) may also be used. Filamentous ascomycetes, such as Neurospora crassa and Aspergillus nidulans may also be used. Plant cells such as those derived from Nicotiana and Arabidopsis are preferred. Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, such as Chinese hamster ovary (CHO) cells, NIH/3T3, COS, 293, VERO, etc (see Kriegler M. in "Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990). Avian e.g., chicken cells also may prove useful. Insect cell systems infected with virus expression vectors

(e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMN) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid) also are contemplated. Ultimately, these cells also may be used as target cells.

An effector may be chosen which modifies and processes the expressed gene products in a specific fashion as desirable. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein in a biochemical pathway. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper and accurate processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be preferred if the donor organism(s) are eukaryotic.

A preferred example of a eukaryotic host organism is the fission yeast, Schizosaccharomyces pombe. First, the molecular biology of S. pombe is highly developed and many major culture and purification processes and manipulations are routinely performed. Second, it is unicellular, and thus can easily be cultured, stored, and manipulated in a laboratory setting. Third, and of particular importance for use in expressing mixed eukaryotic DΝAs, it is capable of properly splicing and expressing genes of other species of fungi, plants, and mammals. Studies of the splicing and processing of heteronuclear RΝA (RΝA which contains introns) have indicated that S. pombe shares with other fungi and higher metazoans a remarkable similarity of pattern and structure of small nuclear RΝA (snRΝA) components needed for splicing. Finally, many non-S. pombe promoters, some of which derive from mammalian and plant viruses, are capable of driving moderate to high levels of gene expression

(Forsburg, 1993, Νuc Acids Res, 21:2955) This feature can allow the shuttling of a fungal DΝA/cDΝA library to mammalian cell expression hosts such as ΝIH3T3 (fibroblasts), GT1-7 (neuronal), or other cell types.

A cloning vector or expression vector may be used to introduce donor DNA into an host cell for expression. An expression construct is an expression vector containing nucleic acid sequences that encode the appropriate expression product operably associated with one or more regulatory regions. The regulatory regions may be supplied by the donor DNA or the vector. A variety of vectors may be used which include, but are not limited to, plasmids; cosmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs, Shizuya et al. 1992, Pro Natl Acad Sci 89: 8794-8797) or modified viruses.

However, it should be noted that the vector must be compatible with the host organism. Non-limiting examples of useful vectors are λgtl 1, pWE15, SuperCosl (Stratagene), pDblet (Brun et al. 1995, Gene, 164:173-177), pBluescript (Stratagene), CDM8, pJB8, pYAC3, pYAC4 (see Appendix 5 of Current Protocols in Molecular Biology, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference).

When the regulatory regions and transcription factors of the host and donor organisms are compatible, donor transcriptional regions will be able to bind host factors, such as RNA polymerase, to effect transcription in the host organism. If the donor and host organisms are not compatible, regulatory regions compatible to the host organism may be attached to the donor DNA fragment in order to ensure expression of the cloned genes.

In cases where the entire operon, including its own translation initiation codon, ribosome binding regions, and adjacent sequences, is inserted into the appropriate cloning or expression vector, no additional control signals may be needed. However, in cases where only a portion of the coding sequence of a gene is inserted, exogenous control signals, including the translation initiation codon (frequently ATG) and adjacent sequences, must be provided. These exogenous regulatory regions and initiation codons can be of a variety of origins, both natural and synthetic. Both constitutive and inducible regulatory regions may be used for expression of the donor DNA. It is desirable to use inducible promoters when the products of the expression library may be toxic. The efficiency of the expression may be enhanced by the inclusion of appropriate transcription enhancer elements, (see Bittner et al. 1987, Methods in Enzymol. 153:516-544). "Operably-associated" refers to an association in which the regulatory regions and the DNA sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence. Such regulatory regions may include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. The non-coding region 3' to the coding sequence may also be retained or replicated for its transcriptional termination regulatory sequences, such as terminators and polyadenylation sites. Two sequences of a nucleic acid molecule are said to be "operably-associated" when they are associated with each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence. A polycistronic transcript may thus be produced. Two or more sequences, such as a promoter and any other nucleic acid sequences are operably-associated if transcription commencing in the promoter will produce an RNA transcript of the operably-associated sequences. In order to be "operably-associated" it is not necessary that two sequences be immediately adjacent to one another.

In addition, the expression vector may contain selectable or screenable marker genes for initially isolating, identifying or tracking host organisms that contain donor DNA. Any antibiotic resistance genes, such as but not limited to ampicillin, kanamycin, chloramphenicol, apramycin or gentamycin (Brau et al., 1984, Mol Gen Genet 193:179-187) and hygromycin (Hopwood et al., 1985, Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, UK) can be used. Universal forward selection based on plasmid stability in a bacterial host, such as the parD/E system (Johnson et al., 1996, J Bacteriol, 178:1420-1429), can also be used, in the absence of antibiotic selection.

The expression vector may also provide unique or conveniently located restriction sites to allow severing and/or rearranging portions of the DNA inserts in an expression construct. The expression vector may contain sequences that permit maintenance and/or replication of the vector in one or more host organism, or integration of the vector into the host chromosome. Such sequences may include but are not limited to replication origins, autonomously replicating sequences (ARS), centromere DNA, and telomere DNA. It may also be advantageous to include in the expression vector, host organism sequences or homologous sequences, especially those that are actively transcribed in the host. Such sequences may facilitate integration of the expression construct into the host chromosome, especially when they are found in positions flanking the cloning site in the cloning vector. The expression construct may be integrated in the host genome or remain episomal in the host organism. As a result, one or more copies of an expression construct may be generated and maintained in a host organism.

It may be advantageous to use shuttle vectors which can be replicated and maintained in at least two host organisms, such as, for example, bacteria and mammalian cells, bacteria and yeasts, bacteria and plant cells, or gram positive and gram negative bacteria. A shuttle vector of the invention is capable of replicating in different species or strains of host organisms, and may contain one or more origins of replication that determine the range of host organism in which the vector can stably maintain itself, and undergo replication in concert with cell growth. In prokaryotes, for example, if a broad host range plasmid replication origin is present, the shuttle vector will be capable of stable inheritance in a very wide range of bacteria, e.g. the origins of replication of RK2 (Pansegrau et al., 1994, J Mol Biol 239:623-663) or

PBBR (Kovach et al., 1994, BioTechniques 16:800-801) are functional in many gram-negative bacteria, such as Pseudomonas, Agrobacterium, Escherichia, and Rhizobium. Many of the bacteria that harbor DNA comprising a broad host range origin of replication are known to produce metabolites of interest. Origin of replication that is functional in a relatively limited range of related hosts can also be used, e.g., the replication origin of pAkijl which functions in four actinomycete genera (J Gen Microbiol 131:2431-2441). Alternatively, a shuttle vector of the invention can comprise two or more replication origins each having a narrowly defined range that permits the vector to be replicated and maintained in the respective hosts, e.g. E. coli and Bacillus. Any origin of replication derived from IncP, IncQ or

IncW plasmids can be used in a vector of the invention. A bacteriophage origin of replication, e.g., fl origin of M13 phage, can also be present in the vector. The coliphage origin of replication can facilitate production of single stranded form of the expression constructs useful for various purposes, such as but not limited to transformation, hybridization. A shuttle vector of the invention may also comprise cis-acting sequences derived from naturally-occurring self-transmissible plasmid, which enable the plasmid to transfer themselves from one species or strain of bacteria to another by means of an interspecies conjugative process (Hayman et al. 1993, Plasmid 30: 251-257). Such sequences, known as origins of transfer, are relatively small (e.g., 200-800 bp) and can be inserted into a shuttle vector of the invention to facilitate the transfer of the shuttle vector between different species or strains of host organisms. Conjugation is a natural process whereby large plasmids are transferred between different species or strains of organism via a conjugation tube at fairly high frequency. The mobilization of transfer origin-containing shuttle vector is mediated by a specific set of transfer proteins which can be provided by expression of function integrated in the host chromosome itself or in trans by a Tra helper plasmid (Ditta et al., 1980, Proc. Natl. Acad. Sci. 77:7347-7351; Knauf et al., 1982, Plasmid 8:45-54). Strains of E. coli harboring integrated Tra functions, e.g., S17-1, are available from the American Type Culture Collection. By using a shuttle vector with the appropriate replication origins, transfer origin(s) and selection mechanisms to construct a library, the DNA sequences of the starting organisms in a library may readily be mobilized from one initial species of host organism to a variety of alternative species of host organisms where the donor DNA sequences can be stably maintained, replicated and expressed. Thus, mobilizable gene expression libraries that are constructed with a shuttle vector, and that can be mobilized into multiple host organisms by conjugation are within the scope of the invention.

For combinatorial gene expression libraries using plant cells as hosts, the expression of the donor coding sequence may be driven by any of a number of promoters. For example, preferred strains are described in Principles of Gene

Manipulation 1985, R. W. OLD and S. B. Primrose 3rd ed. Blackwell Scientific Pub.; Nectors: A survey of molecular cloning vectors and their uses 1988, R. L. Rodriquez, D. T. Denhardt, Butterworths Pub.; A Practical guide to molecular Cloning 1988, B. Perbal, John Wiley and Sons, viral promoters such as the 35S RΝA and 19S RΝA promoters of CaMN (Brisson et al. 1984, Nature 310:511-514), or the coat protein promoter of TMN (Takamatsu et al. 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RuBISCo (Coruzzi et al. 1984, EMBO J. 3:1671-1680; Broglie et al. 1984, Science 224:838-843); or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al. 1986, Mol. Cell. Biol. 6:559-565) may be used. Both plant cells and protoplasts may be used as host cells. Plant hosts may include, but are not limited to, those of maize, wheat, rice, soybean, tomato, tobacco, carrots, peanut, potato, sugar beets, sunflower, yam, Arabidopsis, rape seed, and petunia. Plant protoplasts are preferred because of the absence of a cell wall, and their potential to proliferate as cell cultures, and to regenerate into a plant. In addition, the recombinant constructs may comprise plant-expressible selectable or screenable marker genes which include, but are not limited to, genes that confer antibiotic resistances, (e.g., resistance to kanamycin or hygromycin) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate). Screenable markers include, but are not be limited to, genes encoding β-glucuronidase (Jefferson, 1987, Plant Molec Biol. Rep 5:387-405), luciferase (Ow et al. 1986,

Science 234:856-859), and B protein that regulates anthocyanin pigment production (Goff et al. 1990, EMBO J 9:2517-2522).

To introduce donor organism DΝA into plant host cells, the Agrobacterium tumefaciens system for transforming plants may be used. The proper design and construction of such T-DΝA based transformation vectors are well known to those skilled in the art. Such transformations preferably use binary Agrobacterium T-DΝA vectors (Bevan, 1984, Νuc. Acid Res. 12:8711-8721), and the co-cultivation procedure (Horsch et al. 1985, Science 227:1229-1231). Generally, the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al. 1982, Ann. Rev. Genet 16:357-384; Rogers et al. 1986, Methods Enzymol. 118:627-641), but it may also be used to transform as well as transfer DΝA to monocotyledonous plants and plant cells, (see Hernalsteen et al. 1984, EMBO J 3:3039-3041; Hooykass-Nan Slogteren et al. 1984, Nature 311 :763-764; Grimsley et al. 1987, Nature 325:1677-1679; Boulton et al. 1989, Plant Mol. Biol. 12:31-40.; Gould et al. 1991, Plant Physiol. 95:426-434). Alternative methods for introducing recombinant nucleic acid constructs into plant cells may also be utilized, e.g., protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al, 1984, EMBO J 3:2717-2722, Potrykus et al. 1985, Molec. Gen. Genet. 199:169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828; Shimamoto, 1989, Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al., 1992, Plant Cell

4:1495-1505). Additional methods for plant cell transformation include microiηjection, silicon carbide mediated DNA uptake (Kaeppler et al., 1990, Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al., 1988, Proc. Nat. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al., 1990, Plant Cell 2:603-618).

For general reviews of plant molecular biology techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section Vm, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. In an insect system, Autographa californica nuclear polyhydrosis virus

(AcNPV) a baculovirus, is used as a vector to express donor genes in Spodoptera frugiperda cells. The donor DΝA sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcΝPV promoter (for example the polyhedrin promoter). These recombinant viruses are then used to infect host cells in which the inserted gene is expressed, (e.g., see Smith et al.

1983, J Nirol 46:584; Smith, U.S. Pat. No. 4,215,051).

In yeast, a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast), Pichiapastoris, and Hansenula polymorpha (methylotropic yeasts). For a review see, Current Protocols in Molecular

Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al. 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. π, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular

Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and H

In mammalian host cells, a variety of mammalian expression vectors are commercially available. In addition, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the donor DNA sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659). The Epstein-Barr virus (EBV) origin (OriP) and EBNA-1 as a transacting replication factor has been used to create shuttle episomal cloning vectors, e.g., EBO-pCD (Spickofsky et al. 1990, DNA Prot Eng Tech 2:14-18). Viral vectors based on retroviruses may also be used (Morgenstern et al. 1989, Ann Rev Neurosci,

12:47-65). Alternatively, the vaccinia 7.5 K promoter may be used. (See, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al. 1984, J. Virol. 49:857-864; Panicali et al. 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

A number of selection systems may be used for mammalian cells, including but not limited to the Herpes simplex virus thymidine kinase (Wigler, et al.

1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al. 1980, Cell 22:817) genes can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler, et al. 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al. 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al. 1981, J. Mol. Biol. 150: 1); and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin

(Santerre, et al. 1984, Gene 30:147).

Either DNA or RNA may be used as starting genetic material for preparing expression libraries which may include cDNA libraries, genomic DNA libraries, as well as mixed cDNA/genomic DNA libraries. DNA fragments derived from a plurality of donor organisms, are introduced into a pool of host organisms, such that each host organism in the pool contains a DNA fragment derived from one of the donor organisms.

For example, E. coli has approximately 4400 kbp of DNA; a cosmid vector can package approximately 40 kbp of DNA. Following these calculations, the entire genome of E. coli can be expected to be thoroughly represented in as few as 504 clones in a cosmid library. Since a typical DNA library can contain 500,000 independent recombinant clones, one such library can effectively represent the genomes of up to 1,000 different bacterial species having a genome size similar to E. coli. Thus, considerable chemical diversity can be generated and assessed efficiently by screening a gene expression library comprising the diverse genetic material of

1,000 or more species of bacteria.

The procedures described in standard treatises, e.g., Maniatis et al. 1989, Molecular Cloning, 2nd Edition, Cold Spring Harbor Press, New York; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York, may be followed to carry out routine molecular biology reactions used in constructing the combinatorial gene expression libraries.

Any cell from a donor organism can potentially serve as the source of nucleic acid for construction of a gene expression library. Genomic DNA, which includes chromosomal DNA as well as DNA of extrachromosomal genetic elements, such as naturally occurring plasmids, may be used. Alternatively, RNA of a donor organism may be used. RNA, preferably messenger RNA (mRNA), may be extracted, purified and converted to complementary DNA (cDNA) by any technique known in the art. An oligo-(dT) primer or random sequence primers may be used for priming first strand synthesis of cDNA. DNA inserts may optionally be amplified by polymerase chain reaction (PCR). Genomic DNA and RNA may be extracted and purified by the procedures that are known in the art. For filamentous fungi and bacteria, such procedures may comprise any of several techniques including a) rapid SDS/high salt lysis of protoplasts prepared from young mycelia grown in liquid culture and immediate extraction with equilibrated phenol; b) rapid lysis of protoplasts in guanidinium isothiocyanate followed by ultracentrifugation in a CsCl gradient; or c) isolation of high molecular weight DNA from protoplasts prepared in agarose plugs and pulsed field gel electrophoresis. For bacteria, an alternative procedure of lysis by lysozyme/detergent, incubation with a non-specific protease, followed by a series of phenol/chloroform/isoamyl alcohol extractions may be useful. For optimal results, large random prokaryotic genomic DNA fragments are preferred for the higher probability of containing a complete operon or substantial portions thereof. The genomic DNA may be cleaved at specific sites using various restriction enzymes. Random large DNA fragments (greater than 20 kbp) may be generated by subjecting genomic DNA to partial digestion with a frequent-cutting restriction enzyme. The amount of genomic DNA required varies depending on the complexity of the genome being used. Alternatively, the DNA may be physically sheared, as for example, by passage through a fine-bore needle, or sonication.

Prior to insertion into an expression vector, such DNA inserts may be separated according to size by standard techniques, including but not limited to, agarose gel electrophoresis, dynamic density gradient centrifugation, and column chromatography. A linear 10-40% sucrose gradient is preferred. The insertion can be accomplished by ligating the DNA fragment into an expression vector which has complementary cohesive termini. The amounts of vector DNA and DNA inserts used in a ligation reaction is dependent on their relative sizes, and may be determined empirically by techniques known in the art. However, if the complementary restriction sites used to fragment the DNA are not present in the expression vector, the ends of the DNA molecules may be enzymatically modified, as for example, to create blunt ends. Alternatively, any site desired may be produced by ligating nucleotide sequences i.e., linkers or adaptors, onto the DNA termini; these ligated linkers or adaptors may comprise specific chemically-synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved expression vector and DNA inserts may be modified by homopolymeric tailing.

After ligation of vector DNA to DNA inserts, the expression constructs are introduced into the host organisms. A variety of methods may be used, which include but are not limited to, transformation, transfection, infection, conjugation, protoplast fusion, liposome-mediated transfer, electroporation, microiηjection and microprojectile bombardment. In specific embodiments, the introduction of bacteriophage or cosmid DNA into an E. coli host is carried out by in vitro packaging the DNA into bacteriophage particles then allowing these particles to infect E. coli cells. Other naturally-occurring mechanisms of DNA transfer between microorganisms may also be used, e.g., bacterial conjugation.

After the host cells containing expression constructs are pooled to form a library, they can be amplified and/or replicated by techniques known in the art. The purpose of amplification is to provide a library that can be used many times. Amplification may be achieved by plating out the library, allowing the bacteria to grow, and harvesting the phage or bacteria for storage.

Alternatively, the library may be stored in an ordered array. The bulk of the library can be plated out at low density to allow formation of single, discrete plaques or colonies, followed by transfer of individual plaques or colonies into the wells of coded multi-well master plates, e.g., 96-well plates or 384-well plates. The individual clones are allowed to grow in the wells under the appropriate conditions.

The coded master plates can be used as an archival source to replicate each clone separately into one or more working plates. Thus, each clone in the library may be handled and assayed individually. The coded archival plates may be sealed and stored for future use. Replication and transfer of the clones may be done with a multi-pin replicator, or multi-channel devices for fluid handling. Preferably, all or most of the transfers and manipulations are performed by laboratory robots (Bentley et al. 1992, Genomics 12:534-541).

The libraries of the invention may be preserved by lyophilization, or cryopreservation in a freezer (at -20° C. to -100^° C.) or under liquid nitrogen (-176° C. to -196° C). Host organisms containing donor DNA in a library may be identified and selected by a variety of methods depending on the host-vector system used. In one approach, such host organisms are identified and selected upon the presence or absence of marker gene functions, e.g., thymidine kinase activity, resistance to antibiotics, such as kanamycin, ampicillin, bleomycin, or thiostrepton, production of pigment, such as melanin, and resistance to methotrexate. Alternatively, a change in phenotype or metabolism of the host organism, indicated by metabolic testing, foci formation in tissue culture, or occlusion body formation in baculovirus may be used. Once selected for the presence of donor DNA, a series of enzymatic assays or metabolic tests may be carried out on the clones for further characterization. To characterize the donor DNA inserts in a library of clones containing donor DNA or a portion thereof, mini preparations of DNA and restriction analysis may be performed with a representative set of clones. The results will provide a fingerprint of donor DNA size and restriction patterns that can be compared to the range and extent of insert DNA which is expected of the library. The present invention also may employ combinatorial expression libraries, wherein the host organisms contain randomly concatenated genetic materials that are derived from one or more species of donor organisms, and are capable of producing functional gene products of the donor organisms. A substantial number of host organisms in the library may contain a random and unique combination of genes derived from one or more species of donor organism(s). Coexpression of the cloned genes may be effected by their respective native regulatory regions or by exogenously supplied regulatory regions. The plurality of gene products derived from the different donor organisms interact in the host organism to generate novel chimeric metabolic pathways and novel compounds. Novel activities and compounds of such chimeric pathways may become more accessible to screening by traditional drug discovery techniques or by methods provided herein. Moreover, some of the heterologous gene products may be modified structurally, and compartmentalized or localized differently during biosynthesis in the host organism. Some of the heterologous gene products may be exposed to a host cellular environment that is different from that of their respective donors. It is envisioned that some heterologous gene products may also act on the host organism and modify the host cellular environment. Elements of the host cellular environment that may affect, or be affected by, the function of heterologous gene products may include but are not limited to concentrations of salts, trace elements, nutrients, oxygen, metabolites, energy sources, redox states, and pH. Some heterologous gene products may also interact with host gene products which can result in the modification of the host's metabolic pathways.

Depending on the combination of heterologous genes, novel classes of compounds that do not exist in nature may be formed in the host organisms of the library. In combinatorial expression libraries, the genetic resources of the donor organisms are multiplied and expanded to provide a diversity of chemical structure that may not be found in individual organisms. The libraries prepared by this method may be screened using traditional methods or methods provided by the present invention. Thus, the novel pathways and compounds are made more accessible to drug screening. Insert DNAs may be complementary DNA (cDNA) derived from mRNA, and/or fragments of genomic DNA, or DNA from an archival or mobilizable combinatorial expression library. The DNA or RNA of different species of donor organisms may be copurified, or they may be isolated separately and then combined in specific proportions. The random mixing of insert DNAs can be done at any stage prior to insertion into the cloning or expression vector. For example, large pieces of

DNA from an archival library can be isolated and digested to give smaller fragments, which are then randomly religated to form insert DNAs for a second combinatorial expression library. Other methods for generating and mixing of random fragments of DNA can also be used, for example, in vitro recombination can be used when the DNA fragments share some sequence homologies.

Methylated nucleotides, e.g., 5-methyl-dCTP, maybe used in cDNA synthesis to provide protection against enzymatic cleavage, and allow directional cloning of the cDN A inserts in the sense orientation relative to the promoter and terminator fragments.

Random fragments of genomic DNA in the range of 2-7 kbp may be generated by partial digestion with a restriction enzyme having a relatively high frequency of cutting sites, e.g., Sau3AI. Partial digestion is monitored and confiπned by subjecting aliquots of the samples to agarose gel electrophoresis.

Exogenous regulatory regions, such as constitutive or inducible promoters and terminators may be provided to drive expression of the cloned genes. When the host and donor expression systems are not compatible, it is essential to provide such regulatory sequences. PCR may be used to generate various promoter and terminator fragments that are specific to a particular expression host, and have defined restriction sites on their termini. Any method for attachment of a regulatory region to the DNA inserts may be used. Treatment with the Klenow fragment and a partial set of nucleo tides, i.e., a partial fill-in reaction, may be used to create insert

DNA fragments which will only ligate specifically to promoter and terminator fragments with compatible ends.

In another embodiment of the invention, a biased combinatorial natural or chimeric expression library may be prepared from preselected fragments of DNA that are pooled together from one or more species of donor organisms. Instead of using only the total pooled genomic DNA or cDNA of the donor organism(s), this approach will reduce the number of clones that need to be screened and increase the percentage of clones that will produce compounds of interest. The preselected fragments of DNA contain genes encoding partial or complete biosynthetic pathways, and may be preselected by hybridizing to an initial or archival DNA library a plurality of probes prepared from known genes that may be related to or are involved in producing the expression products of interest.

The initial DNA library, preferably a cosmid or bacterial artificial chromosome (BAC) library, and not necessarily an expression library, may contain DNA from one or more species of donor organisms. For further pre-screening, if the initial library is an expression library, DNA in the positive clones may be transferred into and expressed in a host for production, such as E. coli or Streptomyces lividans. More than one initial library may be pre-screened, and DNA from all the positive clones can be pooled and used for making the biased combinatorial gene expression library. The initial or archival library may be amplified so that DNA of the donor organisms can be pre-screened in a variety of host organisms. In one aspect of the invention, the cloning vector or expression vector can contain the appropriate replication origins and/or transfer origin(s) as described in section 5.1.3, such that the entire initial or archival library can be transferred or mobilized into various compatible host organisms via conjugation. The transfer can also be effected by isolating the donor genetic materials from the archival library and introducing the genetic material into another species or strain of organism by any means, such as but not limited to transformation, transfection and electroporation. For example, once a gene expression library in Streptomyces lividans is generated, it can be introduced into specialized host organisms for expression and screening, such as S. rimosis that produces oxytetracycline, or S. parlus that produces actinomycin D.

In another aspect of the invention, combinatorial gene expression libraries can be prepared from genetic materials derived from a plurality of organisms, wherein the genetic materials have been manipulated by homologous or homeologous recombination.

B. Screening Combinatorial Expression Libraries

The present invention provides methods for genetic elements that encode biologically active products that exert their biological activity from the outside the target cell as a method for drug discovery.

The methods claimed herein enables the management of large sample numbers with minimal handling to permit efficient and high-throughput detection and isolation of productive clones in the library. The libraries may be pre-screened for a broad range of activities, for the production of a class of compounds or for the presence of relevant DNA sequences. The libraries may also be used directly with a target cell in both in vivo and in vitro assays. The identified or isolated population of host cells which contain the nucleic acid that encodes the candidate biologically active expression product may readily be cultured, expanded in numbers, and subjected to further analysis for the production of novel compounds. The genes encoding the biologically active expression product that exerts an extracellular effect on the target cell may be identified by characterizing the genetic material that was introduced into the isolated clones. Information on the genes and the pathway, and the clones, will greatly facilitate drug optimization and production.

As used herein, the terms "library clones" or "library cells" refer to host cells or organisms in a combinatorial gene expression library that contain at least one fragment of donor DNA that may encode a candidate biologically active expression product that exerts an extracellular effect on it target cells. The term "positive clones" or "positive cells" refers to library clones or cells that produce a signal that correlates with the presence or expression of the nucleic acid that encodes the candidate biologically active expression product. The term "productive cells" or "productive clones" refers to host cells or organisms in the library that produce an activity or compound of interest, in distinction from the remainder "non-productive cells" in the library.

The term "pre-screen" refers to a general biological or biochemical assay which indicates the presence of an activity, a compound or a gene of interest. The term "screen" refers to a specific biological or biochemical assay which is directed to a specific condition or phenotype that the candidate expression product induces in the target cells. The use of both pre-screens and screens generally embodies visual detection or automated image analysis of a colorigenic indicator, fluorescence detection by fluorescence-activated cells sorting (FACS) or the use of a magnetic cell sorting system (MACS) performed on a population of library cells in the presence of a reporter regimen. Alternatively, one may monitor cell death e.g., through an apoptosis assay such as a caspase activity assay, a BCL₂ assay, a BAX assay, or a DNA fragmentation assay. An assay particularly useful in monitoring cell death is the CYQUANT cell number assay. The methods of the invention provide alternative but not mutually exclusive approaches to generation of detectable signal associated with productive cells for the purpose of detecting and isolating these cells of interest. A reporter can be a molecule that enables directly or indirectly the generation of a detectable signal. For example, a reporter may be a light emitting molecule, or a cell surface molecule that may be recognized specifically by other components of the regimen. A reporter regimen comprises a reporter and compositions that enable and support signal generation by the reporter. The reporter regimen may include live indicator cells, or portions thereof. Components of a reporter regimen may be incorporated into the host organisms of the library, or they may be co-encapsulated with individual or pools of library cells in a permeable semi-solid medium to form a discrete unit for screening. To facilitate detection of compounds of interest as described in the following text, absorptive materials such as neutral resins, e.g., Diaion HP20 or Amberlite XAD-8 resin, may be added to cultures of library cells (Lam et al. 1995, J Industrial Microbiol 15:453-456). Since many secondary metabolites are hydrophobic molecules, the release or secretion of such metabolites may lead to precipitation on the cell exterior. Inclusion of such resins in the culture causes the sequestration to occur on the resin which may be removed from the culture for elution and screening.

In one embodiment, a physiological probe can be used which generates a signal in response to a physiological change in individual target cells as a result of the presence of a desirable activity or compound generated by expression of the library in the host cells. Such a probe may be a precursor of a reporter molecule that is converted directly or indirectly to the reporter molecule by an activity or candidate expression product expressed from the libraries of the present invention. Upon contact with a target cell, the physiological probe or reporter precursor generates a detectable signal which enables identification and/or isolation of the productive cell that produces the candidate expression product. Contact may be effected by direct addition of the target cells to the library cells. In such assays, the library cells are cultured in a format appropriate for screening, e.g., 96-well plates or multiples thereof, and then the expression products produced by the library cells are contacted with the target cells. This may simply involve adding the target cells to the plates containing the library cells. Alternatively, in instances where the expression product is secreted into the media of the library cells after expression, such media may be added to target cells presented in a format amenable to screening. In the event that the expression product is one that is not secreted into the media of the library cells, the library cells may be ruptured to release the intracellular contents to render the contents accessible to the target cells. Alternatively, contact may be effected by co-encapsulation of the target cells with the library cells during screening. Whole live or fixed target cells may be mixed or co-encapsulated with individual or pools of library cells. Target cells are selected for their biological properties which is responsive to the presence of the desirable activity or compound. The target cells may be the natural target cells of the desirable compound. Alternatively, target cells may be used in conjunction with a reporter to generate a detectable signal. In a further alternative, the target cells may be engineered to be responsive to the candidate expression product.

The present invention contemplates encapsulation as an efficient high-throughput method for growing cells in a confined space, replacing the classic method of growing bacteria in petri dishes. Growing cells in a plate format is both labor- and materials- intensive, while encapsulated cells can be grown easily in a liquid culture with the advantage that dividing cells are kept together, and thus ^' facilitating detection of interesting secondary metabolites. Another advantage of encapsulation is the ability to co-encapsulate components of the reporter regimen and/or other target cells with library cells so that pre-screening or screening may be performed in a discrete unit. Encapsulation of cells can be performed easily by means of thermal or ionic gelation using materials such as agarose, alginate or carrageenan. FACS is a well-known method for separating particles (1-130 μm in size) based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151 :150-165). FACS works on the basis of laser excitation of fluorescent moieties in the individual particles. Positive fluorescence results in addition of a small electrical charge to the particle. The change allows electromagnetic separation of positive and negative particles from a mixture. Separated particles may be directly , deposited into individual wells of 96-well or 384-well plates. MACS is a well-known method for separating particles based on their ability to bind magnetic microspheres (0.5-100 μm diameter) (Dynal, 1995). A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody which specifically recognizes a cell-surface antigen or hapten. Alternatively, for magnetization of encapsulated cells, a reporter regimen can be incorporated into host cells that generate magnetogenic reporter proteins, such as ferritin. In this case, encapsulated cells that generate a positive signal act as magnetic microspheres. The selected microspheres can be physically manipulated by exposure to a magnetic field. For example, the selected microspheres may be sequestered by application of a magnet to the outside of the reaction vessel.

A physiological probe as used herein is a fluorescent or colorigenic agent which upon contact or entry, generates a signal in response to changes in physiological and/or metabolic parameters of a library cell or target cell. The probe can be an enzyme substrate linked to a fluorogenic agent.

Fluorescent probes may be selected for detection of changes in the following physiological and metabolic parameters such as, but not limited to, those described in Shechter, et al. (1982, FEBS Letters 139:121-124), and Bronstein et al.

(Anal Biochem 219:169-81).

The screening assays of the present invention assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted. Contemplated functional "read-outs" include the binding of a substrate to a component of a target cell, in which the substrate is the entity generated from the expression library; candidate expression product binding to a receptor located on the target cell, or any functional assay normally employed to monitor the activity of the class of compound that the candidate expression product. Screening protocols depend on specifics of each individual selection. For example, host cells may be lysed either separately or in the presence of target cells. Lysis may be done by different approaches such as antibiotic treatment (for prokaryotic host cells), osmotic shock, freezing and thawing, etc. Lysates can be added either directly to target cells or subjected to certain modifications to avoid nonspecific biological effects or increase the efficacy of selection. Number of clones per batch is determined by the complexity of the library, expression levels, projected biological activity of the products. In our work, we were able to screen E. coli expression plasmid library for TNF and IL3-expressing clones in batches, each containing 100 clones.

Selection of target cells subjected to the library products may involve any selectable phenotype or any other detectable features, such as: stimulation of growth, inhibition of growth, cell killing, morphological changes of cells or intracellular components, changes in expression of certain target cell components, binding of library products detected by any indirect approach (i.e., immunohistochemistry). Selection may not necessarily be limited to cell systems and may involve the whole organism (i.e. Drosophila, nematode, Zebrafish, other animals, plants, etc.).

Every clone from the batch that showed activity on target cells is then analyzed separately to identify the genetic element with the desired activity. For this purpose, the batches of clones are preserved and stored till the end of selection.

The combinatorial gene expression libraries of the invention may be pre-screened or screened by a variety of methods, including but not limited to, visual inspection, automated image analysis, hybridization to molecular beacon DNA probes (Tyagi et al. 1996, Nature Biotechnol, 14:303-308) fluorescence activated cell sorting (FACS) and magnetic cell sorting (MACS). Screening may be performed on bulk cultures of unamplified or amplified libraries. Libraries which are found to be positive in a pre-screen or screen can be recovered by culturing the droplet by placing it either on appropriate agar or liquid growth media or by dissolving the droplet in sodium citrate. After a period of culturing, the positive cells may grow out of the droplet. For convenience in handling and storage of droplets, the subsequent culturing may be done in multi-well plates. Pre-screened positives which have been reduced to a smaller population can then either be frozen and stored in the presence of glycerol or grown in multi-well plates. These can be used to transfer groups of clones using multi-pin replicators onto various types of assay plates (e.g. differential media, selective media, antimicrobial or engineered assay lawns). Specific assays can also be performed within these microtiter plates and read by a standard plate reader or any other format used in current high-throughput screening technologies. a. Formulations of Libraries in Screening Assays

Individual or pools of library cells may be encapsulated in an inert, stable and porous semi-solid matrix in the form of droplets during pre-screening or screening. The semi-solid matrix is permeable to gas, liquid, as well as macromolecules, and permits the growth and division of encapsulated cells. Examples of suitable matrices may include but are not limited to agarose, alginate, and carrageenan. The encapsulated library cells may be cultured and tested in the droplets, and remain viable so that the cells may be recovered from the droplets for further manipulations. The matrix may optionally be exposed to substances, such as an antibiotic, which can select for library cells that contain a selectable marker. The droplets may also be exposed to nutrients to support the growth of library cells. The following examples are offered by way of illustration and are not intended to limit the invention in any manner.

Encapsulation may be performed in one of many ways, producing either macrodroplets (droplets from 0.5 to 2.5 mm) or microdroplets (droplets from 10 to 250 μm) depending upon the method of detection employed during subsequent pre-screening or screening. The size and the composition of the droplets may be controlled during formation of the droplets. Preferably, each macrodroplet or microdroplet will contain one to five library cells. For example, macrodroplets may be prepared using sodium alginate, dissolved in sterile water at a concentration of 1%. A volume of library cells e.g., E. coli or yeast, such as Schizosaccharomyces pombe and Saccharomyces species; or spores for Streptomyces species; Bacillus subtilis; and filamentous fungus such as Aspergillus and Neurospora species is added to the sodium alginate solution so that 1-5 cells are encapsulated per droplet. The mixture is allowed to sit for at least 30 minutes to degas, and is then extruded through any device that causes the formation of discrete droplets, e.g., a syringe with a 25 gauge needle. The droplets are formed by adding the sodium alginate solution drop-wise into a beaker of gently stirring 135 mM calcium chloride solution. Droplets are allowed to solidify for 10 minutes, and are then transferred to a sterile flask where the calcium chloride solution is removed and replaced with a suitable growth media. Encapsulated library cells can be grown under standard conditions.

Microdroplets may be generated by any method or device that produces small droplets, such as but not limited to, two-fluid annular atomizer, an electrostatic droplet generator, a vibrating orifice system, and emulsification. Other methods for preparing semi-solid droplets are well known in the art; see for example, Weaver,

U.S. Pat. No. 4,399,219, and Monshipouri et al. 1995, (J. Microencapsulation, 12:255-262). The size of the droplets can be examined by phase microscopy. For the purpose of sorting by FACS or MACS, if the droplets are outside of the desired size range necessary for sorting, the droplets can be size selected using a filter membrane of the required size limit.

According to the invention, components of the reporter regimen or the target cells of a drug screen may also be co-encapsulated in a drop with library cell(s). Whole target cells, enzymes, or reporter molecules may be mixed with library cells suspended in the medium prior to formation of macro- or micro-droplets as previously described. Compounds of interest produced by the library cells may accumulate and diffuse within the droplet to reach the co-encapsulated target cells and generate a signal. The co-encapsulated indicator cell may be a live target of the desirable compound, e.g. pathogens for anti-infectives, or tumor cells for anticancer agents. Any change in metabolic status of the indicator cells, such as death, or growth inhibition, constitutes a signal and may be detected within the droplet by a variety of methods known in the art. Such methods may include but are not limited to the use of physiological probes, such as vital stains, or measurement of optical properties of the drop.

Macrodroplets can be sorted using a colorigenic reporter either by screening by eye or by using any device that allows the droplets to pass through a screening point, and which has the capacity to segregate positives. Microdroplets can be sorted using either FACS or MACS. FACS services are performed by a qualified operator on any suitable machine (e.g. Becton-Dickinson FACStar Plus). Particle suspension densities (cells or droplets) are adjusted to 1. times.10. sup.6 particles/ml. In all cases, positives can be sorted directly into multi-well plates at 1 clone per well.

MACS is performed using an MPC-M magnetic tube rack following the manufacturer's instructions (Dynal, 5 Delaware Drive, Lake Success, New York 11042).

b. Assay Formats The present invention provides methods of screening for candidate expression products by monitoring the activity/effect of such an expression product on a target cell. Generally such an assay may be performed in the presence and absence of the candidate substance and comparison of the results will reveal whether or not the expression product has an effect on the target cell. It is contemplated that this screening technique will prove useful in the general identification of compounds that may be useful in the treatment of various disorders. For example, agents that affect chemokine function will be useful in disorders resulting from impaired chemokine production, such as for diseases such as microbial infections; allergic or asthmatic responses; mechanical injury associated with trauma; arteriosclerosis; autoimmune diseases; and leukemia, lymphomas or carcinomas.

In these embodiments, the present invention is directed to a method for determining the ability of a candidate substance to exert an extracellular effect on target cells that either naturally are responsive to compounds of the class to which the candidate expression product belongs or have been engineered to be responsive to such agents. The method includes generally the steps of:

(i) providing an expression library that encodes the candidate expression product; (ii) contacting said expression product with a target cell in a manner that allows the expression product to exert its biological effect on said target cell; and

(iii) identifying expression constructs from the library which encode a biologically active product that exerts a detectable biological effect on the target cell.

The screening step may be repeated multiple time in order to produce an enriched library containing population of expression product of interest that have a particular activity. Such techniques for enriching expression libraries are well known to those of skill in the art.

c. Candidate substances.

As used herein the term "candidate expression product" refers to any molecule that is expressed by the expression libraries described herein and will be tested for its effect on a target cell. The candidate substance may be a protein or fragment thereof, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assay of the present invention will be compounds that are structurally related to other known therapeutically useful proteins, peptides or nucleic acids. The biologically active expression products may include fragments or parts of naturally-occurring compounds or may be generated through combinatorial techniques which produce active combinations of known compounds. It will be understood that ' such expression products could be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be polypeptide, polynucleotide, that may be designed through rational drug design starting from known compounds.

Term "biologically active product" includes polypeptides and RNA encoded by the genetic elements as well as other cellular products (peptides, hormones, etc.), the production of which can be induced in the cell expressing the isolated genetic element. Term "biological activity" includes: (i) the ability of the isolated bioproduct to cause specific alteration of the phenotype of the cell or the organism (stimulation or inhibition of growth, resistance or sensitivity to certain treatments or conditions, induction of morphological changes, cell differentiation, ability to secrete certain factors, etc.), (ii) to specifically recognize and bind certain molecules or structures on the cell surface. "From the outside of the target cell" means that the selected bioproducts does not require an intracellular expression or delivery for the induction of the desired biological activity.

Significant changes in the phenotype or activity of the target cell in response to the presence of the candidate expression product are represented by an increase/decrease in activity or phenotype of at least about 30%-40%, and most preferably, by changes of at least about 50%, with higher values of course being possible. The biologically active expression products identified by the present invention also maybe used for the generation of antibodies which may then be used in analytical and preparatory techniques for detecting and quantifying further such inhibitors. As indicated above, the expression products identified may be any therapeutically or diagnostically useful protein, peptide or polynucleotide. For example the expression products may be chemokines, cell death inducers, tumor growth suppressors, oncogenes, cell surface receptors and the like. The activity or function of chemokines, an exemplary expression product of the instant invention, may be monitored, e.g., as measured using leukocyte migration assays, myeloid, lymphoid or erythroid proliferation assays, HIV proliferation assays, receptor binding, and the like

There are a number of different libraries used for the identification of small molecule modulators including chemical libraries, natural product libraries and combinatorial libraries comprised or random or designed peptides, ohgonucleotides or organic molecules. Chemical libraries consist of structural analogs of known compounds or compounds that are identified as hits or leads via natural product screening or from screening against a potential therapeutic target. Natural product libraries are collections of products from microorganisms, animals, plants, insects or marine organisms which are used to create mixtures of screening by, e.g., fermentation and extractions of broths from soil, plant or marine organisms. Natural product libraries include polypeptides, non-ribosomal peptides and non-naturally occurring variants thereof. For a review see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides ohgonucleotides or organic compounds as a mixture. They are relatively simple to prepare by traditional automated synthesis methods, PCR cloning or other synthetic methods. Of particular interest will be libraries that include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial and polypeptide libraries. A review of combinatorial libraries and libraries created therefrom, see Myers Curr. Opin. Biotechnol. 8: 701-707 (1997). d. In vitro assays.

In one particular embodiment, the invention encompasses various binding assays. These can include screening for complexes of expression product with the cellular receptor for the expression product on a target cell. In such assays, the expression product may be either free in solution, fixed to a support, expressed in or on the surface of the host cell. In certain embodiments, the expression product will be released from the host cell to become available for testing on target cells after they are released from host cells. This can be achieved either naturally (natural secreted products or lytic virus-based expression systems) or artificially (induced lysis: in the case of expression non-lytic vectors

Such assays are highly amenable to automation and high throughput. High throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the target cells and washed. Bound polypeptide is detected by various methods.

Combinatorial methods for generating suitable peptide test compounds are specifically contemplated.

Of particular interest in this format will be the screening of a variety of different variants of a known therapeutically active polypeptide. These variants or mutants, including deletion, truncation, insertion and substitution mutants, will help identify which domains of the peptide are involved with the activity of the expression product. Once this region has been determined, it will be possible to identify which of these mutants, which have altered structure but retain some or all of the desirable functions of peptide. Other forms of in vitro assays include those in which functional readouts are taken. For example molecular analysis may involve assays such as those for protein expression, enzyme function, substrate utilization, mRNA expression (including differential display of whole cell or polyA RNA) and others.

C. Examples

The present invention is described in more detail with reference to the following non-limiting examples which represent preferred embodiments of the invention. Those of skill in the art will understand that the techniques described in these examples represent techniques described by the inventors to function well in the practice of the invention, and as such constitute preferred modes for the practice thereof. However, it should be appreciated that those of skill in the art should in light of the present disclosure, appreciate that many changes can be made in the specific methods which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1. Optimization of conditions of bacterial induction and lysis

The dynamic and efficacy of release of plasmid-encoded protein product into bacterial culture medium was studied on E. coli transformed with pET29-LacZ vector, expression bacterial β-galactosidase gene in IPTG-inducible fashion. Although IPTG caused fast and strong induction of intracellular β-gal, spontaneous release of β-gal into the medium was ineffective during log phase of bacterial growth (FIG. 2A). Therefore, ampicillin treatment was used to improve the release of bacterial proteins into media. By comparing β-gal activity in the samples of cell media and in the equivalent amount of bacterial lysates at different time points after IPTG induction and treatment with varying concentrations of ampicillin we optimized the conditions of induction/lysis allowing for most effective release of the product into medium. "Extracellular" β -gal activity (determined in the supernatant) reaches maximum (60-70% of total β-gal activity, determined in bacterial pellets) in 2 hours after adding ampicillin (50 μg/ml, the dose that dose not affect growth of mammalian cells) and then stays at constant level (FIG. 2B and 2C).

Example 2. Application to functional identification of TNF as an example of isolation of pro-apoptotic factors from a cDNA expression library

Tumor necrosis factor (TNF) is one of the secreted pro-apoptotic factors affecting many mammalian cells. It is known to active recombinant protein produced in E. coli. Testing the activity of the lysates prepared from bacterial mixtures containing different proportions of TNF-producing cells was used as a model allowing to 1) prove the principle of selection of soluble factors by the methods of the invention, 2) to develop the condition of selection of apoptosis- inducing soluble factors. cDNA corresponding to mature peptide of mouse TNF (mTNF) was cloned into pET29 prokaryotic expressing vector, in which expression of the inserts is controlled by T7 polymerase promoter. E. coli, strain BL21(DE3), containing T7 polymerase under the IPTG-inducible promoter, were transformed by pET29-mTNF. Inducible expression of mTNF was confirmed by gel- electrophoresis of the total bacterial protein lysate. As target cells, we used mouse fϊbroblast cell line NIH3T3, known to respond to TNF treatment in the presence of low concentration of cyclophosphamide (CHI) by induction of apoptosis. As an control plasmid-encoded bacteria-produced protein cytokine that supposed to have no effect on these cells we used human IL-3. We mixed bacterial populations (OD 0.8 each), carrying pET-mTNF and pET-hIL-3 in the following proportions: 100% of mTNF expressing cells, 10% of mTNF expressing cells and 90% of hIL-3- expressing cells, 1% of mTNF expressing cells and 99% of hIL-3-expressing cells and finally 100% of hIL-3-expressing cells. The same amounts of each these mixtures (10 μl) were added per well of 96 well plate containing growing NIH3T3 cells in 150 μl of IPTG-containing DMEM medium without antibiotics. In 1 hour ampicillin was added (final concentration 50 μg/ml) followed by CHI (1 μg/ml, after 1 hour in ampicillin) to inhibit NF-κ induction associated with TNF receptor activation, interfering with apoptosis. NIH3T3 cells in parallel well were treated with 2 and 20 ng/ml of TNF in the presence of CHI. As controls, as used NIH3T3 cells incubated in regular media, containing ampicillin and CHI and NIH3T3 cells in the same medium contaminated with non-transformed E. coli, and E. coli transformed with the insert-free pET29 plasmid. After overnight incubation, cells were washed with PBS, fixed with methanol and cell numbers were estimated using methylene blue assay. The results of one representative experiment are demonstrated in FIG. 3. They show that (i) bacterial lysates do not affect cell survival under the experimental conditions used, and (ii) even those bacterial lysates that contained the lowest proportions of TNF-expressing bacteria showed detectable biological effect and could be detected if this would apply this procedure for screening a cDNA library.

Example 3. Application to functional identification of interleukin-3 (IL-3) as an example of isolation of a survival factor from a cDNA expression library IL-3 is a secreted growth factor effective for the cells of lympho- and hematopoietic lineages with broad spectrum of activities. Among cancerous cell lines there are few exhibiting complete dependence on IL-3 for their growth and survival. Human cells TF-1 require IL-3 for their growth and respond by induction of apoptosis on the lack of this cytokine in the medium dying between 24 and 48 hours after removal of the cytokine. cDNA corresponding to the mature peptide of human IL-3 (hIL-3) was cloned into pET29 vector, in which expression of the inserts is controlled by T7 polymerase promoter. BL21 (DE3) cells, containing T7 polymerase under IPTG-inducible promoter, were transformed by pET29-hIL-3. Inducible expression of hIL-3 was confirmed by gel-electrophoresis of the total bacterial protein lysate before and after induction (FIG. 4A). As a control biologically inactive protein for this system we used mTNF, that has no effect on growth and survivability of human TF-1 cells. Both type of constructs were transformed into BL21 (DE3) cells. Then bacterial populations, transformed by pET-mTNF and pET-hIL-3 were mixed in the following proportions: 100% of hlL- 3 expressing cells, 10% of hIL-3 expressing cells and 90% of mTNFa-expressing cells, 1 % of hIL-3 expressing cells and 99% of mTNFa-expressing cells and finally 100% of mTNFa-expressing cells. The same amounts of each of these mixtures were added to the 96 well plate containing growing TF-1 cells in IPTG-containing medium without IL-3. 50 μl/ml of ampicillin were added in 1 hour and live cells were calculated 48 hours after. As negative control, we used TF-1 cells growing in regular medium with 5 ng/ml of IL-3 and TF-1 cells growing in the same conditions but in the presence of non-transformed E. coli and transformed with the insert-free pET29 vector. As positive control, we used TF-1 cells growing in the medium without IL-3 and TF-1 cells growing in the same medium plus E. coli transformed with empty pET29 vector. The results of a representative experiment (FIG. 4B) show that lysates of bacterial mixtures containing even the lowest proportion of IL- 3-expressing bacteria protected TF-1 cells from death in IL-3-deprived medium. They also indicate that we would be capable of isolation of IL-3 -producing clones from a cDNA library using this experimental design.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the processes described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention. Techniques used for the production expression libraries and for the production and isolation of recombinant peptides are well known to those of skill in the art and may be used in conjunction with the present invention.

Claims

What is Claimed is:

1. A method of isolating a polynucleotide encoding a biologically active product comprising; '

a) culturing host cells comprising an expression library, wherein said expression library comprises a pool of expression constructs, said expression constructs each comprising a nucleic acid which encodes a candidate biologically active product operably linked to an expression control sequence, to effect the expression of said biologically active product in said host cell;

b) contacting a target cell with the expression products obtained from said host cell under conditions which permit candidate biologically active expression products to exert a biological effect on said target cell, wherein said target cell is different from the host cell of step (a); and

c) identifying a subpool of expression constructs from the pool of expression constructs of step (a) wherein said subpool comprises an expression construct comprising the polynucleotide which encodes the biologically active product that exerts a detectable biological effect on said target cell in step (b).

2. The method of claim 1, wherein said host cells are Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Pseudomonas aeruginosa, Myxococcus xanthus, Saccharomyces cerevisiae, Schizosaccharomyces pombe,

Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, Nicotiana tabacum, insect cells, chicken cells, COS cells, 293 cells, NERO cells, ΝIH/3T3 cells, or CHO cells.

3. The method of claim 1, wherein said host cells are transformed with an expression vector comprising said expression constructs.

4. The method of claim 3, wherein said expression vector is selected from the group consisting of a plasmid, a bacteriophage, a viral vector, a cosmid vector or an artificial chromosome.

5. The method of claim 1, wherein said contacting step comprises contacting the target cells with supernatant from the media of said host cells.

6. The method of claim 5, wherein the supernatant of said host cells is collected after said host cells have been lysed.

7. The method of claim 5, wherein said supernatant comprises candidate biologically active expression products that have been secreted from said host cells.

8. The method of claim 1, wherein said target cells are selected from the group consisting of bacterial cells, yeast cells, invertebrate cells and vertebrate cells.

9. The method of claim 1, wherein said expression product is an agent that has an extracellular effect on said target cell.

10. The method of claim 1 , wherein said target cell comprises receptor for said candidate biologically active expression product, wherein said receptor is exposed to the extracellular environment of said cell.

11. The method of claim 9, wherein said extracellular effect is a receptor mediated effect and said target cell comprises a receptor for said expression product.

12. The method of claim 9, wherein said expression product is a cytokine, a, chemokine, a tumor suppressor, a growth factor, a hormone, a toxin or a survival factor.

13. the method of claim 1, wherein said biological effect is selected from the group consisting of cell death, cell survival, phosphorylation, cell proliferation, expression or suppression of a reporter gene or a selectable marker.

14. The method of claim 1, further comprising repeating steps (a) through (c) on said subpool of expression constructs identified in step (c) to produce an enriched population of said expression construct.

15. The method of claim 14, further comprising isolating an expression construct that encodes an expression product that exerts a desired biological effect.

16. The method of claim 15, further comprising identifying said expression product.

17. The method of claim 15, wherein said identifying comprises sequence said expression product or sequencing said expression construct.

18. A method of screening for a biologically active product comprising;

a) culturing an expression library in a host cell, wherein said expression library comprises a pool of expression constructs, each expression construct comprising a nucleic acid which encodes a candidate biologically active product operably linked to an expression control sequence, to effect the expression of said biologically active product in said host cell;

b) contacting a target cell with the expression products obtained from said host cell under conditions which permit candidate biologically active expression products to exert a biological effect on said target cell, wherein said target cell is different from the host cell of step (a);

c) monitoring the biological effect of said expression products on said target cell to identify an expression product that exerts biological effect; and d) identifying a subpool of the pool of expression constructs of step (a) which subpool comprises an expression construct which encodes the biologically active product that exerts a biological effect on said target cell.

19. A method of producing a biologically active compound comprising: a) isolating a polynucleotide encoding a biologically active product according to the method of claim 1 ,

(b) preparing an expression construct comprising said polynucleotide operably linked to an expression control sequence;

(c) expressing said expression construct in an appropriate host cell to produce the biologically active compound.

20. The method of claim 19, further comprising recovering said biologically active compound from said host cell.