HUT60523A - Improved process for expressing polypeptides - Google Patents

Abstract

This invention relates to processes and intermediates for improving the level of production of a desired polypeptide in a recombinant host whose genome has integrated into it an island of expression comprising in the 5' to 3' direction, a 5' flanking region, the heterologous polypeptide encoding sequence and a 3' flanking region. The island of expression of this invention permits the expression of the integrated heterologous DNA sequence to be substantially dependent on its copy number and to be substantially independent of its position of integration in the host genome.

Description

The present invention relates to methods and intermediates for improving the level of production of a desired polypeptide in a recombinant host. More particularly, the present invention relates to an expression island, a DNA segment comprising a DNA sequence encoding a heterologous polypeptide, and to the use of an expression island to infect a host organism. Host organisms comprising this island of the term produce surprisingly high levels of the desired heterologous polypeptide. Incorporation of the expression island into a host allows the desired polypeptide to be expressed to be substantially independent of its site of integration in the host genome, but substantially dependent on the number of copies of the expression island that integrate in the host genome.

It is well known in the art that polypeptides can be expressed and selected by host organisms that are transfected or transformed with the DNA sequence encoding said polypeptide. For example, Gilbert et al., U.S. Patent 4,565,785 (1986); VillaKomaroff L. et al., Bacterial Clone Synthesizing Proinsulin, Proc. Natl. Acad. Sci. USA 75, 3727-31 (1978), disclose that a selected polypeptide can be synthesized within a bacterial host and can be secreted through the cell membrane. A similar workflow can be performed in animal cells. £ Doehmer, J., et al., Introduction to Rat Growth, Hormone Gene, Ind. Natl. Acad. Sci. USA 79: 2268-72 (1982). Recombinant proteins expressed at even em stem orchards a transgenic expression systems incorporating a fertilized embryo pronuclei ^ 0. Bucchini et Pancreatic Expression Of Human Insulin Gene In Transgene _c Mice, Proc. Natl. Acad. Sci. USA, 8J5, 2511-15 (1986); Gordon, K., et al., 1987, Production of Hdman Tissue Plasminogen Activator In Transgenic Mousg Milk, Bio / Technology 5/11).

However, at present none of these techniques is consistently successful in the sense that it allows large amounts of a desired heterologous polypeptide to be expressed by a host integrating a heterologous polypeptide coding sequence into its genome. This is particularly surprising when one considers the high level of native protein production resulting from identical expression control sequences in the native environment. so e.g. milk-specific expression control sequences allow large amounts of protein, e.g. casein is formed in and secreted from mammalian glands. However, identical milk-specific expression control sequences have not yet been shown to induce large amounts of heterologous polypeptide when operatively linked to sequences encoding heterologous polypeptide. Pittins C.W. et al., The Milk Protein Gene Promoter Directs The Expression of Human Tissue Plasminogen Activator cDNA To The Mammary Gland In Transgenic Mice, Proc. Natl. Acad. Sci. USA 85: 5874-78 (1988)]. The level of expression in the latter constructs also depends on the copy number of the sequence encoding the heterologous polypeptide integrated into the host genome. In addition, the level of expression is also subject to local effects, that is, the level of expression depends on the heterologous polypeptide. · · * ···· where the coding sequence is integrated into the genome ^ Lee KF et al: Tissue-Specific Expression Of The Rat Beta Casein Gene In Transgenic Mice, Nucleic Acids

Gap. 16/3), 1027-41 (1988)].

Consequently, there is a need for a method that increases expression of a DNA sequence encoding a heterologous protein or polypeptide, regardless of the site of integration into the host genome. In addition, such methods should provide an expression that depends on the copy number integrated into the host genome, so that the level of expression can be controlled.

The present invention solves these problems by providing an expression island comprising a DNA sequence encoding a desired heterologous polypeptide. The expression island of the present invention provides, for the first time, high-level, site-independent but copy-dependent expression of a DNA sequence encoding a heterologous polypeptide.

As shown in Figure 1, the expression island of the present invention comprises in the 5 'to 3' direction: a 5 'flanking region, a coding sequence for a heterologous polypeptide (encoding the desired heterologous protein or polypeptide), and a 3' border area. The 5 'flanking region in the 5' to 5 'direction comprises 5' expressing control sequences and a 5 'untranslated region. Expression control sequences are operably linked to the heterologous polypeptide coding sequence. The 5 'untranslated region begins at a translation initiation site and ends at the translation start site of the sequence encoding the heterologous polypeptide. The 3 'flanking region in the 5' to> 3 'direction includes a 3' untranslated region and a 3 'expressing region

5 control sequences, wherein the control sequences are operably linked to a coding sequence for a heterologous polypeptide. Finally, the 5 'and 3' flanking regions of the expression island of the invention are characterized by an appropriate size and structure that effectively alter the level of production of the desired protein or polypeptide substantially depending on the copy number integrated into the host genome and substantially independent of their site of integration.

The present invention relates to the use of an expression island for the transfection of a host organism and relates to these transfected host organisms. Hosts that have integrated the expression island into their genome produce high levels of the heterologous polypeptide encoded by a DNA sequence within this expression island. This signal sequence coding region is fused to the 5 'end of the heterologous polypeptide coding sequence and has the same reading frame. The coding region of the signal sequence is also operably linked to expression control sequences such that the host whose genome bears this expression island is capable of producing, selecting for and preferably processing the desired protein or polypeptide of the combined signal heterologous polypeptide. a pre-protein or a pre-polypeptide encoded by a coding sequence.

The following is a brief description of the figures.

Figure 1 is a schematic representation of a typical expression island (A) and a preferred expression island (B) of the present invention.

Figure 2 depicts the construction of a plasmid (CAS 1288) containing the 5 'and 3' flanking regions of bovine alpha S-1 casein.

• ··· ··· · · · · · · · · · · · · ·

Figure 6 depicts the introduction of the urokinase structural gene CAS 1288 to form CAS 1295, the expression island.

In order that the invention disclosed herein may be more readily understood, the following detailed description is set forth.

The following terms are used in this description:

Expression control sequences --- sequences that control and regulate the expression of gene products both at the transcription (transcription) and translation levels when operatively linked to a structural gene (DNA encoding a polypeptide). These include promoter and enhancer regions, ribosome binding sites, polyadenylation signals, and other sequences useful for the expression of genes.

Coupling operably linked --- 5 'and 3' expression control sequences to a coding sequence for a heterologous polypeptide such that the expression control sequences are capable of controlling and controlling the expression and production of the heterologous polypeptide.

Heterologous polypeptide coding sequence - a coding sequence for a desired protein or polypeptide inserted into the genome of a host. This DNA sequence encodes a polypeptide that is heterologous to the host organism, flanking sequences, or both. The heterologous polypeptide coding sequence optionally contains its own translation stop codon at the 3 'end. The heterologous polypeptide coding sequence may also comprise its own signal sequence coding region.

Signal sequence coding region --- a DNA sequence that encodes a sequence typically called a hydrophobic amino acid called a signal peptide. The signal peptide allows a polypeptide that binds to it to pass through a biological membrane.

An expression island --- DNA construct comprising a 5 'flanking region, a sequence encoding a heterologous polypeptide, and a 3' flanking region in the 5 'to 3' direction. The 5 'and 3' flanking regions are sized and structured such that the level of production of the desired protein or polypeptide is substantially dependent on the copy number of the expression island construct incorporated into the host genome, but substantially independent of the site of integration of the expression island within the host genome.

5 'flanking region --- that portion of the expression island 5' to the heterologous polypeptide coding sequence. This includes, in the 5 'to> 3' direction, the following: 5 'expression control sequences and a 5' untranslated region; the expression control sequences are operably linked to a sequence encoding the heterologous polypeptide. The 5 'untranslated region typically ranges from a transcription initiation site to a translation start site of a sequence encoding a heterologous polypeptide.

3 'flanking region --- that portion of the expression island 5' to the heterologous polypeptide coding sequence. This includes, in the 5 '-> 3' direction, a 3 'untranslated region and a 3' expression control sequence. The 3 'flanking region may comprise all or part of the coding sequence originally associated with the structural gene to the 3' flanking region.

The present invention is described in detail below.

While not intended to be bound by any theory, we believe that the expression island allows 5 'and 3' • · · · • *. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·Bar - eng

- 8 undefined factors within the flanking regions to operate the expression control sequences and allow the heterologous polypeptide coding sequence to be expressed in higher production. The expression depends on the copy number of the expression island construct incorporated into the host genome, thus allowing the level of polypeptide production to be varied.

The expression island of the present invention also provides a large 5 'and 3' flanking region with a buffer zone such that expression control sequences are isolated from host expression control sequences which can then be used by the surrounding DNA to which the expression island is integrated. Therefore, it does not matter where the expression island is integrated into the host genome; the sequence encoding the heterologous polypeptide is nonetheless expressed at high levels. It carries its own genomic structure, making it an expression island.

While not wishing to be bound by any theory, it is believed that most of the DNA regions that may enhance expression from expression control sequences are located in the 5 'and 3' flanking sequences of a particular structural gene. Therefore, after isolation of a structural gene, together with their 5 'and 3' flanking regions, the structural gene may be partially or completely excised and replaced with a coding sequence for any heterologous polypeptide so as to allow expression at a level corresponding to that of the original structural gene. Alternatively, the sequence encoding the heterologous polypeptide may be inserted at the 5 'end of the structural gene without simultaneous deletion of the gene. In this embodiment, the sequence encoding the heterologous polypeptide can also be expressed at a level such as β ························································ · · · ·

- 9 corresponding to the level of expression of the original structural gene.

Among the expression control sequences used in the various embodiments of the present invention are those that direct expression at a high level in certain types of cells or at particular stages of cell growth or differentiation or in specific culturing conditions. Tissue specific expression control sequences are preferred in the transgenic host of the present invention.

When mammalian host cells are used, useful expression control sequences may be derived from the host cell itself, namely native sequences encoding a highly expressed product. However, they may also be derived from other high-level eukaryotic genes, e.g. genes encoding p-actin, collagen, myosin, albumin, metallothionein or human growth hormone.

A preferred embodiment of the present invention provides for the production of proteins in transgenic mammals. Preferably, this embodiment utilizes expression control sequences that regulate and control expression of gene products in mammalian tissues (such as control sequences, e.g., casein promoters and beta-lactoglobulin promoter. Casein promoters include, for example: alpha casein promoter, beta casein promoter or kappa casein promoter The casein promoter and associated expression control sequences are preferably of bovine origin and most preferably these are the alpha S1 casein promoter and associated expression control sequences.

Expression control sequences may also be derived directly from cells intended to be used as host for the expression island construct. First, the promoter with the desired level of activity in the host and the associated expression control sequences must be identified. The expression island should be designed such that the expression island construct, which is integrated into the host genome, is expressed in a copy-dependent but independent manner.

The following describes methods for identifying expression control sequences, cloning the required flanking regions containing these sequences, adding a sequence encoding the heterologous polypeptide, and testing whether the resulting construct is an expression island of the invention.

The first step is to determine the host and the requirements to allow the host to express at a high level or at specific times. For tissue culture, CHO cells growing on collagen beads in the VERAX system are preferred.

In order to isolate expression sequences for a homologous gene that are expressed at high levels in well-selected host cells, a highly expressed RNA species must be identified. This can be achieved by constructing a cDNA library of polyA RNA isolated from selected host cells under selected induction and growth conditions. The cDNA library is then screened using a labeled aliquot of RNA from which the cDNA library was constructed. The most positive signals indicate the cDNAs whose RNA is most abundant in the host cells under the selected induction and growth conditions. Selected cDNAs can then be used to screen for genomic DNA libraries prepared from selected host cells to find the genomic DNA sequences that correspond to the richest RNAs. These genomic sequences, typically in cosmids ΓManiatis T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982) J, can then be analyzed to determine restriction sites, amount of flanking sequences and regions encoding the polypeptide in the cosmid.

In another but less preferred method, expression control sequences may also be isolated by screening host cells grown and induced under selected conditions on a richly produced protein or polypeptide. This can be achieved by analyzing all polypeptides produced in the host either by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE) or by two-dimensional gel electrophoresis. The most abundant polypeptide was identified as the strongest band in SDS-PAGE or as the largest spot on the two-dimensional gel. Once identified, the band or spot is excised from the gel, eluted and subjected to automated protein sequence analysis. Based on the amino acid sequence obtained from the protein sequence analysis, the corresponding oligonucleotides are then synthesized. These oligonucleotides can then be labeled and used as probes to identify appropriate genomic sequences from the cosmid library of the host cell DNA.

Once a sufficiently detailed map of this richly expressed gene has been determined, the coding sequences and intermediate sequences of the structural gene can be removed from the cosmids, e.g. with appropriate restriction enzymes and may be replaced by a coding sequence for the heterologous polypeptide. Another Method • · · · 4 «· • · * 4 * • f · ··· ·

- 12, the sequence encoding the heterologous polypeptide may be inserted 5 'to the structural gene. In this procedure, the structural gene does not have to be excised. In a preferred embodiment, the heterologous polypeptide is a urokinase whose DNA sequence is isolated and cloned from a genomic library using probe published sequences [Riccio A. et al., The Human UrokinasePlasminogen Activator Gene And Its Promoter, Nucleic Acid Slit. 13 (8): 2759-71 (1985)].

The resulting construct has the DNA sequence encoding the heterologous polypeptide flanked on both sides by the genomic sequences of the abundantly expressed gene originally isolated from the host cells. 5 'and 3' flanking sequences of different lengths should be screened to determine what size flanking regions are required to direct expression of the heterologous polypeptide coding sequence in a copy-dependent but location-independent manner.

In order to determine whether the isolated cosmos contains the corresponding 5 and 3 'flanking regions, the sequence encoding an inserted heterologous polypeptide is expressed at substantially the same level as the highly expressed homologous DNA sequence in the cells of tissue culture. or transfected into the genome of an embryo to produce transgenic animals. Preferably, cells or embryos that are used in the final production are used in this step. The transformed hosts are then assayed for expression of the heterologous protein by any of the well known assays. These include, but are not limited to, radioimmunoassay, ELISA, immuno-staining, and assays that provide the desired polypeptide · 4 · · 4 · * • · · ml · * 1 ·· • ···································································································. Another preferred method is to use mRNA levels measured under a variety of growth conditions. This can be accomplished by the Northern blot technique using probes as previously described (and corresponding to the polypeptide sequences) or previously identified cDNAs.

Because the expression control sequences selected from host cells show the ability to direct high levels of expression of the homologous gene under known conditions (e.g., collagen beads, growing CHO cells in the VERAX system), expression of the heterologous polypeptide is expected to occur at substantially the same level. DNA sequence does not provide any other 5 'cosmids of different lengths and should only be analyzed between appropriate DNA sequences. If the cosmid has any level of expression, the 3 'flanking regions will be held in substantially the same manner until localized.

The level of production of the heterologous protein produced by this sequence is then compared to the copy number of the integrated expression island. The copy number was determined by appropriate restriction enzyme analysis. Expression constructs that show a location-independent but copy-dependent expression are the optimal expression islands according to the present invention.

In a preferred embodiment, a host comprising an expression island of the invention selects the desired polypeptide. Selection of polypeptides is achieved by fusing a DNA sequence encoding a signal peptide with DNA encoding the heterologous polypeptide in the appropriate reading frame. The size of the signal peptide is not critical in this invention. All it takes is just that the signal * · «« «« * 4 4 • '······ 4 ··· • 4 ···· 99 *

- 14 peptides of sufficient size and sequence to efficiently promote the selection of the heterologous polypeptide. Examples of signal sequences encoding a signal peptide may include, in nature, signal sequences associated with expression control sequences, in nature, signal sequences associated with a desired heterologous protein or polypeptide, native signal sequences for the source of the heterologous polypeptide, and expression sequences for any of the expression sequences. another sequence encoding functional signal peptides.

Many of the proteins to be expressed are normally secreted and have their own signal peptide, which should be appropriate for directing the selection. In this case, the DNA encoding this signal may be included in the coding sequence for the heterologous polypeptide, which is inserted into the expression island. When producing a polypeptide that is not normally secreted, it is possible to use a signal sequence derived from polypeptides normally secreted from host cells or other secreted polypeptides.

A preferred embodiment of the present invention utilizes sequences encoding milk-specific signal peptides or sequences of other signal peptides useful for maturation and selection of proteins in mammalian tissues. If the heterologous polypeptide to be selected is naturally associated with its own signal sequence, in nature, the signal sequence associated with the heterologous polypeptide coding sequence is the most preferred signal sequence.

The required 5 'and 3' flanking regions are characterized by their ability to induce expression in a site-independent but copy-dependent manner. The length of the flanking sequences is not critical as long as the desired properties are given to the expression construct. The upper limit is determined by the ease with which the DNA can be manipulated. In the original source of expression control sequences (animal or cell line), expression control sequences are theoretically flanked by the entire chromosome. Current techniques allow for easy manipulation of a 40-50 kb DNA segment. This requires the use of well known cosmid techniques. There may be a limit on the size of DNA to be injected through needles used for embryo manipulation. A preferred technique is to use as large 5 'and 3' flanking areas as large as possible to provide sufficient isolation space to transmit copy number dependency and site independence.

The coding sequence of the desired heterologous polypeptide may be derived from cDNA, genomic sequences, synthetic DNA or semi-synthetic DNA. Among the polypeptide products obtainable by the process of the present invention are, for example, VIII. and IX. clotting factors, human or animal serum albumin, tissue plasminogen activator (tPA), urokinase, alpha-1 antitrypsin, animal growth hormones, Mullerian Inhibiting Substance (MIS)], cell surface proteins, insulins, , interleukins, milk-lipases, antiviral proteins, peptide hormones, immunoglobulins, lipocortinins, and other heterologous protein products.

The desired heterologous polypeptide may also be prepared as a fusion protein that contains additional amino acids in addition to the amino acids present in the desired or native polypeptide. so e.g. the desired heterologous polypeptide of the invention may be prepared as part of a larger heterologous protein or polypeptide in order to stabilize the desired polypeptide or to make purification easier and / or faster. This can be accomplished by inserting the heterologous polypeptide coding sequence into the expression island from the 5 'or 5' site of the structural gene that was originally associated with the expression control sequence; it is installed in a suitable reading frame. Obviously, such a construct requires removal of the termination codon of the heterologous polypeptide before insertion into the expression island.

Alternatively, the fusion protein coding region may be constructed prior to insertion into the expression island. The fusion protein construct may contain sequences or portions encoding two or more heterologous proteins as long as only the sequences are in the same reading frame. Such constructs can be made by methods known in the art. The fusion protein can then be cleaved, if necessary, and the desired protein isolated. The desired heterologous polypeptide may be prepared as a fragment or derivative of the polypeptide that is originally associated with the expression control sequences. Any of these variants can be easily accomplished by selecting and / or manipulating the correct DNA sequences. Such manipulations are well known in the art.

The expression island constructs described above may be prepared using techniques well known in the art. so e.g. conventional switches, restriction sites, etc. employing various ligation techniques with good efficiency. The expression islands of the invention are larger.

It is prepared as part of plasmids. Such compositions allow cloning and selection of correct constructs in an efficient manner, as is well known in the art, and allow convenient production of large amounts of expression island constructs.

The nature of the plasmid is not critical to the practice of the present invention. Any plasmid known in the art to be capable of replication, selection and carrying larger pieces of DNA may be a suitable carrier into which the expression islands of the invention can be inserted. Most preferably, the expression islands of the present invention are located between convenient restriction sites in the plasmid so that they can be readily isolated from other parts of the plasmid sequences for insertion into the desired host.

The selection of a suitable host for the expression island can be controlled by a variety of factors known in the art. These include eg. compatibility with the vector of choice, toxicity of the polypeptide products, ease of obtaining the desired heterologous polypeptide, expression characteristics, specific processing needs of the heterologous polypeptide, biosafety and cost.

Absolute certainty in the selection of the host for a particular desired protein or polypeptide is not possible by any of the above factors. The balance of these factors leads to the realization that not all hosts are equally capable of effectively expressing a particular heterologous polypeptide.

Suitable mammalian host cells include B and T lymphocytes, leukocytes, fibroblasts, hepatocytes, pancreatic cells, and non-differentiated cells. Preferably, immortalized (immortalized) mammalian cell lines may be utilized. It is found among suitable mammalian cell lines

E.g. 3T3, 3T6, STO, CHO, Ltk, FTO2B, Hep2B, AR42J and MPC1L. The most preferred mammalian cell lines are CHO, 3T3 and Ltk.

Embryos from various mammals may be used to produce transgenic animals. The choice of the host embryo will depend on factors such as the desired final fate of the heterologous polypeptide in the animal. so e.g. one preferred embodiment of the expression of heterologous polypeptides is the expression of polypeptides in mammalian milk, wherein the preferred host embryos are derived from animals that have already been bred for high volume milk production; such as cows, sheep, goats and pigs.

There are several standard protocols available for introducing the expression construct DNA into animal cells. Commonly used transfection methods include electroporation. H. Potter et al., Enhancer-Dependent Expression of Human Kappa Immunoglobulin Genes Introduce Mouse Pre-B Lymphocytes By Electroporation, Proc. Natl. Acad. Sci. USA 77 (7), 4216-4200 (1980), protoplast fusion, Sandri-Goldin, R.M., et al. Mole. Cell. Biol., 743-52 (1981) J, Calcium phosphate co-precipitation by J.H. and van Aér Eb Eb: The New Technique For Assay Of Infectivity Of Human Adenovirus 5 DNA, Virology 52 (2), 456-67 (1973); Miller A.O. et al., c-fos Protein Can Induce Cellular Transformation: The Increased Mechanism Of Activation Of Cellular Oncogene Cell 36 (1), 51-60 (1981) 2, and DEAE-dextran sulfate-mediated sessions. In addition, there are many variants of DEAEdextran sulfate and calcium sulfate processes in Toueen C. and · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Baltimore D .: Immunglobulin Gene Transcription Is Activated By Downstream Sequence Elements, Cell 33 (3), 741-48 (1983); Gorman C.M. et al., Recombinant Genomes Which Express Chloramphenicol Acetyl-Transferase In Mammalian Cells, Mol. Cell. Biol. 2/9), 1044-41 (1982); Mclvor R.S. et al., Expression Of A cDNA Sequence Encoding Human Purine Nucleoside Phosphorylase In Rodent And Human Cells, Mol. Cell. Biol. 5 (6), 1349-57 (1985) J, which provide numerous advantages. so e.g. calcium phosphate co-precipitation sessions are particularly effective for mammalian cells, including CHO cells.

Together with the expression island construct, a selectable marker is also generally introduced into mammalian cells as a distinct piece of DNA so that cells that have incorporated the expression construct can be readily detected. Suitable selectable markers include dihydrofolate reductase, metallothioenein, neo, gpt and hisD. The selected cells are then screened for expression of the heterologous protein.

Standard techniques are available for introducing an expressive construct into the genome of a mammalian embryo. One technique for transgenic alteration of mammals is microinjection of the expression island construct into the pronucleus of a fertilized mammalian ovum; this results in one or more copies of the construct being integrated into the genome and retained in the developing mammalian cells. Briefly, a microinjection session consists of isolating the fertilized ovum, visualizing the pronucleus, and then introducing the DNA into the pronucleus by attaching the ova to a blunt support pipette (about 50 ^ 4m in diameter) and apply a sharpened pipette (about 1.5 / qm in diameter) · · · · · · · · · · · · · · · · · · · · · · · ·

20 acids are injected into the pronucleus in the DNA containing buffer. Kraemer, D., et al., Gene Transfer Inton Pronuclei Of Cattle And Sheep Zygotes, 221-27 in Genetic Manipulation of the Early Mammalian Embryo. pages, Cold Spring Harbor Laboratory (Publisher) (1985); R. G. Hammer et al., Production Of Transgenic Rabbits, Sheep and Pigs By Mi Force Injection, Natur 315, 680-83 (1985); and Gordon J.W. and Ruddle, F.H., Gene Transfer Intro Mouse Embryos (1983), Production of Transgenic Mice By Pronuclear Injection Methods 101, 411-33.

Microinjection is preferably performed on a single-cell embryo to maximize the chance that the injected DNA is incorporated into all cells of the animal and the DNA is incorporated into the germ cells so that the offspring of the animal will be equally transgenic. Generally, at least 40% of mammals that have developed from injected egg cells contain at least one copy of the cloned construct in somatic tissues, and these transgenic mammals generally carry the gene through the germ line to the next generation. DNA isolated from the tissues of the resulting transgenic mammals may be analyzed by Southern blot analysis for the presence of the expression island. If one or more copies of the expression island remain stably integrated into the genome of such transgenic mammals, it is possible to establish a permanent transgenic mammalian line carrying the expression island construct.

The offspring of transgenically altered mammals may be examined after birth to incorporate the expression island construct into the genome. This assay is preferably performed by Southern hybridization of the chromosomal material of the offspring * 4 * 4 · 4 »· · 4 · 4

- 21, using a probe corresponding to a portion of the heterologous polypeptide coding sequence. Mammalian offspring that are found to contain at least one copy of the construct in their genome are raised to maturity. In a preferred embodiment of the present invention, the females of these offspring produce the desired heterologous polypeptide in or together with milk. Alternatively, the transgenic mammals are further cultured to generate additional transgenic progeny that are also capable of producing the desired heterologous polypeptides.

EXPORTING EXAMPLES

EXAMPLE 1 - CREATING THE BEEF ALFA S-l CASEIN EXPRESSION ISLAND

One example of this technology is the use of the expression island construct in the production of a heterologous protein in specific tissues or organs of an intact animal. In this case, the high level expression of the heterologous protein is directed to a mammalian mammary gland.

The gene construct described herein contains an expression island in which the large 5 'and 3' flanking regions derived from the bovine alpha casein gene direct the expression of the human urokinase genomic clone. The flanking region 5 'consists of a 21 kb upstream alpha-casein sequence including the first non-coding exon and the non-coding portion of the second exon. The 9 kb 3 'flanking region contains exons encoding the COOH-terminal half of alpha casein, the polyadenylation signal, and 2 kb of further downstream flanking sequences.

From the calf thyme DNA cosmid library, bovine alpha ·· · ·

The 5-1 casein gene was cloned in the cosmid vector HC79 (Boehringer Mannheim) as described by Hohn B. and Collins 3. LA Small Cosmid Forage Cloning Of Large ONA Fragments, Gene 11 (3-4),

291-98

The thyme is obtained from a slaughterhouse and the DNA is isolated by methods well known in the art. Maniatis T. et al., Molecular Cloning: A Laboratory Manual, p.

Cold Spring Harbor Laboratory (Publisher) (1982). The cosmid library is constructed using standard techniques. F. Grosveld, F., et al., Isolation Of Beta-Globin-Related Genes From The Human Cosmid Library, Gene 13 (3), 227-31 (1981). Calf thyme DNA was partially digested with Sau3A (New England BioLabs) and run on a NaCl gradient (1 mol / l to 5 mol / l) to enrich the 30 to 40 kb fragments. The partially digested DNA fragments are then ligated into Bam HI digested cosmid vector HC79 followed by in vitro packaging with lambda extracts (Amersham Corporation, Arlington Heights, Illinois) according to the manufacturer's instructions. The in vitro packaged material is then used to infect E. coli strain K-12 HB101. Integrated clones of this vector were selected by growing on LB plates containing 50 µg / ml ampicillin (Sigma Chemical Co., St. Louis, Missouri).

The resulting library was screened using a 45 base pair oligonucleotide probe, CAS-1. This CAS-1 sequence (5'-ATGGCTTGATCTTCAGTTGATTCACTCCCAATATCCTTGCTCAG3 ') is provided by alpha S-1 casein Willis I.M. et al. (1982), 375-86, based on the sequence of the partial cDNA described in Part et al., et al. (4), 375-86. This sequence is shown on pages 20-35 of mature bovine casein. corresponding to its amino acids. As a result of this screening, three clones (C9, D4 and E1) containing cosmids were isolated.

The 5 'and 3' flanking sequences were obtained from cosmid clones E1 and C9. Restriction mapping and Southern blot analysis: Southern E. Detection Of Specific Sequences Among DNA Fragments Separated By Gel Electrophoresis, 0. Mole. Biol. 98 (3), 503-517 (1975)] using oligonucleotide probes corresponding to known sequence regions of the casein cDNA. Stewart A.F. et al., Nucleotide Sequences Of Bovine Alpha Sl-And Kappa-Casein cDNAs, Nucleic Acids Slit. 12 (9): 3895-3907 (1984); Nagao, M., et al., Isolation and Sequence Analysis of Bovine Alpha Sl-Casein cDNA Clone, Agric. Biol. Chem. 48 (6), 1663-67 (1984) 3, states that the cosmids D4 and E1 contain a portion of the casein structural gene (i.e., the DNA sequence encoding the casein protein), and contain 21 kb of upstream or 5 'flanking sequences. Cosmid C9 contains a portion of the casein structural gene and extends approximately 7 kb downstream of the polyadenylation site. Sequence analysis of the cosmids E1 and D4 is carried out in the region corresponding to the transcription start of the casein structural sequence and determined that the sequence corresponds to the published sequence for the same region. Yu-Lee L, Y. et al .: Evolution Of The Casein Multigene Family · Conserved Sequences In The 5 'Flanking And Exon Regions, Nucleic Acid Slit. 14 (4): 1883-1902 (1986)].

The construction of the expression island of the present invention is shown in Figure 2. From cosmid C9, the 9 kb BamHI fragment is subcloned which starts at the BamHI site within the intron following the 98th amino acid residue of alpha casein and the alpha casein polyadd.

* »················· · · ·

- Continues from 24 nil signal to 2 kb downstream to another BamHI site. This fragment is designated c-term in Figure 2. This 9 kb fragment was cloned into BamHI-cleaved pUC19 to give pCAS 947. The downstream BamHI site was converted to SalI site by partial digestion of pCAS 947 with BamHI and ligated with a SalI linker, CAS 10, having the sequence 5'-GATCGTCGAC-3 '. The resulting plasmid is called pCAS 1238. This 9 kb BamHI-SalI fragment is used as the 3 'flanking sequence of the island. It contains the 3'-untranslated region and the 3 'expression control sequences, as well as a portion of the structural gene derived from alpha S-1 casein.

The next step is to design the 5 'flanking area. This region, which contains the transcription start site, also contains a non-coding exon and a second exon, some of which is also non-coding. A 4 kb SmaI / BamHI fragment was isolated from cosmid E1 and subcloned into BamHI and SmaI cleaved pUC19; This gives pCAS 2276. This plasmid was cleaved with BglII to remove the coding portion of the second exon, and the BglII site was converted to a BamHI site by ligation to a CAS 12 linker having the sequence 5 '-GATCTTGGATCCAA-3'. The resulting plasmid, pCAS 1181, was then digested with SmaI and BamHI to remove a 3 kb fragment of cosmid E1. The fragment was isolated, ligated to the 9 kb BamHI-SalI fragment from pCAS 1238 and inserted into SmaI and SalI digested pUC19 to give pCAS 1276.

The resulting construct links the transcription start site to the downstream genomic sequence; among them is a unique BamHI cloning site into which the tilting sequence encoding the heterologous polypeptide can be inserted. Because the final constructs contain several other BamHI sites in the genomic sequences, the sequence cloning site encoding the heterologous polypeptide must be changed to both the XhoI site and the NotI site by the addition of the CAS 30 linker sequence 5'-GATCTCGAGCGCGGC CGCGCT-3 '. The resulting vector, pCAS 1277, contains XhoI and NotI sites as cloning sites, including the transcription starter of alpha casein and the C-terminal genomic portion of alpha casein.

The transcription start and c-term from pCAS 1277 are then used to replace appropriate portions of the alpha casein genomic sequence in cosmid E1. Because the construct is 39 kb in length, the cosmid technique is used to manipulate plasmids. The original cosmid E1 was partially digested with Xmal and then completely digested with SalI to remove most of the 3 'portion of the alpha casein gene in the cosmid. The Smal and Xmal enzymes share the same recognition site, except that Xmal leaves a 5 'overhang while Smal leaves a blunt end. The 12 kb Xmal-SalI fragment from pCAS 1277 is then inserted into the XmaI and SalI-cleaved cosmids to replace the deleted portion.

The ligated products are then subjected to in vitro packaging using an in vitro packaging kit (Amersham Corporation), and the packaged DNA is used to infect E. coli DH5 cells; followed by selection on LB plates containing 50 µg / ml ampicillin (Sigma Chemical Co.). Plasmids from ampicillin-resistant colonies were screened using specific probes for the 3 'end of casein. We identify and • · · · «♦ • · · i ····« · «···

- 26 features plasmids containing 21 kb upstream of the transcription start and containing the XhoI / NotI cloning site along with the genomic 3 'end of the casein gene. One of these plasmids, CAS 1288, is then used to express the heterologous DNA sequence.

The genomic clone of human urokinase is isolated from a genomic library using probe sequences (see Riccio A., cited above). From the disclosed sequence, it can be seen that there is an Apai site upstream of the translation start site of the gene and downstream of the polyA transcriptional signal. Oligonucleotide adapters (URO 8 having 5'-CGT CGACG-3 'and URI 9 having 5'-GTACCGTCGACCGGCC-3') were used to add SalI sites to these two flanking Apa sites. This allows the genomic clone to be inserted downstream of the SV40 early promoter into an animal cell expression vector so that expression can be examined before insertion into the alpha casein expression island. The resulting plasmid, pUK0409, directs the expression of authentic human urokinase in transfected tissue culture cells. We therefore know that the genomic clone is functional. The next step is to insert the urokinase genomic clone into the CAS 1288 Xhol cloning site. These steps are illustrated in Figure 3.

The urokinase genomic clone was isolated as an 8 kb Sal I fragment from pUK0409. The protruding ends can ligate to the Xhol cloning site found in CAS 1288. However, there is another Xhol site in the 21 kb upstream areas of alpha casein. Mj is therefore subjected to partial Xhol digestion followed by ligation with the isolated SalI urokinase fragment (see Figure 3). Plasmids were isolated from the colonies and screened for the presence of the urokinase DNA sequence ························································ · and orientation. One of these plasmids, CAS 1295, contains the urokinase gene in the correct orientation, as determined by restriction analysis. This plasmid contains, in the 5 'to 3' orientation: the 21 kb downstream region, the first non-coding exon and intron sequences of the casein, the genomic sequence encoding the urokinase, and the 9 kb 3 'genomic alpha. casein area.

EXAMPLE 2 TRANSGENIC INSTALLATION OF EXPRESSION ISLAND CONSTRUCTIONS IN MICE

For the purpose of performing transgenic experiments, the prokaryotic vector sequences present in CAS 1295 are removed prior to injection into embryos. This is accomplished by digesting CAS 1295 with Clal and SalI and then gel electrophoresis on 1¾ agarose TBE (see Maniatis, supra). The 41 kb fragment corresponding to the eukaryotic sequences of the expression island construct was excised from the gel and the DNA was purified by electroelution. The DNA was then centrifuged overnight on a balanced CsCl gradient. The DNA was removed from the gradient and vigorously dialyzed against TNE buffer at 5 mM Tris pH 7.4, 5 mM NaCl and 0.1 mM EDTA pH 8.0.

The process of transgenic incorporation of the desired genetic information into a developing mouse embryo is already well established in the art. The procedure of B. Hogan et al., J. Manipulating The Mouse Embryo, Cold Spring Harbor Laboratory Manual (1986) J. The FI generation (Sloan Kettering) of the cross between C57B1 and CB6 mice (Jackson Laboratories) was used. Six-week-old females were superovulated by injection of Gestile (pregnant mare serum), which was injected with human chorionic ganadotropin 2 days later, ························• follow. Treated females were mated with C57B1 breeding males 24 hours later. Already fertilized embryos are removed within 12 hours of mating for microinjection with DNA and for implantation in pregnant females.

After embryo isolation, the cumulus cells surrounding the ovum are first digested with hyaluronidase. The expression island construct is then injected into the pronucleus of the embryo until it is swollen by 30-50% of its size. The injected embryos are then implanted into the fallopian tube of pregnant F1 female. The ONS from the tail of the resulting live offspring was screened using probe nick-translated CAS 1295 DNA to identify the animals carrying the expression island construct. Three transgenic animals were identified. These animals are mated and the offspring examined for the presence of the expression island construct as described above.

One of the transgenic lines carrying 2-3 copies of the expression island construct carries the genetic material according to Mendelian rules. The females of this transgenic line carrying the CAS 1295 insert all produce human urokinase in their milk at approximately 1 mg / ml as determined by enzymatic assay. Urokinase is inhibited by monoclonal antibody 394 which is specific for human urokinase (Americana Diagnostica, Inc., New York, New York).

The other two transgenic lines carry 20-50 copies of the construct but do not pass ONS to the next generation of mice. We believe that high copy number lines are unable to transmit these genes because of the fact that during embryogenesis, ··· · 99 r · · 49 • · 9 9 · 9 ··

9 9 ··· 999 · 9

9 9 9 9 9 · 999 High baseline urokinase. Urokinase is normally expressed in embryonic tissues (embryonic stem cells) and functions during development. Low baseline urokinase expression from casein expression control sequences does not interfere with development in embryos that inherit two copies of the gene. However, if the expression is dependent on copy number, lines with 20-50 copies have a baseline of 20-50 times higher and therefore express enough urokinase to interfere with proper development. These results indicate that / expressed urokinase levels are dependent on copy number.

EXAMPLE 3 REFLECTING THE EXPRESSION ISLAND CONSTRUCTION IN ANIMAL CELLS

The expression island construct and the pSV2-DHFR selectable marker available at American Type CultureCollection (ATCC 37146) J, which codes for the production of dihydrofolate reductase in mammalian cells, are co-introduced into DHFR CHO cells by electroporation. This technique was chosen because it is capable of producing host cells characterized by high copy number of stably integrated foreign DNA. EP 0 343 783 describes this technique in detail; this application is incorporated herein by reference.

Prior to electroporation, plasmid pSV2-DHFR was linearized by digestion with AatlI overnight. The expression island sequences were isolated from the vector sequences by restriction digestion as described in Example 2, followed by gel electrophoresis to allow isolation and isolation (see Maniatis, cited above). Salmon sperm DNA (200 µg / tg) previously digested into 300-1000 bp fragments by sonication is added to the mixture of 200 µg / g of linearized pSV2-DHFR and

Contains 0.5 mg / ml expression island construct. In order to precipitate the DNA, NaCl was added to the mixture to a final concentration of 0.1 M. Subsequently, 2.5 volumes of ethanol were added and the mixture was incubated on dry ice for 10 minutes. After centrifugation at 4 ° C for 10 minutes, ethanol is aspirated and the DNA pellet is air-dried for 15 minutes in a tissue culture vessel. The DNA pellet was then resuspended in 800 µl HeBS buffer E20 mM Hepes / NaOH pH 7.05; 137 mM NaCl; 5 mM KCl; 0.7 mmol / lΝΝΗΡΗΡ ^ ^; 6 mM glucose) at least 2 hours before electroporation.

About 2.10 ⁷ DHFR ^- CHO cells are subcloned from Qa clone CH0-DUKX-B1 described by Urlaub and Charis in Isolation of Chinese Hamster Cell Mutants Deficient In Dihydrofolate Reductase Activity, Proc. Natl. Acad. Sci.USA

77, 4216 are applicable to each electroporation. THE

DHFR ^- CHO cells were passaged three days prior to electroporation and at the time of harvest for electroporation, the culture was confluent at about 50% on 10 cm plates. DHFR CHO cells were removed from the plates by trypsin treatment and then 8.0 ml <Z of trypsin per plate. medium supplemented with 1 M MEM alpha supplemented with ribonucleotides and deoxyribonucleotides (10-10 ng / L adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxyguanosine and 2'-deoxythymidine; 11 ng / L 2'-deoxycytidine hydrochloride (Gibco Laboratories, Grand Island, New York)] _; It is inactivated by the addition of 10¾ calf embryo serum (Házelton, Lenexa, KS) and 4 mM glutamine (MA Bioproducts, Walkersville, Maryland). The cells isolated from the plates are then harvested31: '··: * ···

♦ · .. ·· · and centrifuged at 1000 rpm for 4 minutes. Most of the medium is aspirated from the cell pellet and resuspended in the remaining medium by plating.

The expression island, pSV2-DHFR, as well as the tubular coccoglazers' sperm DNA, suspended in 800 µl x HeBS, are then added to the DHFR CHO cell suspension. The resulting mixture is immediately transferred to an electroporation cuvette. Set it on the electroporation device.

rpm, its capacitor is 960 µF, and its voltage is 300 µg individual stroke, which is added to about 10 milliliters of the contents of the cuvette at room temperature.

The cells are then incubated for 8-10 minutes at room temperature and transferred to a tube containing 14 ml of cL ⁺ medium. The cells are centrifuged as above. After the medium was aspirated, the wet cell pellet was resuspended by jogging the tube and then fresh * Á ⁺ medium was added. The suspended cells are then seeded on culture plates for 2 days in non-selective medium to allow for the recovery of cells after electroporation and expression of the selective gene. About 20-30 'of viable CHO whole cells are believed to have incorporated the expression island / pSV2-DHFR and thus survived the selection workflow. Therefore, a 10 cm plate of about 1.10 ^ cells were seeded into and cultured at 37 ° C, containing ₂ per inkubáto'rban 5,5¾ co.

After a two-day recovery period, cells are removed from the stem plate by trypsin treatment as described above, counted and inoculated into 6 x 10 cm plates.

1.10 cells / plate in << medium (Sigma Chemical Co.).

Cells containing the expression island and pSV2-DHFR are selected after incubation for 4 days in oC medium. The Winds • · »

Tested cells are then assayed for urokinase expression by standard techniques, e.g. with a commercially available Spectrozyme Colorimetric Assay Kit (Americana Diagnostica, Inc.)

Numerous clones have been isolated which express urokinase at different levels. DNA and RNA were isolated from these clones and Northern and Southern analysis was performed to determine the transcriptional level and copy number of the expression island construct. This analysis reveals whether the expression of the urokinase message is dependent on copy number and independent of the site of integration of the integrated construct.

An embodiment of the present invention comprising plasmid CAS 1288 is exemplified by a culture deposited with In Vitro International, Inc. (Linthicum, Maryland) on February 1, 1990; this identifies CAS 1288 and contains plasmid CAS 1288 in E. coli DH5. It is deposited under IVI 10232. It: <x strain kc / 'tl a L hi ^ ez-tık, .vz í \ ^ cΙ'ΐί-Λπ CliILmi'c í c? ílect lu í'i - ₍ 4 / ho ¹ of Alii Kill £ ·

A second construct of the present invention comprising the plasmid CAS 1295 is exemplified by a culture deposited with In Vitro International, Inc. (Linthicum, Maryland) on February 1, 1990; this plasmid CAS 1295 is in E. coli DH5. Its deposit number is IVI 10231> / 7i; <λ lake>.> / lyfí: ίζΙτζίΎ ( _v ; ij 'Ili :, t ά' A h ó 'l û''\ j í * - O í í t' s Z. - ^Ί ? tk iA j · í

Although a number of embodiments of the present invention have been described herein, it is obvious that our basic construction is variable. Thus, other embodiments may be provided that employ the procedures and compositions of the present invention. Thus, the scope of the invention is not limited to the specific embodiments shown in the examples, but to the appended claims.

Claims (13)

PATENT CLAIMS 1.) A method of producing high levels of a desired polypeptide in a host comprising: i. ) integrating at least one expression island into said host genome, wherein said expression island 5 'to 3' comprises a 5 'flanking region, a heterologous polypeptide coding sequence and a 3' flanking region; said 5 'flanking region comprising a 5' expression control sequence operably linked to a coding sequence for said heterologous polypeptide and a 5 'untranslated region; said 3 'flanking region comprising a 3' untranslated region and a 3 'expression control sequence operably linked to said heterologous polypeptide coding sequence; and the flanking regions 5 'and 3' of said expression island are sized and structured to efficiently depend on the production of the desired heterologous polypeptide for copy number integrated into the host genome and substantially independent of the expression site integration site in the host genome; and ii. ) culturing said host under conditions that allow expression of the desired heterologous polypeptide. 2. The method of claim 1, wherein said heterologous polypeptide coding sequence further comprises a coding region for a functional signal sequence. 3. The method of claim 2, wherein said region encoding said signal sequence is derived from a milk-specific protein gene. • 4 «· • * 4 4 · • 4 ·· • 4 44 4. The method of claim 3, wherein the milk-specific protein gene is a casein gene. 5.) In steps 2-4. The method according to any one of claims 1 to 4, wherein the host is a dairy mammal, selected from the group consisting of mice, cattle, sheep, goats and pigs; and the 5 'and 3' flanking sequences are derived from a milk-specific protein gene. 6. The method of claim 1, wherein the heterologous polypeptide coding sequence is one of the following polypeptides: tPA, urokinase, Mullerian Inhibiting Substance, interferon, VIII. and IX. clotting factor, animal growth factor, insulin, an interleukin, an immunoglobulin and a lipocortin. 7.) An expression island comprising a 5 'flanking region, a coding sequence for a heterologous polypeptide and a 3' flanking region in the 5 'to 3' direction; said 5 'flanking region comprising a 5' expression control sequence operably linked to a coding sequence for said heterologous polypeptide and a 5 'untranslated region; said 3 'flanking region comprising a 3' untranslated region and a 3 'expression control sequence operably linked to said heterologous polypeptide; a coding sequence; and the 5 'and 3' flanking regions of said expression island are sized and structured to efficiently depend on the production of the desired heterologous polypeptide for copy number integrated into the host genome and substantially independent of the expression site integration site in the host genome. ··· «·· ·· 8. The expression island of claim 7, wherein said heterologous polypeptide coding sequence further comprises a coding region for a functional signal sequence. Expression island according to claim 7, characterized in that the 5 'and 3' flanking sequences are derived from a milk-specific protein gene. 10. The expression island of claim 8, wherein the coding region for said signal sequence is derived from a milk-specific protein gene. 11. The expression island of claim 9, wherein the milk-specific protein gene is a casein gene. 12. The expression island of claim 7, wherein the heterologous polypeptide encoding sequence is one of the following polypeptides: tPA, urokinase, Mullerian Inhibiting Substance, interferon, VIII. and IX. clotting factor, animal growth factor, insulin, an interleukin, an immunoglobulin and a lipocortin. 13.) A transformed host, characterized in that its genome has a sequence from 7 to 12. An integrated expression island according to any one of claims 1 to 6.