METHOD OF MAKING A PROTEASE DEFICIENT HOST
Detailed Description ofthe Preferred Embodiments The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All published work, including patents, and patent applications cited herein are hereby incorporated by reference.
The invention can be better understood in light ofthe following definitions incoφorated herein.
Definitions
A "nucleic acid molecule" or a "coding sequence," as used herein, refers to either RNA or DNA that encodes a specific amino acid sequence or its complementary strand. The term "an expression control sequence" or "regulatory sequence" refers to a sequence that is conventionally used to effect expression of a gene that encodes a polypeptide and includes one or more components that affect expression, including transcription and translation signals. Such a sequence includes, for example, one or more ofthe following: a promoter sequence, an enhancer sequence, an upstream activation sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation in mammalian cells, and a Shine-
Dalgarno sequence. In eukaryotes, for example, such a sequence can include a promoter sequence, and a transcription termination sequence. If any necessary component of an expression control sequence is lacking in the nucleic acid molecule ofthe present invention, such a component can be supplied by the expression vector to effect expression. Expression control sequences suitable for use herein may be derived from a eukaryotic source, or a virus or viral vector or from a linear or circular plasmid.
The term "leader sequence" refers to either a translated amino acid sequence situated 5' to the N-terminus ofa polypeptide sequence to be expressed, or an untranslated nucleotide sequence. This term includes at least one ofthe following and can be a combination thereof: a secretion leader sequence, as defined below, a fusion protein leader sequence, and an untranslated nucleotide sequence. The translated amino acid leader sequence can be used herein to optimize secretion, as in a secretion leader
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promoters selected for use herein, or can be a synthetic sequence, or partly synthetic or partly derived.
The promoters suitable for use herein can be any promoter, including those that are constitutively active or those that are inducible or regulatable. The promoters can be naturally derived or synthetically made. They can be derived from any genes, viral, prokaryotic or eukaryotic. The eukaryotic genes can be yeast or other fungal, insect, mammalian or avian genes. Examples of suitable promoters are described below in the portion relating to expression systems.
Although the methodology described below is believed to contain sufficient details to enable one skilled in the art to practice the present invention, other items not specifically exemplified, such as plasmids, can be constructed and purified using standard recombinant DNA techniques described in, for example, Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2d edition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), and Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1994), (Greene Publishing Associates and John Wiley & Sons, New York, N.Y ). under the current regulations described in United States Dept. of HEW, NATIONAL INSTITUTE OF HEALTH (NIH) GUIDELINES FOR RECOMBINANT DNA RESEARCH. These references include procedures for the following standard methods: cloning procedures with plasmids, transformation of host cells, cell culture, plasmid DNA purification, phenol extraction of DNA, ethanol precipitation of DNA, agarose gel electrophoresis, purification of DNA fragments from agarose gels, and restriction endonuclease and other DNA-modifying enzyme reactions.
Details of specific host systems are described below. The non-yeast systems may be useful for contructing the defective or mutated promoters or defective or mutated genes which are then transformed in yeast hosts. In addition to an application for constructing the defective or mutated promoters or genes, the yeast host systems are also applicable to the method of the invention as host systems that can be rendered protease deficient. Expression in Bacterial Cells
Control elements for use in bacteria include promoters, optionally containing operator sequences, and ribosome binding sites. Useful promoters include sequences
virus A, human preproinsulin, and bovine growth hormone, among others. Further details regarding secretion leader sequences are provided below.
A "regulatory sequence" refers to a nucleic acid sequence encoding one or more elements that are capable of affecting or effecting expression ofa gene sequence, including transcription or translation thereof, when the gene sequence is placed in such a position as to subject it to the control thereof. Such a regulatory sequence can be, for example, a minimal promoter sequence, a complete promoter sequence, an enhancer sequence, an upstream activation sequence ("UAS"), an operator sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 51 leader sequence to optimize initiation of translation, and a Shine-Dalgarno sequence. Alternatively, the regulatory sequence can contain a combination enhancer/promoter element. The regulatory sequence that is appropriate for expression ofthe gene of interest differs depending upon the host system in which the construct is to be expressed. Selection of the appropriate regulatory sequences for use herein is within the capability of one skilled in the art. In eukaryotes, for example, such a sequence can include one or more ofa promoter sequence and/or a transcription termination sequence. If any necessary component ofa regulatory sequence that is needed for expression is lacking in the collision construct, such a component can be supplied by a vector into which the collision construct can be inserted for transformation or reintroduction into a host cell. Regulatory sequences suitable for use herein may be derived from any source including a prokaryotic source, an eukaryotic source, a virus, a viral vector, a bacteriophage or a linear or circular plasmid. The regulatory sequence herein can also be a synthetic sequence, for example, one made by combining the UAS of one gene with the remainder of a requisite promoter from another gene, such as the GADP/ADH2 hybrid promoter. A selectable marker gene is a gene which confers resistance to the cell that expresses it. Selectable markers useful for the method ofthe invention include gentamycin that confers resistance to the antibiotic G418.
The regulatory sequences suitable for use herein can be any regulatory sequence that is compatible for use with the promoters for expression in a desired host cell. For example, for expression in yeast, a regulatory sequence derived from yeast systems would be desirable. The regulatory sequence can be a sequence naturally associated with the
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Prokaryotic cells used to produce the target polypeptide of this invention are cultured in suitable media, as described generally in Sambrook et al., cited above.
Expression in Yeast Cells Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, among others, the following yeasts: Saccharomyces cerevisiae ,as described in Hinnen et al., Proc. Natl. Acad. Sci.
USA (1978) 75: 1929; Ito et al, J. BacterioL (1983) 153: 163; Candida albicans as described in Kurtz et al, Mol. Cell. Biol. (1986) 6: 142; Candida maltosa, as described in Kunze et al., J. Basic Microbiol. (1985) 25: 141; Hansenula polymorpha, as described in Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459 and Roggenkamp et al., Mol. Gen. Genet. (1986) 202 :302); Kluyveromyces fragilis, as described in Das et al., J. BacterioL (1984) 158: 1165; Kluyveromyces lactis, as described in De Louvencourt et a , J. BacterioL (1983) 154: 131 and Van den Berg et aL,
Bio Technology (1990) 8: 135; Pichia guillerimondii, as described in Kunze et al., J.
Basic Microbiol. (1985) 25: 141; Pichia pastoris, as described in Cregg et al., Mol.
Cell. Biol. (1985) 5: 3376 and U.S. Patent Nos. 4,837,148 and 4,929,555;
Schizosaccharomyces pombe, as described in Beach and Nurse, Nature (1981) 300: 706; and Yarrowia lipolytica, as described in Davidow et al., Curr. Genet. (1985) 10:
380 and Gaillardin et al., Curr. Genet. (1985) 10: 49, Aspergillus hosts such as A. nidulans, as described in Ballance et aL, Biochem. Biophys. Res. Commun. (1983)
112: 284-289; Tilburn et al., Gene (1983) 26: 205-221 and Yelton et al., Proc. Natl.
Acad. Sci. USA (1984) 81 1470-1474, and A. niger, as described in Kelly and Hynes, EMBO J. (1985) 4: 475479; Trichoderma reesia, as described in EP 244,234, and filamentous fungi such as, e.g, Neurospora, Penicillium, Tolypocladium, as described in WO 91/00357.
Control sequences for yeast vectors are known and include promoters regions from genes such as alcohol dehydrogenase (ADH), as described in EP 284,044, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate- dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3- phosphoglycerate mutase, and pyruvate kinase (PyK), as described in EP 329,203.
derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the β-lactamase (bla) promoter system, bacteriophage λPL, and T7. In addition, synthetic promoters can be used, such as the tac promoter. The β-lactamase and lactose promoter systems are described in Chang et al., Nature (1978) 275: 615, and Goeddel et al., Nature (1979) 281: 544; the alkaline phosphatase, tryptophan (tφ) promoter system are described in Goeddel et al. , Nucleic Acids Res. (1980) 8: 4057 and EP 36,776 and hybrid promoters such as the tac promoter is described in U.S. Patent No. 4,551,433 and deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25. However, other known bacterial promoters useful for expression of eukaryotic proteins are also suitable. A person skilled in the art would be able to operably ligate such promoters to the coding sequences of interest, for example, as described in Siebenlist et aL, Cell (1980) 20: 269, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also generally will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the target polypeptide. For prokaryotic host cells that do not recognize and process the native target polypeptide signal sequence, the signal sequence can be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria. The foregoing systems are particularly compatible with Escherichia coli. However, numerous other systems for use in bacterial hosts including Gram-negative or Gram-positive organisms such as Bacillus spp. , Streptococcus spp. , Streptomyces spp., Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescans, among others. Methods for introducing exogenous DNA into these hosts typically include the use of CaCl2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al. (1989), cited above. These examples are illustrative rather than limiting. Preferably, the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro methods of cloning, e.g. , PCR or other nucleic acid polymerase reactions, are suitable.
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U.S. Patent Nos. 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO 89/02463. Alternatively, leaders of non-yeast origin, such as an interferon leader, also provide for secretion in yeast, as described in EP 060,057.
Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.
Transformations into yeast can be carried out according to the method described in Van Solingen et al., J. Bact. (1977) 130:946 and Hsiao et al., Proc. Natl. Acad. Sci. (USA) (1979) 76:3829. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used as described generally in Sambrook et al., cited above.
For yeast secretion the native target polypeptide signal sequence may be substituted by the yeast invertase, α-factor, or acid phosphatase leaders. The origin of replication from the 2μ plasmid origin is suitable for yeast. A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid described in Kingsman et al., Gene (1979) 7: 141 or Tschemper et aL, Gene (1980) i0:157. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 Gene. For intracellular production of the present polypeptides in yeast, a sequence encoding a yeast protein can be linked to a coding sequence of the ob polypeptide to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression. An example, of such a yeast leader sequence is the yeast ubiquitin gene.
Expression in Insect Cells
Baculovirus expression vectors (BEVs) are recombinant insect viruses in which the coding sequence for a foreign gene to be expressed is inserted behind a baculovirus promoter in place of a viral gene, e.g., polyhedrin, as described in Smith and Summers, U.S. Pat. No. , 4,745,051. An expression construct herein includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, optionally but
The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences, as described in Myanohara et aL, Proc. Natl. Acad. Sci. USA (1983) 80: 1. Other suitable promoter sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase, as described in Hitzeman eta , J. Biol. Chem. (1980) 255: 2073, or other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase, as described in Hess et al., J. Adv. Enzyme Reg. (1968) 7_ 149 and Holland et aL, Biochemistry (1978) 77:4900. Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions, include from the list above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP 073,657. Yeast enhancers also are advantageously used with yeast promoters. In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Nos. 4,876,197 and 4,880,734. Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GALIO, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 164,556. Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
Other control elements which may be included in the yeast expression vectors are terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al., J. Biol. Chem. (1981) 256: 1385, and leader sequences which encode signal sequences for secretion. DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene as described in EP 012,873 and JP 62,096,086 and the α-factor gene, as described in
al., Mol. Cell. Biol. (1988) 8: 3129; human IL-2, as described in Smith et aL, Proc. Natl. Acad. Sci. USA (1985) 52:8404; mouse IL-3, as described in Miyajima et aL, Gene (1987) 58:213; and human glucocerebrosidase, as described in Martin et aL, DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (cateφillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori host cells have been identified and can be used herein. See, for example, the description in Luckow et aL,
6: 47-55, Miller et al., in GENETIC ENGINEERING (Setlow, J.K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al., Nature, (1985) 315 592-594. A variety of such viral strains are publicly available, e.g. , the L-l variant oi Autographa californica NPV and the Bm-5 strain oi Bombyx mori NPV. Such viruses may be used as the virus for transfection of host cells such as Spodoptera frugiperda cells. Other baculovirus genes in addition to the polyhedrin promoter may be employed to advantage in a baculovirus expression system. These include immediate-early (alpha), delayed-early (beta), late (gamma), or very late (delta), according to the phase of the viral infection during which they are expressed. The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes. Some delayed-early gene products, in turn, induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes. One relatively well defined component of this regulatory cascade is IEI, a preferred immediate-early gene of Autographo californica nuclear polyhedrosis virus (AcMNPV). IEI is pressed in the absence of other viral functions and encodes a product that stimulates the transcnption of several genes of the delayed-early class, including the preferred 39K gene, as described in Guarino and Summers, J. Virol. (1986) 57:563-571 and J. Virol. (1987) 67:2091-2099 as well as late genes, as described in Guanno and Summers, Virol. (1988) 162:444-451.
preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a desired protein, and a site for insertion of the foreign gene encoding the foreign protein, the signal DNA sequence and the foreign gene being placed under the transcriptional control of a baculovirus promoter, the foreign gene herein being the coding sequence of the ob polypeptide.
The promoter for use herein can be a baculovirus transcriptional promoter region derived from any of the over 500 baculoviruses generally infecting insects, such as, for example, the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo californica MNPV, Bombyx mori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV, Aedes aegypti, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. Thus, the baculovirus transcriptional promoter can be, for example, a baculovirus immediate-early gene IEI or IEN promoter; an immediate-early gene in combination with a baculovirus delayed-early gene promoter region selected from the group consisting of a 39K and a HmdIII fragment containing a delayed-early gene; or a baculovirus late gene promoter. The immediate-early or delayed-early promoters can be enhanced with transcriptional enhancer elements.
Particularly suitable for use herein is the strong polyhedrin promoter of the baculovirus, which directs a high level of expression of a DNA insert, as described in Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression" in: THE
MOLECULAR BIOLOGY OF BACULOVIRUSES (W.Doerfler, ed.); EP 127,839 and EP 155,476; and the promoter from the gene encoding the plO protein, as described in Vlak et al., J. Gen. Virol. (1988) 69:765-776.
The plasmid for use herein usually also contains the polyhedrin polyadenylation signal, as described in Miller et al., Ann. Rev. Microbiol. (1988) 42: 111 and a procaryotic ampicillin-resistance (amp) gene and an origin of replication for selection and propagation in E. coli. DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene, as described in Carbonell et al., Gene (1988) 75:409, as well as mammalian signal sequences such as those derived from genes encoding human α-interferon as described in Maeda et aL, Nature (1985) 375:592-594; human gastrin-releasing peptide, as described in Lebacq-Verheyden et
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expression of secretory glycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely processed form.
While it has been recognized that an insect signal sequence can be used to express a foreign protein that can be cleaved to produce a mature protein, the present invention is preferably practiced with a mammalian signal sequence appropriate for the gene expressed.
An exemplary insect signal sequence suitable herein is the sequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide. The AKH family consists of short blocked neuropeptides that regulate energy substrate mobilization and metabolism in insects. In a preferred embodiment, a DNA sequence coding for a Lepidopteran Manduca sexta AKH signal peptide can be used. Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can also be employed to advantage. Another exemplary insect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPl, CP2, CP3 or CP4.
Currently, the most commonly used transfer vector that can be used herein for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, can also be used herein. Materials and methods for baculovirus/insect cell expression systems are commercially available in a kit form from companies such as Invitrogen (San Diego CA) ("MaxBac" kit). The techniques utilized herein are generally known to those skilled in the art and are fully described in Summers and Smith, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experiment Station Bulletin No. 1555, Texas A&M University (1987); Smith et al., Mol. Cell. BioL (1983) 3: 2156, and Luckow and Summers (1989). These include, for example, the use of pVL985 which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHl cloning site 32 basepairs downstream from the ATT, as described in Luckow and Summers, Virology (1989) 77:31.
Thus, for example, for insect cell expression of the present polypeptides, the desired DNA sequence can be inserted into the transfer vector, using known techniques. An insect cell host can be cotransformed with the transfer vector containing the inserted desired DNA together with the genomic DNA of wild type
Immediate-early genes as described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes. The combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the HmdIII fragment of the baculovirus genome. In the present instance, the 39 K promoter region can be linked to the foreign gene to be expressed such that expression can be further controlled by the presence of IEI, as described in L. A. Guarino and Summers (1986a), cited above; Guarino & Summers (1986b) J. Virol., (1986) 60:215-223, and Guarino et al. (1986c), J. Virol. (1986) 60:224-229.
Additionally, when a combination of immediate-early genes with a delayed-early gene promoter region is used, enhancement of the expression of heterologous genes can be realized by the prescence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region. Such enhancer sequences are characterized by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited. For example, the hr5 enhancer sequence can be linked directly, in cis, to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA as described in Guarino and Summers (1986a), (1986b), and Guarino et al. (1986).
The polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this delayed expression of the polyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers in, for example, U.S. Pat. No., 4,745,051 will express foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculovirus infection progresses. Thus, gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is potentially diminished for certain proteins such as human tissue plasminogen activator. As a consequence, the
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described in Boshart et al., Cell (1985) 41:521. A leader sequence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the gene of interest such that the leader sequence can be cleaved either in vivo or in vitro. The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
Once complete, me mammalian expression vectors can be used to transform any of several mammalian cells. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Patent No. 4,399,216.
The method of the invention is practiced as follows. The protease to disable is determined. Thus, for example, where a Pichia host is used, Pep4, Protease A and Protease B are candidates for disablement. So that, for example, where a host with a disabled Pep4 is desired, the promoter or gene sequences of Pep4 are identified, cloned and placed in plasmids. Plasmids suitable for this process are described herein. The method of the invention can be practiced using either the gene for the protease or its promoter. In either case, the method is basically the same. For example, where the goal is to disable the promoter for Pep4, the 5' and 3' end of the promoter remains intact, or sequences 5' and 3' of the promoter can also be used. Either the middle region of the promoter is replaced by an integrating event or the entire promoter can be replaced. The replacement can be made with a selectable marker such as, for example, a gene that conveys G418 resistance to host cells, and regulatory sequences for that gene. The promoter construct is cleaved from its plasmid and transformed into a Pichia host as described in Rothstein, (1991) Methods in Enzymoiogy, v. 194 p. 281-301, herein incoφorated by reference. The Pichia host cells are selected by resistance to G418.
Alternatively, the gene of interest, for example IGF-I, or any other gene of interest, such as those described herein, can be constructed for integration into the gene
baculovirus, usually by cotransfection. The vector and viral genome are allowed to recombine resulting in a recombinant virus that can be easily identified and purified. The packaged recombinant virus can be used to infect insect host cells to express a desired polypeptide. Other methods that are applicable herein are the standard methods of insect cell culture, cotransfection and preparation of plasmids are set forth in Summers and Smith (1987), cited above. This reference also pertains to the standard methods of cloning genes into AcMNPV transfer vectors, plasmid DNA isolation, transferring genes into the AcmMNPV genome, viral DNA purification, radiolabeling recombinant proteins and preparation of insect cell culture media. The procedure for the cultivation of viruses and cells are described in Volkman and Summers, J. Virol. (1975) 79:820-832 and Volkman, aL, J. Virol. ( 1976) 79:820-832.
Expression in Mammalian Cells Typical promoters for mammalian cell expression of the polypeptides of the invention include the SV40 early promoter, the CMV promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the heφes simplex virus promoter, among others. Other non-viral promoters, such as a promoter derived from the murine metallothionein gene, will also find use in mammalian constructs. Mammalian expression may be either constitutive or regulated (inducible), depending on the promoter. Typically, transcription termination and polyadenylation sequences will also be present, located 3* to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the ob polypeptide coding sequence, is also present. Examples of transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al. (1989), cited previously. Introns, containing splice donor and acceptor sites, may also be designed into the constructs of the present invention.
Enhancer elements can also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al.,, EMBO J. (1985) 4:761 and the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and human cytomegalovirus, as
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WHAT IS CLAIMED IS:
1. A cell comprising a deficient protease activity, wherein the deficiency is not a result ofa defective protease structural gene.
2. The cell of claim 1, wherein the deficiency results from a defective promoter for regulation of protease gene.
3. The cell of claim 1, wherein the deficiency results from absence ofa promoter for regulation ofa protease gene.
4. The cell of claim 1, wherein the defective promoter results from interruption ofthe promoter sequence.
5. The cell of claim 2, wherein the deficiency results from an alteration ofthe promoter sequence and the alteration is produced by a process selected from the processes consisting of site directed mutagenesis, and PCR.
6. The cell of claim 4, wherein the interruption results from insertion of a polynucleotide that encodes a polypeptide.
7. The cell of claim 6, wherein the polypeptide is one selected from the group consisting ofa hormone, a growth factor, a cytokine, a haematopoietic factor, an j-mmunoglobulin, an enzyme, a repressor, a cell differentiation factor, a binding protein, and a transcription factor or a fragment thereof.
8. The cell of claim 6, wherein the polypeptide is one selected from the group consisting of growth hormone, luteinizing hormone, thyroid stimulating hormone, oxytocin, insulin, vasopresin, renin, calcitonin, follicle stimulating hormone, prolactin, insulin-like growth factor (IGF-I, IGF-II), an IGF-binding protein, epidermal growth factor (EGF), platelet derived growth factor (PDGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), nerve growth factor (NGF), TGF-beta, vascular
or the promoter for Pep4 and transformed into a Pichia host as just described, thereby disabling the protease or its promoter and simaltaneously introducing the gene of interest. The construct for the gene of interest, for example IGF-I, should include flanking regions at the 5' and 3' end of the piece of DNA into which the gene will be integrated. So that, for example, where IGF-I is to be integrated into the coding sequence of the protease Pep4, regions of DNA at the 5' and 3' ends of the protease are ligated into the construct for integration puφoses. In addition, IGF-I gene is accompanied in operable linkage by a leader sequence, a promoter sequence, and a terminator sequence, such as, for example, those disclosed herein. These regulatory regions must be operable in the host cells, and the promoters are preferably inducible such as AOX-I or the ADH-GAP promoters, both inducible and functional in Pichia. The construct also includes a selectable marker gene, and its regulatory sequences, such as a gene that confers resistance to G418 antibiotic. The construct of IGF-I with its flanking regions for integration into the protease Pep4 and its regulatory regions and selectable marker is then cleaved from the plasmid in which it was constructed and transformed as described above in the Pichia host. The cell line constructed serves the function of producing IGF-I by expressing the protein, and because it is deficient in the protease into which the IGF-I integrated.
The method of the invention can be practiced by integrating the gene of interest into any region of the promoter or gene of the protease sought to be disabled.
Alternatively, as described above, a selectable marker construct can be integrated into the protease or its promoter to disable the protease, and create a protease deficient cell line. The advantage of the approach in which the gene of interest is transformed in a manner that disables the protease is that in one transformation the protease is disabled and the gene of interest is incoφorated.