WO1993025663A1

WO1993025663A1 - USE OF mtr GENE SEQUENCES FOR EXPRESSION OF FOREIGN GENES

Info

Publication number: WO1993025663A1
Application number: PCT/US1993/005866
Authority: WO
Inventors: W. Dorsey Stuart; Kenneth Koo; Morton Mandel; Elie K. Kato; Fred C. Greenwood
Original assignee: University Of Hawaii
Priority date: 1992-06-17
Filing date: 1993-06-17
Publication date: 1993-12-23
Also published as: AU4641393A; IL106049A0

Abstract

Expression systems useful in recombinant hosts are provided which permit selection of homokaryotic recombinants capable of producing a desired foreign gene. The expression system contains its own selectable marker. A first nucleotide sequence encoding the desired foreign gene is included in an expression system containing a second nucleotide sequence derived in an illustrative embodiment from the mtr gene of N. crassa which is disposed with respect to the first sequence so as to effect homologous recombination of both the first and second sequences into the mtr locus of the host cell, thus disrupting the endogenous locus. Since the functional product of the mtr gene is required in order to make the cells susceptible to certain poisonous substances, homokaryotic recombinants are resistant to these substances. Successful recombinants can therefore be efficiently selected. The inserted expression system is capable of effecting the production of the desired protein.

Description

USE OF mtr GENE SEQUENCES FOR EXPRESSION OF FOREIGN GENES

This invention relates to the use of cloned sequences of the mtr gene of Neurospora crassa to express DNA encoding heterologous proteins, and to use of recessive traits to assure homokaryotic transformation in polynucleated cells.

BACKGROUND OF THE INVENTION

The translocation of aliphatic and aromatic amino acids across the plasma membrane of the ascomycete, Neurospora crassa, requires a functional gene product of the genomic mtr locus. Perkins, D.D. et al., Microbiol Rev (1982) 46.=426-570. Mutations at this locus result in defective transport of neutral aliphatic and aromatic amino acids and the fungus exhibits altered cell-surface glycoproteins . These mutations can be selected by resistance to 4-methyltryptophan (4-MT) and p-fluorophenylalanine

(pFPA) . These substances are poisons, which cannot be transported into the cell in the absence of the mtr gene product.

The mtr locus has been mapped to the right arm of linkage group IV by Stadler, H., Genetics (1966) 54:677-685. Intralocus recombination of allelic mutants in the mtr locus has been shown by Larimer, F.W. and DeBusk, A.G. , J Bacteriol (1977) 129:1636-1638. A restriction map of a 2.9 kb fragment of genomic DNA encompassing the mtr coding region has been produced from a cosmid library of genomic DNA of Neurospora crassa by Stuart, .D., et al . , Genome (1988) 3_0:198-203. In a later paper, Koo, K. and Stuart, .D., Genome (1991) 3_4.:644-651, the complete nucleotide sequence of the open reading frame and the upstream and downstream regulatory sequences of the mtr locus of an IL. crassa strain were described, along with the location of useful restriction sites. The disclosure of these articles is incorporated herein by reference.

The 1991 article contains two minor errors in the DNA sequence there provided. They are in the upstream region of what is reported as the open reading frame. Correction of these errors results in extension of the open reading frame further into the 5' region; however, this change does not materially affect the utilization of this gene in the invention described herein. The disclosure of the parent application herein, U.S. serial no. 07/899,689 filed 17 June 1992 is also incorporated herein by reference.

Other transport related genes that confer susceptibility to certain amino acid analogs in N__- crassa are also known, such as the pmb locus, which confers canavanine susceptibility.

The present invention provides materials and methods to take advantage of the necessity for a functional mtr gene product to confer susceptibility to certain metabolic poisons such as 4-MT and pFPA.

By using as host a cell which is capable of providing functional mtr gene products, successful homologous recombination of an expression system for a desired protein into all functional mtr loci present in the host can be selected for directly.

DISCLOSURE OF THE INVENTION

The invention provides materials and methods for efficient transformation and selection of hosts that provide functional mtr or other recessive gene products, especially filamentous fungal hosts, which are modified to contain an expression system for a desired recombinant foreign protein. Expression systems are used for transformation which are simultaneously capable of producing the desired protein and providing a selectable marker for successful homokaryotic transformants . The expression systems are designed so as to effect homologous recombination into native mtr loci or analogous loci in the host.

Thus, in one aspect, the invention is directed to an expression system for a gene encoding a desired protein. The expression system consists of a DNA molecule which comprises a first nucleotide sequence encoding the desired protein and a second nucleotide sequence derived from a gene that confers a recessive susceptibility to a poison, such as the mtr gene of J _ crassa. The first and second sequences are disposed in relationship so as to permit homologous recombination of the first and second sequences into the target locus of the host in such a way as to incapacitate normal expression of the locus. This prevents the pioduction of functional protein from that locus. The first, coding, nucleotide sequence is operably linked to control sequences which effect expression of the coding sequence when contained in a recombinant host cell. When all functional native, recessive loci are destroyed, the transformed host can be selected by growth on a medium containing a substrate which is poisonous to the cell requiring the gene product for transport. This general method for selecting homokaryotic transformed cells is illustrated herein using the mtr locus of N_j. crassa.

In other aspects, the invention is directed to filamentous fungal cells modified to contain the expression system of the invention, to methods to obtain such modified cells, and to methods to produce the desired protein using the modified cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the nucleotide sequence of an approximately 2.9 kb fragment of the N_ crassa mtr gene containing the entire open reading frame as well as the promoter and transcription terminating signals.

Figure 2 shows nucelotide and deduced amino acid sequences of the open reading frame of the mtr locus. MODES OF CARRYING OUT THE INVENTION

The present invention provides, in a preferred embodiment, the relevant DNA of a wild type Neurospora crassa mtr gene locus. Figure 1 shows the complete nucleotide sequence of this wild type mtr; an approximately 2.9 kb segment of DNA derived from a cosmid library of N_-_ crassa FGSC No. 3043 is depicted. Because of considerable homology among various strains of N__j_ crassa and across various filamentous fungi, both allelic variants of the nucleotide sequence in

Figure 1 and variants derived from alternative strains of filamentous fungal species and other organisms which exhibit unicellular growth may also be used in the invention. Provision of the sequence information of Figure 1 is adequate to permit retrieval of these alternative mtr genes. Modifications of the depicted sequence can be made by site-directed mutagenesis and portions or all of this or other retrieved sequences may be synthesized using well known solid phase nucleotide synthesis techniques. Alternate sequences can also be derived from cosmid libraries prepared from other fungi in a manner analogous to that described for the retrieval of the gene disclosed herein by Koo, K. et al. , Genome (1991) J34_.:644-651, cited above.

Thus, as used herein, the phrase "a nucleotide sequence derived from the mtr gene of N. crassa" refers to a nucleotide sequence as shown in Figure 1, or a functional portion thereof, or a modified form thereof which contains sufficient homology to the sequence depicted to be functional in effecting homologous recombination with a native mtr locus, allelic variants thereof, corresponding loci from other organisms which are retrievable using the depicted sequence as a probe or using relevant portions of the sequence shown in Figure 1 as primers for PCR cloning. Functional portions of all of the foregoing may be used. These nucleotide sequences may be obtained by cloning the retrieved genes, modifying retrieved genes and amplifying them, or partially or completely synthesized using standard polynucleotide synthesis techniques.

The expression systems of the present invention contain, in addition to a nucleotide sequence derived from the mtr gene of N_j. crassa, a gene encoding a desired protein to be produced recombinantly in a host cell. Any desired protein can be produced using the expression systems of the invention. Such proteins include both plant and animal proteins and proteins derived from microorganisms. Thus, genes encoding enzymes such as thrombin, tissue plasminogen activator, streptokinase, urokinase, alcohol dehydrogenase, various proteases such as trypsin, chymosin, enterokinase, and the like are suitable for production using the expression systems of the invention. Such enzymes may be useful as pharmaceuticals, as reagents, or as industrial enzymes. Other exemplary types of proteins whose production may be desired include growth factors and cytokines such as interleukin-2, bovine growth hormone, human growth hormone, fibroblast growth factor, TGF-o:, TGF-3, bone morphogenic protein, IL-3, IL-6, and the like. In addition, the expression system may be used for the recombinant production of antibodies or fragments thereof, including chimeric antibodies as well as hormones such as insulin, ACTH, the gonadotropins, and the like. Any protein for which a practical means of production is desired and the coding sequence for which is available can be employed in the expression systems and methods of the invent

The host cells which are useful in the invention method for production of recombinant protein using the expression systems containing nucleotide sequences derived from the mtr gene of N-j. crassa are cells containing genes encoding functional mtr products which confer susceptibility on the native host to substances toxic to the cells when internalized using the mtr system, such as the 4-MT and pFPA described above. The host cells can be derived from any species, provided the relevant gene is present in these cells, and provided the cells can be grown in culture. Filamentous fungal host cells are preferred. N_^. crassa host cells are particularly preferred.

Cells subject to the general method described herein (selection for homokaryotic recombinants by effecting destruction of an endogenous recessive gene conferring susceptibility to a poison) may be any cells which contain such recessive genes. In a manner analogous to that described herein using a nucleotide sequence derived from the mtr locus, sequences derived from the relevant recessive gene locus are used to effect homologous recombination of a desired DNA into the genome of the host cell. The recombination results in destruction of the functionality of the native gene and thus converts the successful homokaryotic transformant into a cell resistant to the poison. Only homokaryotic transformants will therefore grow on media containing the poison.

Other recessive genes of this type, besides the mtr locus, are known. For example, N,. crassa also contains a locus encoding a "pmb" product which effects the transport of basic amino acids into the cells. Canavanine, a basic substance which is toxic to native J crassa, is then not toxic to cells with each such locus destroyed. Successful transformants can then be identified by successful growth on a medium containing canavanine.

The method disclosed herein for efficient selection of successful homokaryotic transformed cells is particularly useful where the cell to be transformed has more than a single copy of a gene locus. Thus, this approach is useful in transformation of diploid or polyploid cells, or, especially, in polynucleated cells such as those found particularly in filamentous fungal species. Since all of the native loci must be destroyed in order to render the cells resistant, the selection method automatically provides homokaryotic transformants. Unless otherwise stated, the present invention employs molecular biology, microbiology and recombinant DNA techniques as described, for example, Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984) ; "Nucleic Acid Hybridization" (B.D. Hames & S.J. Higgins eds. 1985); "Transcription And Translation" (B.D. Hames &• S.J. Higgins eds. 1984); "Animal Cell Culture" (R.I. Freshney ed. 1986); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984) .

The expression systems of the invention provide for insertion of heterologous DNA into cells (including spheroplasts or protoplasts) that can be ultinuclear, so that a selection of homokaryotic transformants is facilitated. Homokaryotic transformants may be selected in one step from a colony or mixture containing multinuclear cells. This is particularly advantageous in dealing with Neurospora or other filamentous fungi having vegetative spores (conidia) which contain one or more nuclei. The transformation of such spores, after digestion of the cell walls to produce spheroplasts, is a random event. If the spheroplasts are multinueleated, the transforming DNA may be inserted into less than all of the nuclei that are present. Other multinucleate cells can also be used as subjects for the invention.

Transformation selection protocols directed to a dominant phenotype, such as the restoration of an enzyme which confers the ability to grow on minimal medium, do not select for homokaryotic transformants. Transformation of any single nucleus in any spheroplast will result in growth of transformed colonies on selective media, even though many of the transformants will also carry untransformed nuclei in a number equal to, or in excess of, the desired transformed nucleus. In the invention method, when a recessive allele is inserted, the recessive genotype can be expressed phenotypically (and thus selected) only if it is the only allele in the culture of transformed strains, thus ensuring that selected transformants will carry only the nuclei which have the constructed gene of interest.

The illustrative expression system herein provides such a recessive allele. Neurospora cells that include wild-type mtr cannot grow on media containing 4-MT or pFPA because the transport system in the host of which the mtr gene product is a part brings these substances into the cell and the cell dies. If the host cells are made homokaryotic mtr negative mutants, the transport system is inoperative so the cell can survive on the media containing these substances, because it is not transported into the cell.

For colonies of multinucleated spheroplasts, transformation of one nucleus to mtr^" by insertion of a vector according to the present invention would still result in selection against that transformant because the nontransformed nuclei would continue to produce the mtr protein, the cell will continue to transport the poison and would therefore die as a result. Only if a transformed spheroplast has all mtr loci disrupted will it be able to resist the poison, survive the selection and grow into a strain where all the nuclei contain the inserted gene. Thus, in selecting for resistant transformants, selection of homokaryotes is assured. The general features for use of the expression systems of the invention can be understood in terms of the diagram showing the features of the mtr gene (common features with genes in general) . 5 ' UT ORF 3 ' UT

The 5' untranslated region (5'UT) transcribed into the mRNA contains features which facilitate translation of the mRNA. This is followed by the open reading frame (ORF) which contains the coding region which is ultimately translated into the protein. In the mtr gene illustrated, the ORF also contains a 59-nucleotide intron which remains in the mRNA but is not translated. (See Figure 2.)

Presumably, the intron contributes to enhanced stability of the mRNA. The transcribed region is completed by the 3' untranslated region (3'UT) which contains the polyadenylation signal also contributing to mRNA stability and facilitating translation.

In the DNA, the transcribed region is preceded by the promoter (P) and followed by the transcription terminating sequences (T) . The transcription terminating sequences are believed to render expression more efficient, but do not appear to be necessary to effect at least some expression.

The expression systems of the invention contain some or all of the mtr sequence just discussed, or the corresponding portions of other recessive loci, and thus provide for the insertion of DNA comprising the sequence _.encoding the desired protein to be inserted somewhere in the overall gene or contiguous with one of its termini, provided sufficient deletion or interruption is made in the gene sequences so as to disrupt expression of the wild type gene when homologous recombination occurs. While homologous recombination can be effected by extension at only one terminus of the desired DNA to be inserted, insertion is more efficient when the DNA to be inserted is bracketed by homologous sequences. The efficiency of homologous recombination is increased with increasing length of nucleotide sequence bearing homology to the target for recombination. It is believed that a minimum of ten nucleotides is required, but preferably 20, more preferably 100, still more preferably 200 and still more preferably 1000 nucleotides can be included in the construct to effect recombination. The length of the nucleotide sequence provided is arbitrary; the frequency of recombination is simply increased as the length of homologous sequence increases. Furthermore, the homologous sequences, such as the mtr sequences, must be modified in some way so as to make them nonfunctional if they are to be simply ligated to one end of the inserted sequence, rather than automatically disrupted by the DNA to be inserted. Thus, expression vectors which result from the insertion into the homologous sequence of the DNA comprising the gene encoding the desired protein are preferred.

It is, of course, not necessary to utilize the complete mtr gene sequences illustrated; insertion can be made into the mtr gene in which deletions have been made. For example, substantial portions of the open reading frame of the mtr gene may be removed prior to (or after) insertion of the desired DNA.

Using the above diagram for mtr as an illustration, the position for insertion of the DNA comprising the desired coding DNA is arbitrary, but will depend on the nature of the DNA to be inserted. If the inserted DNA consists essentially of the desired coding sequence, to be placed under control of the mtr promoter, the insert must be made so that the coding sequence is in operable linkage with the mtr promoter. Similar comments apply to utilization of the mtr transcription terminator. Thus, the insert is made into the transcribed region of the mtr gene, preferably into the open reading frame. It is possible, but not necessary, to provide for the production of a fusion protein by ligating the desired coding region in open reading frame with the mtr ORF. Alternatively, the DNA comprising the desired coding sequence can be inserted under the control of its own promoter and/or transcription terminator which, themselves, may either be homologous with the coding sequence or heterologous to it. The promoter must, of course, be functional in the host. An additional copy of the mtr promoter could also, of course, be used.

Similarly, DNAs comprising coding sequences for two or more desired heterologous proteins can be inserted, some or all under control of the promoter and/or transcription terminating sequences in the mtr sequence and some or all under control of their own transcription control sequences.

The functional features of the expression system in effecting homologous recombination can be illustrated in the diagrams below which show but two of many options for the construction and function of such vectors:

In the first illustration, the vector contains the transcribed portion of a mammalian gene as a replacement for the transcribed portion of the mtr gene. Upon homologous recombination, the transcribed sequences of the mammalian gene are integrated into allele replacing the corresponding regions of the mtr gene and thus disrupting its function. This is shown diagrammatically below:

vector: PPPP-MAMMALIAN GENE-TTTTTT

integrates into

genome: PPPPP-MTR GENE-TTTTTT-

at the mtr locus disrupting the mtr gene and producing the allele: PPPPP-MAMMALIAN GENE-TTTTTT .

In a second alternative, multiple genes under control of their own promoters and terminators can be inserted between the controls of the host mtr gene as shown in the following diagram: vector: PPPPP-GENEt-pGENEt-pGENEt-pGENEt-TTTTT

integrates into

genome: PPPPP-MTR GENE-TTTTTT

at the mtr locus disrupting the mtr gene and producing the allele:

PPPPP-GENEt-pGENEt-pGENEt-pGENEt-TTTTTT .

In this illustration, only the first gene in the sequence is under control of the mtr promoter; the remaining genes bear their own control sequences. Again, the function of the wild type mtr gene will be destroyed.

Of course, substantial portions of the open reading frame of the mtr gene can also be retained, if desired, so long as insufficient sequences are provided to effect production of a functional protein gene product. Indeed, deletions, additions and alterations in the sequence encoding the mtr protein product may be made in the expression system constructed so long as the remaining sequences fail, when integrated, to provide a functional product, but are functional to effect integration.

Thus, the mtr gene sequences contained in the expression vector have at least two functions:

(1) they effect homologous integration of the inserted or contiguous DNA comprising the coding sequence for the desired protein, and

(2) they are of such a nature that when homologous combination occurs, no functional mtr protein is produced. Thus, they cannot themselves be sufficient to restore mtr protein function.

The construction of expression systems of the invention are illustrated by the initial construction of a host vector, designated herein pXpress, which includes the mtr gene sequence shown in Figure 1. This vector is suitable for amplification in EL. coli, and provides endogenous insertion sites for the DNA containing the desired coding sequence. Also illustrative of host vectors is pDXpress, an analogous vector which is identical to pXpress except that an additional polylinker in the original host vector, from which pXpress was constructed, is deleted.

The illustrative vector pXpress is constructed from the vector pBN3 which is described in Stuart, W.D., et al. , Genome (1988) 3_0.198-203. pBN3 contains the 2783 bp Nj_ crassa genomic DNA containing the mtr gene shown in Figure 1, which is bracketed by a Bglll site in the gene and a BamHI site contained in the vector. pBN3 was digested with BamHI and Bglll and the segment containing the mtr gene was inserted into the BamHI site of the commercially available vector, pTZ18R, obtained from Pharmacia. This produced clones pN807 and pN816 wherein the EcoRI site contained in the polylinker of the pTZ18R vector is upstream of the ORF; clones in the opposite orientation were designated pN846 and pN839. pXpress is a version of pN846 wherein the 5' polylinker of pTZ18R is deleted. The pXpress vector has useful cloning sites for insertion of the desired DNA in the upstream region just downstream of the mtr promoter (Sall/AccI/HincII) (position 307 in Figure 1) and also in the latter third of the ORF (Hindi at position 1406 and Accl at position 1920) . Thus, in one illustrative embodiment, pXpress may be used directly as an insertion vector to construct the expression system of the invention by cleaving with Accl, which deletes most of the open reading frame while retaining both the promoter and transcription terminator of the mtr gene. In another embodiment, the vector may first be digested with Sail, which recognizes only the upstream post-promoter site, and then with Hindi which, since the upstream recognition sites overlap, now recognizes only the downstream site to produce a SalI-blunt opening into which the desired DNA may be inserted in a directionally controlled way. pDXpress is a modified form of pXpress in which the linker provided in the host vector pTZ18R at the 3' end of the pN846 insert is deleted, thus eliminating additional restriction sites duplicate to those available in the sequence of the mtr gene.

After construction of a suitable expression vector by insertion of the desired coding sequence into the host vector containing the nucleotide sequence derived from mtr, cells of the desired recombinant host are treated with the vector under conditions which favor the uptake of the vector DNA into the cells. These transformation conditions are standard and depend on the nature of the cells to be transformed. For transformation of filamentous fungi, the preferred hosts of the invention, spheroplasts or protoplasts are generally used and the vector is linearlized prior to treating the cells. The transformants are then selected on medium containing a substance to which mtr confers susceptibility, such as pFPA or 4-MT. Successful homokaryotic transformants can survive in this medium and can be cloned. The cloned cells lines are then used in culture for production of recombinant protein encoded by the inserted DNA. The protein thus produced may be contained intracellularly or secreted into the medium, depending on the expression system construction used, as is understood in the art. After culturing under conditions which encourage the production of the protein, the protein produced is recovered from the culture and, if desired, purified using standard techniques known in the art. Alternatively, the cultures themselves or the culture media may provide the protein in sufficient purity for its intended use, as may be the case, for example, for the production of industrial enzymes or enzymes used for organic synthesis steps. Of course, if the protein produced is intended for pharmaceutical use, it may be desirable to purify the protein produced to a suitable level for formulation.

The following examples illustrate, but do not limit the invention.

Example 1 Preparation of pBN3 11.3 μg of Bglll digested 5:4H DNA (DNA from cosmid 5:4H in the Vollmer-Yanosky library, described in Vollmer et al. , PNAS (1986) 11=4869-4873), was electrophoresed in a preparative 1% LMT-agarose gel. 15.0 μl (70 ng of DNA) of the liquified agarose gel, containing the 15.0 kbp fragment was mixed with 1.5 μl H₂0, 2.0 μl 10X T4 ligase buffer at 37°C and maintained at this temperature for 15 min. The cosmid 5:4H is capable of transforming an mtr negative host strain, as are 5:3B and 5:10H from the same library. Then 1.5 μl 1.4 U/μl T4 DNA ligase was added. The contents were briefly mixed by flicking the tube and brought down with a quick pulse in a microcentrifuge. The reaction was incubated at 15°C overnight.

At the end of the incubation, 150 μl Tris . Cl/CaCl₂ was added to the ligation reaction which was heated to 65-70°C for 5 minutes to liquify the agarose. The condensation was brought down with a rapid pulse, and the tube was immersed into an ice bath. The contents, after sufficient cooling, were dispensed into a microfuge tube containing 200 μl competent Ej. coli LE392 cells. The transformation and plating of the cell/media mix was the same as previously described.

Example 2 Generation of pN807 and pN846

6 μg pTZ18R DNA as digested to completion with 17 U BamHI and the final concentration adjusted to 60 ng/μl. 12.0 μg pBN3 DNA was double digested with 36 U Bglll and 34 U BamHI. The sample was electrophoresed in a 1% LMT-agarose gel and the 2.9 kbp fragment was excised. The agarose plug was immersed in H₂0 at room temperature (60 min.) to remove excess EtBr and IX TBE buffer. The agarose plug, containing 600 ng of the 2.9 kbp fragment was liquified by heating and mixed with 120 ng of BamHI digested pTZ18R vector. 5X T4 ligase buffer and 2 U T4 DNA ligase was mixed into the solution at 37°C for a final volume of 90 μl. The reaction was incubated at room temperature (overnight) .

A half volume aliquot of the reaction mix was liquified and mixed with an equal volume of Tris Cl/CaCl₂ solution, and cooled in an ice bath. The solution was used to transform 200 μl competent E__s_ coli NM522 cells. The transformed cells were spread on X-gal indicator plates to detect recombinant DNA molecules. The numbered clones pN389 and pN846 are the same clones picked and numbered independently from different colonies. The numbered clones pN807 and pN816 are the same clones picked and numbered independently from different colonies, and contain the pBN3 insert in opposite orientation from that of pN839/846?

Example 3

Preparation of pXpress and pDXpress Five micrograms of pN846 DNA were isolated from E_;_ coli NM522 by standard methods (Koo and Stuart, 1991) . The DNA was double digested with Xbal and Hindlll, treated with Klenow and NTPs, cleaned with Geneclean (Bio 101) , and ligated with 400 units of DNA T4 polymerase at room temperature overnight . The ligation mixture was used to transform E_^_ coli NM522 host cells and selected for Amp^r. Transformed colonies were picked and grown in 1.5 ml liquid cultures in tubes overnight. Plasmid DNA was isolated and tested for the presence of Hindlll, Xbal and Pstl restriction sites. Isolates which had lost the three sites were then tested for the remaining sites expected to be in pN846. One plasmid which had lost the expected sites and retained the expected sites was designated plasmid pXpress.

To prepare pDXpress, pXpress was digested with BamHI and EcoRI, the ends polished with Klenow, and the DNA is relegated (in the presence of Smal, to reduce the number of copies of partially cut and relegated vector) . The ligation mixture is transformed into I _ coli host cells and transformants selected for resistance to ampicillin. Plasmid DNA from resistant colonies is tested for loss of EcoRI, BamHI and Sad sites. A plasmid meeting these criteria and also retaining the internal Kpnl sites of pXpress was designated pDXpress (plasmid downstream express) .

Example 4 Production of Chvmosin The gene encoding bovine chymosin (Moir et al., Gene (1982) 21:127-138) was inserted into pXpress as a Xhol/HindiII-blunted fragment. pXpress was digested with Sail and then with Hindi, and blunted. This deletes the portion of the mtr gene between positions 307 and 1406 shown in Figure 1. A sample of 0.5 μg of the chymosin open reading frame was ligated into 0.5 μg of the cleaved vector using 40 units of T4 ligase incubated overnight at 16°C. Some fragments ligate Sail to Xhol and blunt Hindlll to blunt Hindi (losing all four restriction sites) .

The ligated fragments were transformed into competent E_j_ coli cells DH-5alpha and selected for Amp^r. Plasmids were isolated and digested with EcoRV to confirm the insert. Plasmids containing the insert in the correct orientation were renamed pNEC for plasmid neutral (mtr) expression of chymosin.

Transformation of pNEC into Neurospora spheroplasts was accomplished by standard methods. See Koo and Stuart (1991) supra. pNEC DNA was linearized by cutting with Sad and 5 μg of linearized pNEC was used to transform 1 X 10⁸ spheroplasts of strain 74a "wild" type Neurospora (Fungal Genetics Stock Center, Dept . of Microbiology, University of Kansas Medical Center, Kansas City, Kansas 66103) .

The mixture was taken up in 15 ml of minimal top agar and spread onto a bottom plate containing 0.05 mg/ml pFPA. Plates were screened 3 days later. Eighteen colonies were picked and grown on solid Vogel's IX media containing 0.05 mg/ml pFPA in tube slants.

Colonies were transformed to liquid cultures of IX Vogel's with 2% sucrose in double distilled H₂0. The liquid was collected and assayed for chymosin activity using 25 μl culture, 125 μl 10 mM Phosphate Buffered Sodium (PBS) , and 150 μl clotting buffer (4.5 mg/ml CaCl₂, 3.0 mg/ml NaOAc in 10 mg/ml nonfat milk) . Samples were tested on a 64 well microtiter plate, incubated at 37°C for 15 min then assayed by absorbance at 630 nm on a Dynatech MR 5000 ELISA machine. Among the 18 transformants, four showed milk clotting ability. Chymosin concentrations ranging from 1.6-4.6 μg/ml were extrapolated from known chymosin standards.

ELISA assay was performed on the pNEC transformants using rabbit antibovine chymosin antibody. Secondary antibody, goat antirabbit Ab conjugated to alkaline phosphatase was used to visualize chymosin concentrations. The ELISA reactions indicated chymosin concentrations ranging from 1-16 μg/ml. These results indicated that not all of the chymosin product was biologically active. Western blots of PAGE gels showed that some of the chymosin in the Neurospora media was in the form of prochymosin which is inactive until converted by incubation at pH4 for several hours. Example 5 Expression of Relaxin Gene A gene coding for the open reading frame of porcine relaxin (Haley et al . , DNA (1982) 1:155-162) was inserted into the pXpress in a manner similar to the protocols used for chymosin. The gene was copied using the PCR reaction using primers designed so that a Kpnl site and the Neurospora translation consensus sequence (Koo and Stuart 1991) were added to the 5' end of the gene and a BamHI site was added to the 3' end. The PCR product was polished with T4 polymerase and inserted in the plasmid pXpress at the Hindi sites in a manner identical to the chymosin construction. Orientation was confirmed by double digestion with EcoRV and BamHI and with single digestion with Kpnl. Inserts with 5' end of the gene following the mtr promoter were used to transform Neurospora spheroplasts as in Example 4. Transformants resistant to pFPA were transferred to liquid cultures and the media screened for production of relaxin by ELISA assay using a rabbit anti-relaxin antibody and goat anti-rabbit secondary antibody. Six transformants secreted a protein which cross reacted with relaxin antibody, but at levels 10 to 100 times lower than chymosin as determined by ELISA.

Claims

1. An expression system for expressing a gene encoding a desired protein in a recombinant host having a functional mtr locus, said expression system consisting of a DNA molecule which molecule comprises : a first nucleotide sequence encoding said desired protein; a second nucleotide sequence derived from the mtr gene of N crassa disposed in relationship to said first sequence so as to permit homologous recombination of said first and second sequences into the mtr locus of said host so as to incapacitate the mtr locus in said host from effecting production of functional mtr protein; wherein said first nucleotide sequence is operably linked to control sequences which effect expression of said encoding sequence, when contained in the recombinant host cell.

2. The expression system of claim 1 wherein at least a portion of said second sequence is disposed at the 5' terminus of the said first nucleotide sequence.

3. The expression system of claim 2 wherein said portion comprises the mtr promoter operably linked to said first nucleotide sequence.

4. The expression system of claim 1 wherein at least a portion of said second nucleotide sequence is disposed at the 3' terminus of said first nucleotide sequence.

5. The expression system of claim 4 wherein said portion comprises the mtr transcription terminator sequences operably linked to said first nucleotide sequence.

6. The expression system of claim 1 wherein said first nucleotide sequence is operably linked to a promoter homologous to said first nucleotide sequence, or wherein said first DNA sequence is operably linked to a promoter which is heterologous both to said first and second nucleotide sequences, or wherein said first nucleotide sequence is operably linked to a transcription terminator homologous to said first nucleotide sequence, or wherein said first DNA sequence is operably linked to a transcription terminator heterologous to both said first and second nucleotide sequences, or wherein said first nucleotide sequence is an open reading frame with the coding region of said second sequence, or wherein said second nucleotide sequence comprises less than the complete sequence of the mtr gene.

7. A recombinant host cell which contains the expression system of claim 1.

8. The cell of claim 7 which is Neurospora crassa.

9. A method to produce a desired protein which method comprises culturing the host cell of claim 7 under conditions wherein expression of the nucleotide sequence encoding the desired protein is effected to as to produce said protein in the culture; and recovering the protein from the culture.

10. The method of claim 9 wherein said desired protein is relaxin or chymosin.

11. A method to select for a recombinant host cell which has, integrated into all mtr loci in said cell, the expression system of claim 1, which method comprises culturing said candidate cells on a medium containing a substance which is lethal to said cell in the presence of a functional mtr gene product; and selecting cells capable of growth on said medium.

12. A method to obtain cells which are homokaryotic transformants with a desired DNA, which method comprises contacting said host cells, in a form susceptible for uptake of DNA molecules and under conditions which are suitable for the uptake of DNA molecules, with a DNA molecule which comprises said desired DNA contiguous to a sequence homologous to a recessive gene that confers susceptibility to a poison, so as to permit homologous recombination of said DNA molecule into the locus of said recessive gene to prevent expression of said gene; culturing the resulting cells in a medium containing said poison; and recovering cells which are capable of growth in the presence of said poison.

13. A method to obtain cells which are homokaryotic transformants with a desired DNA, which method comprises culturing cells that have been treated, in a form susceptible for uptake of DNA and under conditions which are suitable for the uptake of DNA molecules with a DNA molecule which comprises said desired DNA contiguous to a sequence homologous to a recessive gene that confers susceptibility to a poison, so as to permit homologous recombination of said DNA molecule into the locus of said recessive gene to prevent expression of said gene; in a medium containing said poison; and recovering cells which are capable of growth in the presence of said poison.

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