IE83566B1 - Pichia pastoris acid phosphatase gene - Google Patents

Description

PATENTS ACT, 1992 Al5$L2l PICHIA PASTORIS ACID PHOSPHATASE GENE RESEARCH CORPORATION TECHNOLOGIES INC.

Field of the Invention This invention relates to the field of recombinant biotechnology utilizing yeast host systems and expression vectors. In one aspect the invention relates to novel DNA fragments containing part or all of the yeast Bighig pastoris acid phosphatase gene (SEQ ID NO:1).

In another aspect the invention relates to novel vectors containing DNA fragments derived from the Bighig pastoris acid phosphatase gene (SEQ ID NO:1). In yet another aspect the invention relates to host cells transformed with vectors containing fragments derived from the gighia pastoris acid phosphatase gene (SEQ ID N0:1). In another aspect the invention relates to using the above-described DNA fragments to facilitate the expression and/or secretion of heterologous proteins in yeast.

Background of the Invention As recombinant DNA biotechnology has developed in recent years, the controlled production by microorganisms of an enormous variety of useful polypeptides has become possible. Many polypeptides, such as for example human growth hormone, leukocyte interferons, human insulin, and human proinsulin have already been produced by various microorganisms. The continued application of techniques already in hand is expected to permit production of a variety of other useful polypeptide products.

The basic techniques employed in the field of recombinant DNA technology are known by those of skill in the art. The elements desirably present for the practice of recombinant DNA technology include but are not limited to: (1) a gene coding for one or more desired polypeptide(s) and functionally associated with adequate control sequences required for expression of the gene in the host organism; ' (2) a vector into which the gene can be inserted; (3) a suitable host organism into which the vector carrying the gene can be transformed; (4) if secretion of the heterologous protein is desired, a signal sequence capable of directing the heterologous protein into the secretion pathway of the host cell, and thereafter out of the cell; (5) a transformation system; and (6) a method of selecting transformants.

Recombinant gene constructs can be designed such that the recombinant protein transits the host's secretory pathway and is secreted into the growth media. Secretion is a desired mode of recombinant expression for several reasons. First, some heterologous proteins have a toxic effect on the host organism. When such heterologous gene products are secreted rather than accumulated within the host, they are less likely to interfere with normal cellular Second, functions. some proteins that are inactive when produced intracellularly are active when secreted. Third, secretion into the medium avoids the necessity of breaking open the host cells in order to Product purification is much easier and cost And, fourth, since the recombinant product is present in the nutrient medium, the recover the product. effective when product is present in the growth medium. desired product can be continuously removed and the media can be recycled.

Host secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form, containing an appended amino terminal extension called a signal peptide. The signal peptide plays an essential role in transporting the appended polypeptide into and/or through the limiting cellular membranes. This signal peptide is then cleaved proteolytically by a signal peptidase during or after secretion to yield a mature protein product.

Secretion of a heterologous or foreign protein can be accomplished by linking the coding sequence of the heterologous DNA to DNA encoding a signal peptide. It would be desirable to isolate a signal sequence encoding this signal peptide, which would facilitate secretion.

Signal sequences are especially useful in the creation of expression vectors. The use of such vectors would make it possible to transform compatible host cells so that they produce and secrete heterologous gene products. Examples of leader sequences which have been used to successfully secrete recombinant proteins from yeast hosts include those from the Saccharomyces cerevisiae alpha mating factor, a mating factor, and killer toxin genes. Isolation of a signal sequence from a methylotrophic yeast, such as gighia pastoris, has not been described.

Conveniently, the promoter which is employed in such vectors to regulate expression of the heterologous gene products may be the promoter natively associated with the signal sequence. It would be especially advantageous if the promoter natively associated with the signal sequence provides for a high level of DNA transcription and is responsive to exogenous environmental stimuli. An example of such a promoter is the 5' regulatory region of the Eighig pastoris acid phosphatase (3391) gene (SEQ ID N022), which is transcribed at a high level in response to the absence of phosphate in the media, and repressed by the presence of phosphate in the media.

It is often desirable to transform a Eighig pastoris host with a recombinant DNA construct that will integrate at a precise position in the gighia pastoris genome. The 5' and 3' sequences which flank the gighig pastoris gag; gene, also known as first and second insertable DNA fragments, respectively, are used in expression vectors to direct the integration of the recombinant sequences at the 359; locus. The ability to integrate recombinant DNAs at the Egg; locus is advantageous for at least two reasons: 1) in the development of Eighia pastoris expression strains having multiple copies of the same or different expression cassettes at the PH01 locus or another Pichia locus, or 2) stable integration of one or more expression cassettes at the gag; locus only, in a host Eighig pastoris strain wherein disruption of an essential gene or a gene of the methanol metabolism pathway is undesirable.

Cells in which the 359; gene has been disrupted show a concomitant loss of acid phosphatase enzyme activity. The Pho- phenotype, indicative of Eflgl gene disruption, may be screened for by plating the cells on low phosphate indicator plates and allowing colonies to grow overnight. Colonies in which the gag; gene is disrupted are white, whereas those colonies having an intact Egg; gene are green. This colorimetric screen provides a rapid and easy method for detecting cells which have integrated expression cassettes correctly at the £59; locus and thus disrupted it.

Thus, it would be a significant contribution to the art to isolate a signal sequence that would facilitate the secretion of proteins from a host cell.

Additionally, it would be advantageous to isolate a ' regulatory region which would provide for high levels of DNA transcription and is responsive to exogenous environmental stimuli.

Currently no 5' regulatory region is known in the art which is transcribed at a high level in response to the absence of phosphate in the media and which can be used with the highly productive fermentation yeast Bighia pastoris.

It would additionally be advantageous to isolate the acid phosphatase (AP) structural gene.

It would also be advantageous to provide novel vectors comprising fragments of the acid phosphatase gene.

It would additionally be advantageous to isolate a 3' transcription termination sequence.

It would also be advantageous to provide integrative vectors which would direct integration at the fighig pastoris gag; locus.

It would additionally be advantageous to provide a method of identifying disruptants.

Thus, it is an object of the present invention to provide a signal sequence which facilitates the secretion of proteins from cells.

It is also an object of this invention to provide a in response to the absence of ' regulatory region transcribed phosphate.

Another object of the present invention is to provide the DNA sequence of the Eighia pastoris acid phosphatase structural gene (SEQ ID N024).

It is a further object of this invention to provide novel vectors comprising a regulatory region and/or signal sequence operably-linked to a heterologous DNA sequence which encodes at least one polypeptide, and means of inducing said regulatory region to facilitate expression of said heterologous DNA sequence.

It is a still further object of this invention to provide a 3' transcription termination sequence from the acid phosphatase gene.

Yet another object of this invention is to provide integrative vectors which would direct integration at the Biggie pastoris gag; locus.

A further object of this invention is to provide a method of identifying disruptants.

Other aspects, objects, and advantages of the present invention will become apparent from the following specification, examples, and claims.

Summary of the Invention In accordance with the present invention, there is provided a novel DNA fragment comprising the signal sequence from the gighig pastoris acid phosphatase gene (SEQ ID N0:3) which facilitates the secretion of proteins from cells.

A further aspect of this invention provides a novel DNA fragment comprising the Eighia pastoris acid phosphatase 5' regulatory region (SEQ ID N022).

In still another aspect of this invention there is provided a novel DNA fragment comprising the DNA sequence of the gighig pastoris acid phosphatase (AP) structural gene (SEQ ID N024).

Yet another aspect of this invention provides novel vectors comprising the regulatory region and/or signal sequence operably-linked to a heterologous DNA sequence which encodes at least one polypeptide, and means of inducing said regulatory region to facilitate expression of said heterologous DNA sequence.

In another aspect of this invention there is provided novel DNA vectors comprising a 3' transcription termination sequence from the Pichia pastoris acid phosphatase gene (SEQ ID NO:S).

Another aspect of this invention provides integrative vectors which direct integration of the vector at the Pichia pastoris PH01 locus.

Yet another aspect of this invention is to provide a method of identifying disruptants.

Brief Description of the Figures Figure 1 provides a restriction map of the Pichia pastoris acid phosphatase gene (SEQ ID N021).

Detailed Description of the Invention The present invention provides a novel isolated DNA fragment comprising an acid phosphatase gene, including its promoter (or S'regu1atory region), signal, transcription terminator, and flanking sequences, derived from Pichia pastoris (SEQ ID N0:l).

Acid phosphatase (AP) is an extracellular enzyme secreted by gighig pastoris and other yeasts under conditions of inorganic phosphate starvation. The secreted acid phosphatase catalyzes the removal of phosphate from organic substrates, thus availing the phosphate for cell growth and survival.

The gene coding for acid phosphatase has been isolated and studied in several yeast species, and the regulatable nature of acid phosphatase gene expression has been localized to the acid phosphatase promoter element. In conditions of low media concentrations of inorganic phosphate, the acid phosphatase promoter (or 5' regulatory region) is turned "on", the acid phosphatase gene is transcribed, and subsequently translated into the acid phosphatase enzyme. The newly synthesized acid phosphatase enzyme releases phosphate from organic substrates and makes the phosphate available to the cell. The subsequent increase in phosphate concentration modulates the acid phosphatase promoter, resulting in decreased acid phosphatase gene transcription concomitant with the lessened need for acid phosphatase protein. Thus, the acid phosphatase promoter can be induced or repressed by low or high phosphate concentrations, respectively.

Identification and isolation of the Bighig pastoris acid phosphatase promoter is significant. Because the regulation of acid phosphatase expression varies among the yeast species for which this expression has been analyzed, it is expected that the tight regulation observed for acid phosphatase expression in fiighig pastoris depends on the availability and use of the homologous promoter sequences.

Identification and isolation of the Bighia pastoris gag; gene signal sequence (SEQ ID NO:3) is significant in that, in addition to being the first signal sequence isolated from a Eighia pastoris gene, it represents the first signal sequence isolated from a methylotrophic yeast. It has been discovered to be equivalent or superior to native gene signal sequences, gigi, the signal sequence from the tPA (Tissue Plasminogen Activator) or invertase genes, in directing secretion of heterologous proteins from Eighig pastoris, gigi, tPA or invertase.

It is often desirable to integrate recombinant expression vectors into the host genome instead of maintaining them as autonomously replicating elements. Integrated vectors are significantly more stable than autonomous vectors, and are preferred for the practice of the present invention; Specifically, linear site-specific integrative vectors as described in U.S. 4,882,279, which is herein incorporated by reference, are preferred. Such vectors comprise a serially arranged sequence of at least 1) a first insertable DNA fragment; 2) a selectable marker gene; and 3) a second insertable DNA fragment.

The first and second insertable DNA fragments are each at least about 200 nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the species to be transformed. The various components of the integrative vector are serially arranged forming a linear fragment of DNA such that the expression cassette and the selectable marker gene are positioned between the 3' end of the first insertable DNA fragment and the 5' end of the second insertable DNA fragment. The first and second insertable DNA fragments are oriented with respect to one another in the serially arranged linear fragment as they are so oriented in the parent genome.

The first insertable DNA fragment may contain an operable regulatory region which may comprise the regulatory region used in the expression cassette. The use of the first insertable DNA fragment as the regulatory region for an expression cassette is a preferred embodiment of the invention. Optionally, an insertion site or sites and a 3' termination sequence may be placed immediately 3' to the first insertable DNA fragment. This conformation of the linear site-specific integrative vector has the additional advantage of providing a ready site for insertion of a structural gene without nessessitating the addition of a compatible 3' termination sequence.

It is also necessary to include at least one selectable marker gene in the DNA used to transform the host strain. This facilitates selection and isolation of those organisms which have incorporated the transforming DNA. The marker gene confers a phenotypic trait to the transformed organism which the host did not have, e.g., restoration of the ability to produce a specific amino acid where the untransformed host has a defect in the specific amino acid biosynthetic pathway or resistance to antibiotics and the like. Exemplary selectable marker genes may be selected from the group consisting of the HISA gene and the ARG4 gene from Pichia pastoris and Saccharomyces cerevisiae, the invertase gene (SUC2) from Saccharomyces cerevisiae, and the G418R If the first insertable DNA fragment does not contain a regulatory region, a suitable regulatory region will need to be inserted operably linked to the structural gene, in order to provide an operable expression cassette. Similarly, if no 3' termination sequence is provided at the insertion site to complete the expression cassette, a 3' termination sequence will have to be operably linked to the structural gene to be inserted.

It is important that integration occurs at a position in the genome that will not have a deleterious effect on the host cell. It was a surprising discovery that integration of a recombinant expression construct at the Eighia pastoris acid phosphatase gene locus (fiflgl) was not deleterious to the Pichia host and led to stable integration of the recombinant sequences. Directed integration at the gag; locus is accomplished by the use of the 5' and 3' sequences which flank the Eighia pastoris gag; gene (which are also refererred to as first and second insertable DNA fragments), in recombinant expression vectors.

A further discovery was a method for screening transformed cells to identify those which integrated the expression vector sequences Cells in which the gag; gene has been disrupted show a concomitant loss of acid phosphatase enzyme activity. by disrupting the PH01 locus.

The Pho- phenotype, indicative of gag; disruption, may be screened for by plating the cells on low phosphate indicator plates and allowing colonies to grow overnight. Colonies in which the gag; gene is disrupted are white, whereas those colonies having an intact gag; gene are green. This colorimetric screen provides a rapid and easy method for detecting cells which have integrated expression cassettes correctly at the ggg; locus.

A partial restriction map of the Eighia pastoris acid phosphatase gene (SEQ ID NO:l) is depicted in Figure 1. This gene has been further characterized by the nucleotide sequence which is provided in Table 1.

Also provided by the present invention are novel DNA fragments comprising the Eighig pastoris acid phosphatase 5' regulatory region (SEQ ID N022), signal sequence (SEQ ID NO:3), structural gene (SEQ ID N0:4), and 3' transcription termination sequence (SEQ ID N025).

The following Tables denote the sequences of the Eighig pastoris acid phosphatase gene (SEQ ID N0:1) and fragments thereof.

Table 1 Acid Phosphatase Gene SEQ ID N021: BamHI -390 -370 -350 GGATCCCTATTGTTACTTTTGCTTAACATTCCAATATTCTTCAACGGTTAATTGATTAAC -330 -310 -290 ACTGTAACCTCTGCCCATGTGCTTCATCCAAATCTGGTAATCTGCTTTCTATTTCTGCCA -270 -250 -230 AAATAGTTAATCTATGAGACATGTGCCCTCAATTGCGCAGTAGATCGAGTGGAAGTCTTC -210 -190 -170 TTTGCGTAACACTCAAAGTATATCCCTGTTAGTCTTTATTCACCTGTTGCTGCATTGGTG -150 -130 -110 TCAGTTACCATTATTGTTTCCACTTGGAAAAGCTTGTTTTTTTTTGATAGCACAGAAACG -90 -70 -50 TGGGCTCCGATAAGCTAAACTTCAACGAGAATATAAAAGCTGAAAAGATTCTTGTCAAGA -30 -10 10 ACTTGTACAACGACCAATAAGTCTTTCAAGGCATCAGACATGTTTTCTCCTATTCTAAGT MetPheSerPro11eLeuSer 70 CTGGAAATTATTCTCGCTTTGGCTACTCTCCAATCAGTCTTTGCGGTTGAGTTGCAGCAC LeuG1u11e11eLeuA1aLeuA1aThrLeuG1nSerVa1PheA1aVa1G1uLeuG1nHis Bell 130 GTTCTTGGAGTCAACGACAGACCCTATCCTCAGAGGACAGATGATCAGTACAACATTCTG Va1LeuG1yVa1AsnAspArgProTyrProG1nArgThrAspAspG1nTyrAsn11eLeu 190 AGACATCTGGGAGGCTTGGGCCCCTACATCGGTTACAATGGATGGGGAATTGCTGCTGAG ArgHisLeuGlyG1yLeuG1yPRoTyrl1eG1yTyrAsnG1yTrpG1y11eA1aAlaGlu 250 TCTGAAATTGAATCCTGTACGATTGATCAGGCTCATCTGTTGATGAGACATGGAGAAAGA SerG1u11eGluSerCysThr1leAspG1nA1aHisLeuLeuHetArgHisG1yG1uArg 310 TACCCAAGTACCAATGTGGGGAAACAACTAGAAGCTTTGTACCAGAAACTACTAGATGCT TyrProSerThrAsnVa1G1yLysG1nLeuG1uA1aLeuTyrG1nLysLeuLeuAspA1a 370 GATGTGGAAGTCCCTACAGGACCATTGTCTTTCTTTCAAGACTATGATTACTTCGTCTCT AspVe1G1uVa1ProThrG1yProLeuSerPhePheG1nAspTyrAspTyrPheVa1Ser 430 GACGCCGCTTGGTACGAGCAAGAAACAACTAAGGGTTTCTACTCGGGGTTAAACACCGCT AspA1aA1aTrpTyrGluG1nG1uThrThrLysG1yPheTyrSerG1yLeuAsnThrAla 490 TTCGATTTTGGTACCACTTTGAGAGAACGATATGAACATTTGATAAACAATAGCGAAGAA PheAspPheG1yThrThrLeuArgG1uArgTyrG1uH1sLeu11eAsnAsnSerG1uG1u 550 GGAAAGAAACTTTCTGTTTGGGCTGGCTCTCAAGAAAGAGTTGTTGACAACGCAAAGTAC G1yLysLysLeuSerVa1TrpA1aG1ySerG1nG1uArgValValAspAsnA1aLysTyr 610 TTTGCTCAAGGATTTATGAAATCTAACTACACCGTTATGGTCGAAGTCGTTGCTCTAGAA PheAlaG1nG1yPheMetLysSerAsnTyrThrVa1HetVa1G1uVa1Va1A1aLeuG1u 670 GAGGAGAAATCCCAGGGACTCAACTCTCTAACGGCTCGAATTTCATGTCCAAACTATAAC G1uG1uLysSerG1nGlyLeuAsnSerLeuThrA1aArg11eSerCysProAsnTyrAsn 730 AGCCATATCTACAAAGATGGCGACTTGGGGAATGACATTGCTCAAAGAGAAGCTGACAGA SerH1s11eTyrLysAspG1yAspLeuG1yAsnAspl1eA1aGinArgG1uAlaAspArg 790 TTGAACACTCTTTCTCCAGGATTTAACATTACTGCAGATGATATTCCAACAATTGCCCTA LeuAsnThrLeuSerProG1yPheAsn11eThrA1aAspAsp11eProThr11eA1aLeu 850 TACTGTGGCTTTGAACTAAATGTAAGAGGTGAGTCATCCTTCTGTGACGTCTTGTCAAGA TyrCysG1yPheG1uLeuAsnVa1ArgG1yG1uSerSerPheCysAspVe1LeuSerArg 910 GAGGCTCTACTGTACACTGCTTATCTTAGAGATTTGGGATGGTATTACAATGTTGGAAAC G1uAlaLeuLeuTyrThrA1aTyrLeuArgAspLeuG1yTrpTyrTyrAsnVa1G1yAsn 970 GGGAACCCACTTGGAAAGACAATCGGCTACGTCTATGCCAACGCCACAAGACAGCTGTTG G1yAsnProLeuG1yLysThr11eG1yTyrVa1TyrA1aAsnA1aThrArgGinLeuLeu 1030 GAAAACACAGAAGCTGATCCTAGAGATTATCCTTTGTACTTTTCCTTTAGTCATGATACC G1uAsnThrGluA1aAspProArgAspTyrProLeuTyrPbeSerPheSerHisAspThr 1090 GATCTGCTTCAAGTATTCACTTCACTCGGTCTTTTCAACGTGACAGATCTGCCATTAGAC AspLeuLeuGinVa1PheThrSerLeuGlyLeuPheAsnVa1ThrAspLeuProLeuAsp Ncol CAGATTCAATTCCAGACCTCTTTCAAATCTACCGAAATAGTTCCCATGGGAGCAAGATTG Gin11eGinPheGinThrSerPheLysSerThrGlu11eVa1ProHetG1yA1aArgLeu 1210 CTTACCGAGAGATTATTGTGTACTGTTGAAGGTGAAGAAAAATACTACGTTAGAACTATC LeuThrG1uArgLeuLeuCysThrVa1G1uG1yG1uG1uLysTyrTyrValArgThr11e 1270 CTCAACGATGCAGTCTTCCCACTGAGTGACTGTTCCTCTGGCCCTGGATTCTCTTGTCCG LeuAsnAspA1aVa1PheProLeuSerAspCysSerSerG1yProG1yPheSerCysPro 1330 TTGAACGATTATGTTTCTAGACTTGAGGCATTGAACGAGGACAGTGACTTTGCGGAAAAC LeuAsnAspTyrVa1SerArgLeuG1uAlaLeuAsnG1uAspSerAspPheA1aG1uAsn 1390 TGTGGAGTTCCTAAAAATGCTTCCTACCCACTTGAACTATCATTCTTCTGGGATGACTTG CysG1yVa1ProLysAsnAlaSerTyrProLeuG1uLeuSerPhePheTrpAspAspLeu 1450 TCATAAAAATGGTAAGGAATGTTTTGCATCAGATACGAGTTCAAAACGATTAAGAAGAGA SerEnd 1510 ATGCTCTTTTTTTTGTTTCTATCCAATTGGACTATTTTCGTTTATTTTAAATAGCGTACA 1570 ACTTTAACTAGATGATATCTTCTTCTTCAAACGATACCACTTCTCTCATACTAGGTGGAG BamHI GTTCAATGGATCC Table 2 ' Regulatory Region SE ID N012: BamHI -390 -370 -350 GGATCCCTATTGTTACTTTTGCTTAACATTCCAATATTCTTCAACGGTTAATTGATTAAC -330 -310 -290 ACTGTAACCTCTGCCCATGTGCTTCATCCAAATCTGGTAATCTGCTTTCTATTTCTGCCA -270 -250 -230 AAATAGTTAATCTATGAGACATGTGCCCTCAATTGCGCAGTAGATCGAGTGGAAGTCTTC -210 -190 -170 TTTGCGTAACACTCAAAGTATATCCCTGTTAGTCTTTATTCACCTGTTGCTGCATTGGTG -150 -130 -110 TCAGTTACCATTATTGTTTCCACTTGGAAAAGCTTGTTTTTTTTTGATAGCACAGAAACG -90 —7o —5o TGGGCTCCGATAAGCTAAACTTCAACGAGAATATAAAAGCTGAAAAGATTCTTGTCAAGA -30 -10 ACTTGTACAACGACCAATAAGTCTTTCAAGGCATCAGAC Table 3 Signal Seguence SEQ ID NO:3: ATGTTTTCTCCTATTCTAAGT PH01 signal sequence --> HetPheSerPro11eLeuSer CTGGAAATTATTCTCGCTTTGGCTACTCTCCAATCAGTCTTTGCG LeuG1u11e11eLeuA1aLeuA1aThrLeuG1nSerVa1PheA1a Table 4 Acid Phosphatase Structural Gene SEQ ID N024: 70 GTTGAGTTGCAGCAC Va1G1uLeuG1nHis 90 110 Bc1I 130 GTTCTTGGAGTCAACGACAGACCCTATCCTCAGAGGACAGATGATCAGTACAACATTCTG Va1LeuG1yVa1AsnAspArgProTyrProG1nArgThrAspAspG1nTyrAsn11eLeu 190 AGACATCTGGGAGGCTTGGGCCCCTACATCGGTTACAATGGATGGGGAATTGCTGCTGAG ArgHisLeuG1yGlyLeuG1yPRoTyr1leG1yTyrAsnGlyTrpG1yl1eA1aA1aG1u 250 TCTGAAATTGAATCCTGTACGATTGATCAGGCTCATCTGTTGATGAGACATGGAGAAAGA SerG1u11eG1uSerCysThr11eAspG1nA1aHisLeuLeuHetArgHisG1yG1uArg 310 TACCCAAGTACCAATGTGGGGAAACAACTAGAAGCTTTGTACCAGAAACTACTAGATGCT TyrProSerThrAsnVa1G1yLysG1nLeuG1uA1aLeuTyrGlnLysLeuLeuAspA1a 370 GATGTGGAAGTCCCTACAGGACCATTGTCTTTCTTTCAAGACTATGATTACTTCGTCTCT AspVa1G1uVa1ProThrG1yProLeuSerPhePheG1nAspTyrAspTyrPheVa1Ser 430 GACGCCGCTTGGTACGAGCAAGAAACAACTAAGGGTTTCTACTCGGGGTTAAACACCGCT AspA1aA1aTrpTyrG1uG1nG1uThrThrLysG1yPheTyrSerG1yLeuAsnThrA1a 490 TTCGATTTTGGTACCACTTTGAGAGAACGATATGAACATTTGATAAACAATAGCGAAGAA PheAspPheG1yThrThrLeuArgG1uArgTyrG1uH1sLeu11eAsnAsnSerG1uG1u 550 GGAAAGAAACTTTCTGTTTGGGCTGGCTCTCAAGAAAGAGTTGTTGACAACGCAAAGTAC G1yLysLysLeuSerVa1TrpA1aG1ySerG1nGluArgVa1Va1AspAsnA1aLysTyr 610 TTTGCTCAAGGATTTATGAAATCTAACTACACCGTTATGGTCGAAGTCGTTGCTCTAGAA PheA1aG1nG1yPheMetLysSerAsnTyrThrVa1HetVa1G1uVa1Va1A1aLeuG1u 670 GAGGAGAAATCCCAGGGACTCAACTCTCTAACGGCTCGAATTTCATGTCCAAACTATAAC G1uG1uLysSerG1nG1yLeuAsnSerLeuThrA1aArg11eSerCysProAsnTyrAsn 730 AGCCATATCTACAAAGATGGCGACTTGGGGAATGACATTGCTCAAAGAGAAGCTGACAGA SerH1s11eTyrLysAspG1yAspLeuG1yAsnAsp11eA1aGinArgG1uA1aAspArg 790 TTGAACACTCTTTCTCCAGGATTTAACATTACTGCAGATGATATTCCAACAATTGCCCTA LeuAsnThrLeuSerProG1yPheAsn11eThrA1aAspAsp11eProThr11eA1aLeu 850 TACTGTGGCTTTGAACTAAATGTAAGAGGTGAGTCATCCTTCTGTGACGTCTTGTCAAGA TyrCysG1yPheGluLeuAsnVa1ArgG1yGluSerSerPheCysAspVa1LeuSerArg 910 GAGGCTCTACTGTACACTGCTTATCTTAGAGATTTGGGATGGTATTACAATGTTGGAAAC G1uA1aLeuLeuTyrThrA1aTyrLeuArgAspLeuG1yTrpTyrTyrAsnVa1G1yAsn 970 GGGAACCCACTTGGAAAGACAATCGGCTACGTCTATGCCAACGCCACAAGACAGCTGTTG G1yAsnProLeuG1yLysThr11eGlyTyrVa1TyrA1aAsnA1aThrArgGinLeuLeu 1030 GAAAACACAGAAGCTGATCCTAGAGATTATCCTTTGTACTTTTCCTTTAGTCATGATACC G1uAsnThrG1uA1aAspProArgAspTyrProLeuTyrPheSerPheSerHisAspThr 1090 GATCTGCTTCAAGTATTCACTTCACTCGGTCTTTTCAACGTGACAGATCTGCCATTAGAC AspLeuLeuGinVa1PheThrSerLeuG1yLeuPheAsnVa1ThrAspLeuProLeuAsp Noel CAGATTCAATTCCAGACCTCTTTCAAATCTACCGAAATAGTTCCCATGGGAGCAAGATTG Ginl1eGinPheGinThrSerPheLysSerThrGlu11eVa1ProHetG1yA1aArgLeu 1210 CTTACCGAGAGATTATTGTGTACTGTTGAAGGTGAAGAAAAATACTACGTTAGAACTATC LeuThrG1uArgLeuLeuCysThrVa1G1uG1yG1uG1uLysTyrTyrVa1ArgThr11e 1270 CTCAACGATGCAGTCTTCCCACTGAGTGACTGTTCCTCTGGCCCTGGATTCTCTTGTCCG LeuAsnAspA1aVa1PheProLeuSerAspCysSerSerG1yProG1yPheSerCysPro 1330 TTGAACGATTATGTTTCTAGACTTGAGGCATTGAACGAGGACAGTGACTTTGCGGAAAAC LeuAsnAspTyrVa1SerArgLeuG1uA1aLeuAsnG1uAspSerAspPheA1aG1uAsn 1390 TGTGGAGTTCCTAAAAATGCTTCCTACCCACTTGAACTATCATTCTTCTGGGATGACTTG CysG1yVa1ProLysAsnA1aSerTyrProLeuG1uLeuSerPhePheTrpAspAspLeu TCATAA SerEnd Table 5 3' Transcription Termination Sequence SEQ ID NO:5: 1450 AAATGGTAAGGAATGTTTTGCATCAGATACGAGTTCAAAACGATTAAGAAGAGA 1510 ATGCTCTTTTTTTTGTTTCTATCCAATTGGACTATTTTCGTTTATTTTAAATAGCGTACA 1570 ACTTTAACTAGATGATATCTTCTTCTTCAAACGATACCACTTCTCTCATACTAGGTGGAG BamHI GTTCAATGGATCC The acid phosphatase gene is recovered from Biggie pastoris cultures such as Eighia pastoris NRRL Y-11430 by methods as set forth in the following Examples. A general method for recovering the giggle pastoris acid phosphatase gene (SEQ ID NO:1) consists of using an acid phosphatase probe, such as the low phosphate (LP) probes described in the following examples, to screen a library of gighia pastoris DNA.

Other probes could be selected or synthesized based on the sequence disclosed in Table 1. any suitable protocol known to those skilled in the art.

Alternatively, the acid phosphatase 5' regulatory region (SEQ ID NO:2), signal sequence (SEQ ID NO:3), structural gene (SEQ ID NO:h), and 3' regulatory region (SEQ ID N025) may be obtained by synthesizing the appropriate sequence as defined in Tables 1-5 using known enzymatic or chemical means. Suitable means include but are not limited to chemical procedures based on phosphotriester, phophite, or cyanoethylphosphoramidite chemistry.

Those skilled in the art will also recognize that the isolated Eighig pastoris acid phosphatase 5' regulatory region (SEQ ID NO:2), signal sequence (SEQ ID N0:3), structural gene (SEQ ID NO:4), and ' transcriptional termination sequence (SEQ ID N0:S) of the present invention as compared to subsequently isolated Eighia pastoris acid phosphatase 5' regulatory region, signal sequence, structural gene, and 3' transcriptional termination sequences may contain a de minimis number of nucleotide differences due to clonal variation or sequencing error which may occur.

Modification of the Eighig pastoris acid phosphatase ' regulatory region (SEQ ID N0:2), signal sequence (SEQ ID NO:3), structural gene (SEQ ID NO:4), and 3' transcriptional termination sequence (SEQ ID N025) can also be performed, such as adding linker DNA, or performing nutagenesis (for example H13 mutagenesis) to provide or remove restriction site(s) and the like.

Once the Eighia pastoris acid phosphatase gene is recovered, it may be maintained or replicated in eucaryotic or procaryotic plasmid-host systems, such as pBR322 maintained in E. coli, or in any other suitable system known in the art.

Those skilled in the art will also recognize that numerous additional DNA sequences can also be incorporated into the vector employed, such as bacterial plasmid DNA, various marker genes, bacteriophage DNA, autonomous replicating sequences, and centromeric DNA, to name only a few representative examples.

The acid phosphatase 5' regulatory region (SEQ ID N0:2) is contained within the DNA fragment extending from nucleotide -399 to about nucleotide 0, as shown in Figure l and Table 2. This fragment is capable of effecting the transcription of DNA to RNA when operably linked to and positioned at the 5' end of a heterologous DNA sequence coding for at least one polypeptide.

To utilize the acid phosphatase 5' regulatory region (SEQ ID N0:2) disclosed herein, the fragment described in Figure 1 and Table 2 can be operably linked to heterologous DNA sequences encoding at least one polypeptide. For the purpose of this specification heterologous DNA sequences are combinations of DNA sequences which do not naturally occur in the host or in association with said regulatory region. Suitable heterologous DNA sequences encoding at least one polypeptide which could be operably linked with the acid phosphatase 5' regulatory region (SEQ ID N0:2) include but are not limited to tissue plasminogen activator, human serum albumin, and invertase. Heterologous DNA sequences used with the present invention should contain a 5' ATG start codon, a 3' stop codon and may additionally include nucleotide sequences which function to stabilize the mRNA, or to direct polyadenylation.

The combination of the acid phosphatase 5' regulatory region (SEQ ID NO:2) operably linked to a heterologous DNA sequence may be inserted in a suitable vector. Numerous yeast vector-host combinations Additional sequences such as marker genes or other sequences which render the are possible and are known to those skilled in the art. vector capable of growth amplification and rapid propagation in bacteria or yeast may also be present.

Suitable host cells which can be transformed with a vector containing the acid phosphatase 5' regulatory region include yeast such as those from the genera of Saccharomyces, gighig and Hansenula, preferably gighia, and most preferably Eighia pastoris.

Transformation of a suitable host cell with a vector containing the acid phosphatase 5' regulatory region (SEQ ID N022) can be accomplished by any suitable transformation technique known to those skilled in the art.

The acid phosphatase 5' regulatory region (SEQ ID N0:2) is controlled by the concentration of phosphate in the media.

Specifically, this regulatory region is derepressed by low concentrations of phosphate. Therefore, the 5' regulatory region is regulated by changing the concentration of phosphate present in the media.

The acid phosphatase signal sequence (SEQ ID NO:3) is contained within the DNA fragment extending from nucleotide 1 to about nucleotide 66 as shown in Figure 1 and Table 3. To utilize the acid phosphatase signal sequence (SEQ ID N0:3) disclosed herein, the fragment described in Figure 1 and Table 3 can be operably linked to heterologous DNA sequences encoding at least one polypeptide. Suitable heterologous DNA sequences include but are not limited to those DNA sequences selected from the group consisting of tissue plasminogen activator, human serum albumin, and invertase.

The combination of the acid phosphatase signal sequence (SEQ ID NO 3) operably linked to a heterologous DNA sequence may then be linked to a suitable promoter. Conveniently, the promoter which is employed may be the promoter associated with the leader sequence.

Alternatively, one may replace the naturally occuring gag; promoter with other heterologous promoters which would allow for transcriptional regulation. An example of a suitable heterologous promoter would be the Pichia pastoris alcohol oxidase (AOXI) promoter (or 5' regulatory region) disclosed in U.S. 4,808,537, published on February 28, 1989.

Suitable vectors into which this DNA fragment containing the acid phosphatase signal sequence (SEQ ID NO:3) could be inserted may be obtained as described above. Additionally, suitable host cells which may be transformed with the resulting vector containing a DNA fragment coding for a signal sequence include yeast such as those from the genera Saccharomyces, Hansenula, and Pichia, with Pichia pastoris being preferred. Transformation of these host cells can be accomplished by any suitable means known to those skilled in the art. The signal sequence that is operably linked to a protein that has been produced by one of these vector/ host systems may direct the secretion of said protein from the host cell.

The acid phosphatase structural gene (SEQ ID N0:h) is contained within the DNA fragment extending from nucleotide 67 to nucleotide 1407 as shown in Figure 1 and Table 4. The acid phosphatase structural gene (SEQ ID NO:4) may be utilized in recombinant biotechnology for a variety of purposes including but not limited to: (a) DNA constructs for performing disruptive homologous recombination (a process for inserting heterologous DNA sequences into the Eighia pastoris genome at the acid phosphatase locus and thus disrupting the acid phosphatase gene activity, and (b) the production of acid phosphatase protein for use in various bioassays.

The acid phosphatase 3' transcription termination sequence (SEQ ID NO:S) terminates the transcription of mRNA or stabilizes mRNA when operably linked to the 3' end of a DNA sequence which codes for the production of a polypeptide. This acid phosphatase 3' transcription termination sequence (SEQ ID N0:S) is contained within the DNA fragment extending from nucleotide 1h08 to about nucleotide 1594 as shown in Figure 1 and Table 5. The acid phosphatase 3' transcription termination sequence (SEQ ID N0:S) may be operably linked to a heterologous DNA sequence which codes for a polypeptide, and used to terminate transcription of, or to stabilize mRNA in yeast such as those from the genera Saccharomyces, Hansenula, and Pichia, but it is particulary well suited for use in Pichia gastoris.

The following non-limiting Examples are provided to further illustrate the practice of the present invention.

Strains Examples The following strains have been used in these Examples: Pichia Pichia Pichia Pichia Bishié Pichia Bishlé £i£hi2 Pichia Pichia Pichia Pichia pastoris pastoris pastoris pastoris pastoris pastoris pastoris pastoris pastoris pastoris pastoris pastoris (strA) KH71 (ggg;, hisfi) cs11s (hish) NRRL Y-15851 cs19o (arga) NRRL Y-18014 GS247 (Ade-) MB102-51 KM71:pPSU216 cunt‘) GS115:pPSU216 (Mut+) E. coli. JM103 delta (lac pro) thi rpsl supE end A sbcB hsdR Bowes melanoma tPA over-expressing cell line ATCC# CRL9607 (human melanoma cells) Media, Buffers, and Solutions The media, buffers, and solutions employed in the following Examples have the compositions indicated below: LP Media biotin calcium pantothenate low hos hate wg/L A00 pg/L folic acid nicotinic acid p-aminobenzoic acid pyridoxine hydrochloride riboflavin thiamine-HC1 boric acid cupric sulfate potassium iodide ferric chloride manganese sulfate zinc sulfate inositol sodium molybdate ammonium sulfate monobasic potassium phosphate potassium chloride sodium chloride calcium chloride magnesium sulfate TE Buffer Tris-HC1, pH 8.0 EDTA SSPE (IX) NaC1 Na,PO., pH 7.7 EDTA 400 S00 40 100 200 .5 1.7 us/L us/L us/L us/L us/L P8/L P8/L H8/L P8/L us/L P8/L P8/L mg/L P8/L 8/L mg/L 8/L AH .68 nM mM IH IH nH mfi NaC1 Na citrate Denhardt's solution (IX) Ficoll polyvinylpyrrolidone bovine serum albumin 200 mM mM mg/L mg/L mg/L BEE LiC1 Tris-HC1, pH 7.4 EDTA B91 phenol chloroform isoamyl alcohol CI chloroform isoamyl alcohol LP Indicator glates, 1 liter mM 100 mM 0.1 mM ml/L £80 ml/L ml/L ml/L 40 ml/L X LP media 22.5 mg citric acid pH 4.3 g dextrose 60 mg 5~bromo,4-chloro, 3-indolyl phosphate (Sigma) g Noble agar (Difco) SCE Buffer 9.1 g sorbitol 1.47 g sodium citrate 0.168 g EDTA pH to 5.8 with HCI in ml dH2O and autoclave YNB Media 6.75 g yeast nitrogen base without amino acids (DIFCO) in 1 L of water Example I Construction of QLPZ4 In order to isolate and characterize the acid phosphatase gene from Eighig pastoris (SEQ ID NO:1), the following experiments were performed. gighig pastoris GS11S (NRRL Y-15851) was grown in both a high phosphate (HP) environment [comprised of yeast nitrogen base minus amino acids (DIFCO), 2% dextrose, and 20 mg/1 histidine] and in a low phosphate (LP) environment [comprised of LP media, 2% dextrose, and 20 mg/1 histidine], each in a 300 ml total volume. The cells were pelleted, washed once with 10 ml REB, and resuspended in & ml REB in a ml Corex tube. 8 g of glass beads and 4 ml PCI were then added. The suspension was mixed on a vortex mixer at high speed, eight times at 20 seconds each, with cooling on ice for 20 seconds between mixings. The suspension was then centrifuged at 10,000 X g for 10 min. The aqueous (top) layer was extracted twice with h ml PCI and 4 ml CI. The RNA was precipitated from the aqueous phase, at -20°C, with 0.1 volume of 3g potassium acetate, pH 5.2 and 2.5 volumes of ethanol. Poly A+ RNA was selected as per Maniatis et al. (Molecular Cloning, A Laboratory Manual), with LiC1 substituted for NaCl. Synthesis of LP or HP mRNA-labeled cDNA probes, using 2 pg of each type of poly A+ RNA, was also according to Haniatis et al. ng of a purified Eighig pastoris plasmid library in YEP13 [Broach et al., gggg: 8121 (l979)] was used to transform E. coli. HClO6l 1970, J. Mol. Biol. S3:lSh).

(Handel and Higa, Approximately 8,000 colonies resulted. The colonies were replicated on duplicate nitrocellulose filters, amplified on LB plates containing 100 ug/ml ampicillin and 170 pg/ml chloramphenicol, lysed by standard protocols 1979, Methods in Enzymology 68, 379-389), baked at 80°C for 90 min., and separate filters were hybridized with either the LP or HP cDNA probes.

Denhardt's solution, 0.2% sodium dodecyl sulfate (SDS), and 2.5 X 10’ (Grunstein and Wallis, Hybridization was performed using 2X SSPE, 1X cpm labeled CDNA probes per ml of hybridization solution. Hybridization was at 55°C for 40 hours in a 25 ml total volume. Following hybridization, the filters were washed twice in 2X SSC, 0.1% SDS at room temperature and twice in 0.2X SSC, 0.1% SDS at 65°C, then dried and exposed to x-ray film. again hybridized with the LP cDNA The fragments encoded These fragments were then restriction mapped using probe. unrelated phosphate-regulated gene segments as determined by the differences in their restriction maps.

The regions identified by this procedure as encoding LP-regulated genes were subcloned into pUC8 or pUCl9 (New England Biolabs). translation (Haniatis).

The resultant plasmids were labeled with 32F by nick The labeled plasmids were used to probe RNA blots of LP and HP RNA (Spg/blot). One of the original 24 clones, identified as pLP2l+, was chosen for further study.

Exanple II Construction of pLP2h11 The plasmid pLP24 generated in Example I and thought to E. coli strain HCl061 was transformed and the correct The correct plasmid was called Example III Construction of pLP2412 PH01-Disruption Vector and Development of GSl90:pLP2412 This fragment contained the Saccharonyces cerevisiae ARG4 gene.

The 3.3 kb fragment was isolated and used to transform Pichia pastoris GSl90 (NRRL Y-18014) to Arg+ prototropy (the transformation procedure is described in Example IV). Arginine prototrophs were identified by their ability to grow in media lacking arginine. They were isolated and screened on LP indicator plates for the presence of acid phosphatase.

The colonies were replica-plated to LP indicator plates and allowed to grow overnight at 30°C. Colonies on the LP indicator plates were either green (PHOI) or white (phol). Vhite colonies were transformants containing the 3.3 kb expression cassette from above, stably integrated by disruption at the PH0l locus of the Pichia pastoris genome.

Genomic DNAs from stable Arg+ strains were analyzed by Southern filter hybridization to determine the location of the expression cassette. The 3.3 kb EEQRI-ggmfll fragment, containing the Saccharomyces cerevisiae ARG4 gene, had specifically integrated and disrupted the genomic sequence which was analogous to the DNA fragment contained in pLP24ll, confirming that this locus coded for acid phosphatase (PHOI). This transformant was designated GSl90:pLP2412.

Example IV Transformation of Pichia pastoris The following protocol was used in the transformation of gighia pastoris.

Yeast cells were inoculated into about 10 ml of YPD medium and shake cultured at 30°C for 16-20 hours. an A... of about 0.01 to 0.1 and maintained in log phase in YPD medium at 30°C for about 6-8 hours.

The cells were then diluted to ml of YPD medium was inoculated with 0.5 ml of the seed culture at an A‘., of about 0.1 and shake cultured at °C for about 16-20 hours. that was about 0.2 to 0.3 (after approximately 16-20 hours) by The culture was then harvested with an A... centrifugation using a DAMON IEC DPR-6000 centrifuge at 1500 g for 5 minutes.

To prepare spheroplasts, the cells were washed once in 10 ml of sterile water (centrifugation was performed after each wash as described above), once in 10 ml of freshly prepared SED, once in 10 ml pl of 4 mg/ml Zymolyase 60,000 (available from Miles Laboratories) was added of sterile 1M sorbitol, and resuspended in 5 ml of SCE buffer. and the cells incubated at 30°C for about 30 minutes.

Spheroplast formation was monitored as follows. 100 pl aliquots of cells were added to 900 pl of 51 SDS and 900 pl of 1M sorbitol before or just after the addition of Zymolyase, and at various times during the incubation period. The incubation was stopped at the point where cells would lyse in SDS but not sorbitol. Once formed, spheroplasts were washed once in 10 ml of sterile 1g sorbitol by centrifugation at 1,000 g for 5-10 minutes, washed once in 10 ml of sterile CaS by centrifugation, and resuspended to 0.6 ml in Gas, For the actual transformation, DNA samples in water or TE buffer were added (up to 20 pl total volume) to 12 X 75 mm sterile polypropylene tubes. (For small amounts of DNA, maximum transformation occurs using about 1 pl of 5 mg/ml sonicated E. coli DNA in each 100 pl of spheroplasts were added to each DNA sample and ml of PEG sample.) incubated at room temperature for about 20 minutes. solution was added to each sample and incubated at room temperature for about 15 minutes. The samples were centrifuged at 1,000 g for 5-10 minutes and the supernatant was discarded. The pellets were resuspended in 150 pl of S08 and incubated at room temperature for 30 minutes. 850 pl of sterile 1M sorbitol was added to each, and the samples were plated as described below. ml of Regeneration Agar was poured per plate at least 30 minutes before transformation samples were ready. 10 ml aliquots of Regeneration Agar were also distributed to tubes in a 45-50° bath during the period that transformation samples were in SOS. Samples were then added to the tubes, poured onto plates containing the solid bottom agar layer, and incubated at 30°C for 3-5 days.

Spheroplast quality at various points was determined as follows. 10 pl of sample was removed and diluted 100 X by addition to pl of the dilution was removed, and an pl of both pl of lfl sorbitol. additional 990 pl aliquot of 1g sorbitol was added. dilutions were spread-plated on YPD agar medium to determine the concentration of unspheroplasted whole cells remaining in the preparation. 100 pl of each dilution was added to 10ml of Regeneration Agar which had been supplemented with 40 pg/ml of all amino acids required by the host to determine the total regeneratable spheroplasts.

Good values for a transformation experiment were 1-3 X 107 total regenerable spheroplasts/ml and about 1 X 10’ whole cells/ml.

Example V Yeast DNA Preparation The following protocol was used in the preparation of Eighlé pastoris DNA.

Yeast cells were grown in 100 ml of YNB medium plus 22 dextrose at 30°C until A,.° equaled 1-2 and then pelleted using a Damon IEC DPR-6000 centrifuge at 2,000 g for 5 minutes. The pellet was washed once in dH,0, once in SED, once in 1H sorbitol and then resuspended in 5 The cells were then mixed with S0-100 pl of a 4 mg/ml solution of Zymolyase 60,000 ml of a solution of 0.15 Tris-Cl, pH 7.0, and 1g sorbitol.

(Miles Laboratories) and incubated at 30°C for 1 hour. The resulting spheroplasts were then centrifuged at 1,000 g for 5-10 minutes and suspended in 5 ml Lysis Buffer [0.l% SDS, 10mg Tris-Cl (pH 7.4), Smg EDTA and 50 mg NaC1].

(Sigma) were each added to 100 pg/ml and the solution incubated at 37°C Proteinase K (Boehringer Mannheim) and RNase A for 30 minutes. DNA was deproteinized by gently mixing the preparation with an equal volume of CI, and the phases were separated by centrifugation at 12,000 g for 20 minutes. The upper (aqueous) phase was drawn off into a fresh tube and extracted with an equal volume of PCI. The phases were separated as before and the top phase placed in a tube containing 2-3 volumes of cold 100% ethanol. The sample was gently mixed and DNA was collected by spooling onto a plastic rod. The DNA was immediately dissolved in 1 mL of TE buffer and dialyzed overnight at 4°C against 100 volumes TE buffer.

Example VI Isolation of the Full Length PH01 Gene To isolate a plasmid containing the entire Eighia pastoris gag; gene (SEQ ID NO:1), the original gighig pastoris genomic library in HCl061 was hybridized, under the conditions described previously, with the 600 bp ggmfll fragment from pLP2h. This procedure identified five positive clones. Plasmid DNA was prepared from these clones, digested within the 2.0 kb gamhl fragment.

Example VII Development of Strains M8102-26:pLP2430-Tl and HBl02-26;pLP2h30-T3 The 2.0 kb figmfll fragment of pLP2420 containing the Egg; gene was ligated into the gamhl site of pYM8, a plasmid which contains the [(pYM8 can be Ten pg of pYA2 (NRRL B-15874) were digested with Ten pg of pBR322 Saccharomyces cerevisiae HIS4 gene and pBR322 sequences. the Saccharomyces cerevisiae HIS4 gene sequence.

GS190:pLP24l2 (Pho-), a Eighia pastoris strain lacking acid phosphatase activity (see Example III) was mated with Richie pastoris strain HDIOO-20 (higfi, Ade-). 4,812,405, Strain HDIOO-20 was developed as follows: The mating protocol used was the same as that disclosed in U.S. which is herein incorporated by reference. cells of Eighig pastoris strain GS1l5 (high) NRRL Y-15851 were mixed with cells of strain GS247 (Ade-) under conditions known to promote zygote formation and diploidization (the protocol for this procedure is found in U.S. ,812,405, published on March 14, 1989 and plated on YNB + dextrose plates to select for the prototrophic diploids. strain was called HD100.

The diploid MDl00 was cultured under conditions known to induce sporulation of Eichia pastoris diploids (also as disclosed in U 5,312,405) and the spore progeny cultured onto YNB dextrose + adenine + histidine plates. Individual colonies were tested for the ability to grow in the absence of adenine or histidine supplements. A strain able to grow without supplemented histidine but unable to grow without supplemented adenine was identified and called MDIOO-20.

Strain MBIOZ-26 was transformed with plasmid pLP243O as described in Example IV. His+ transformants were selected and screened for acid phophatase expression on LP indicator plates (see Example Ill).

Five transformants were positive for acid phosphatase expression.

MBl02-26:pLP2430-T1 and Two of these transformants, expression of acid phosphatase.

Table I -fold Strain Genotzge Units AP/OD‘°° Induction HB102-252pLP2430-Tl ade-, H154, PHO1 707 30,244 43 HB102-26!pLP2430-T3 ade‘, H154, PHO1 852 28,198 33 HB102-51 ade—, H154, PHO1 61.4 1,975 32 MB102-28 ade-, HIS4, phol o.o2 o.o3 1,5 Example VIII Secretion of Invertase open reading frame from the PHOI signal sequence (SEQ ID NO:3) into the SUC2 structural gene: PHOI sequence ...GTGTTCGCT linkers sequence from pSEYC306 CGA GAA TCC CCC GGG GAT CCG TCG ACC TGC AGC CCA GCT TTG SUC2 seguence ACA AAC GAA...

Ten pg of each plasmid, pAPINV1 or pAPINV2, were digested with figlll and used to transform GS1lS as in Example IV. Transformants containing the flglll fragment of pAPINV1, designated GS11S:pAPINV1, were selected for histidine prototrophy and screened for the Nut. phenotype, which denotes proper integration and disruption at the AOXl locus. The Hut screen was performed by replica plating colonies from glucose-containing media to methanol-containing media and evaluating growth rate on methanol. Slow growth on methanol was indicative of the Mut- phenotype. Transformants with the gglll fragment of pAPINV2, designated GS1lS:pAPINV2, were also selected for histidine prototrophy _ but were screened for the Phol- phenotype, which denotes proper integration and disruption at the PHOI locus (see Example III).

Transformants of each class were identified and cultured in YNB+ 2% glycerol, pastoris KH7l:GSlO2(Hut-) and GSllS:GS102(Mut+) which contain integrated plasmids in parallel with Pichia strains identical to pAPINVl, except that the SUC2 gene contains its native signal sequence and lacks the PH0l signal sequence. These two strains are described in EP 256,421.

KM71:pPSV216(Mut-) and GSllS:pPSV2l6(Mut+), which contain integrated Also cultured in parallel were strains plasmids identical to pAPINV2, except that sggg sequences are lacking.

Each culture was grown in YNB+2Z glycerol to an A‘°° of approximately 3.0. An aliquot of each was removed, washed in sterile water, and resuspended in 10 mls of YNB+ 0.5% MeOH. Mut+ cultures were resuspended at A‘°°=0.02, and Nut‘ cultures at A‘°°=0.2, and grown at °C for 24 hours to approximately A‘°°=0.3-0.h. At this time, approximately 1.0 mOD‘°° was assayed for invertase activity. The results are shown in Table II.

Table II Signal Secreted Total Secretion Culture 591 Sequence Invertasea Invertaseb Efficiencyc GSIIS: ' PH01 20.3 3.05 0.67 pAPINV1 KH71: - SUC2 12.5 18.9 0,56 GS102 KH7l: ‘ PH01 0 0 NA pPSV216 GS11S: + PH01 6.1 6.7 0.91 pAPINV2 GS115: + SUC2 5.3 5.7 0.93 GS102 GS11S: + PH01 0 0 NA pPSV216 secreted invertase - measured as per Goldstein and Lampen°. without Triton, expressed as units of invertase/A‘°° of culture assayed. 1 Unit = 1 pmole of glucose released/minute, at 37°C. total invertase - measured in the presence of 0.2% Triton X-100. secretion efficiency - secreted invertase/total invertase assayed.

PH01 signal present, no SUC2 sequences Goldstein and Lampen, Methods in Enzymolology 42:504-511, 1975. invertase secretion from Pichia pastoris.

Example IX Secretion of tPA (Tissue Plasminogen Activator) This Example demonstrates that the PHO1 signal sequence (SEQ ID NO:3) functions when operably linked to heterologous genes.

. Isolation of tPA-encoding CDNA The Bowes melanoma tPA~overexpressing cell line, ATCC# CRL9607, was the source of RNA used to construct a cDNA library.

This cell line was used by others to clone tPA sequences (Pennica gt used to make CDNA using a commercial cDNA cloning kit (available from Poly A+ RNA was isolated and oligo dT-primed, Laboratory Manual; Cold Spring Harbor Laboratory, 1982).

Amersham), and inserted into the Xgtll cloning vector. Xgtll phage containing inserts were infected into E. coli host Yl088, using a commercial packaging mixture (available from Stratagene).

The library was triplicate plated and probed with three different oligonucleotides designed from the published sequence of the human tPA cDNA (Pennica et al., supra). The oligonucleotides corresponded to the 5', middle, and 3' sections of the cDNA and were of the following sequences: ' probe:S' GAC TGG ATT CGT GAC AAC ATG CGA CCG TGA 3' middle probe:s' TCA CAG TAC TCC CAC GTC AGC CTG ccc TTC 3' s‘ probe:5' GAG ccc TCT CTT CAT TGC ATC CAT GAT TGC T 3' identified by EEQRI digestion.

Positive transformants were A clone carrying the insert in the sense orientation was identified by a gstl restriction pattern of h20bp, bp, 760bp, and 2.7kb fragments and was called pUCl8#3. A clone carrying the insert in the antisense orientation was identified by a Estl restriction pattern of 420 bp, 624 bp, and 3.46 kb, and was called pUCl8#2. acid of mature tPA through 1972 nucleotides of 3' noncoding sequence.

The insert encodes from two nucleotides before the first amino . Construction of vector pT37 (PH01ss-tPA-pUC) The tPA insert from pUC18#2 was cloned into the Eighia pastoris expression plasmid pAO8l0. Plasmid pAO810 is comprised of the gighig pastoris gggi promoter, the gighig pastoris gag; signal sequence, the gggi transcription terminator, the Biggie pastoris fllfifi gene for selection, an fl origin of replication, and sequences necessary for replication and selection in bacteria. The construction of pAO8l0 is described in Example XI.

The mutagenizing oligonucleotide was of sequence: ' CAA TCT GTC TTC GCT TCT TAC CAA GTG ATC 3' are shown in Table III.

Table III *tPA pg[L Effici- Runfl Strain Signal Mode Internal External Total ency** 1 RM7l:pT7 tPA continuous 255 60 315 .19 2 KM71:pT7 tPA fed batch 651 30 681 .04 3 KM71:pT37 PHOI continuous 840 A20 1260 .33 A KH71:pT37 PH01 fed batch 1856 1200 3056 .39 * tPA assayed by ELISA using human tPA as standard ** efficiency of tPA secretion = external tPA/total tPA produced The results of this experiment show that regardless of the mode of fermentation the Egg; signal sequence (SEQ ID N013) was more efficient at promoting tPA secretion in fiighia pastoris than was the native signal sequence.

In addition, micro—Edman degradation analysis confirmed that the N-terminal amino acid sequence of the recombinant, secreted tPA from strain KM7l:pT3, using the 339; signal sequence (SEQ ID NO:3) was identical to the native tPA purified from a cultured human tissue (melanoma) source.

. Construction ofgpT7 (tPAss-tPA-no pUC) modified 3' end was sequenced and shown to be of the correct sequence with no extra pUC18 sequences. This plasmid was called pT&9.

Oligonucleotides encoding the authentic t-PA signal sequence were added to the Xbgl site of plasmid pT49, and then two mutageneses were performed to I) delete the extra 8 nucleotides of sequence remaining between the t-PA signal sequence and the sequence coding for mature t-PA, and 2) delete the 359; signal sequence. These manipulations were accomplished as follows.

An oligonucleotide of the following sequence was synthesized (109 nucleotides): ' CTA GAT GGA TGC AAT GAA GAG AGG GCT CTG CTG TGT GCT GCT GCT GTG TGG AGC AGT CTT CGT TTC GCC CAG CCA GGA AAT CCA TGC ccc ATT CAG AAG AGG AGC CAG A3‘ The complementary oligonucleotide needed for the second strand was synthesized as three oligonucleotides, of 33, 37, and 39 nucleotides in length, respectively, which were of the following sequences: ' CTG GCT GGG CGA AAC GAA GAC TGC TCC ACA CAG 3' ' CTA GTC TGG CTC CTC TTC TGA ATC GGG CAT GGA TTT C 3' ' CAG CAG CAC ACA GCA GAG CCC TCT CTT CAT TGC ATC CAT 3' The full three oligonucleotides were kinased and then annealed together by heating in a length oligonucleotide and the complementing boiling water bath for 3 min, and then slowly cooled to room temperature. Annealed oligonucleotide was separated and isolated from a 52 polyacrylamide gel. ng of partially Xbal-linearized pTh9 was ligated to 1 pg of the annealed double-stranded oligonucleotide. The ligation reaction was transformed into E. coli HC1061 cells and ampR colonies were selected. Clones containing the correctly altered plasmid were identified by an additional 700 bp band upon digestion with Asull. A correct plasmid was called pTS44.

The first mutagenesis was accomplished by standard fl-based site-directed mutagenesis using pTSAh DNA (5 pg) and an oligonucleotide (1 pg) of the following sequence: ' GA TTC AGA GGA GCC AGA TCT TAC CAA GTG ATC TGC AG 3' The correct plasmid was screened for by figlll digestion. The correct restriction pattern (1.1, 2.8 and 5.3 kb) was indicative of correct plasmid, which was called p'l‘64. .8 and 6.3 kb bands.) (Incorrect plasmid showed a pattern of The second round of mutagenesis was performed with plasmid pT64 to delete the PHOl signal sequence. An oligonucleotide of the following sequence was used: ' ACT AAT TAT TCG AAA CGA TGG ATG CAA TGA AGA GAG G 3' pg of pT6& and 0.4 pg of the oligonucleotide above were used in the mutagenesis. Mini-preps were screened by digestion with gglll. The correct plasmid was identified by three DNA fragments of 1, 2.8, and 5.3 kb in size (incorrect plasmid showed a smallest band of 1.1 kb instead of 1 kb). The mutagenesis was confirmed by sequencing, and the correct plasmid was called pT7.

Example X Construction of pAPB101: PH01 promoter-1acZ expression plasmid fragment was isolated from a 0.8% agarose gel. of pBR322 DNA, "1075 bp of the Egg; 5' flanking DNA including the 359; promoter (SEQ ID N0:2), and 123 bp of 359; coding sequence was isolated from a 1% agarose gel. 100 ng of the EEQRI-ggmfll fragment of pSAOH5 and 60 ng of the 1.6 kb EEQRI-ggll fragment of pLP2&20 were ligated in a 20 pl mixture of ligase buffer with 1 mM ATP and IU of T4 DNA ligase (available from Boehringer Mannheim). The ligation mixture was transformed into E. coli JHIO3, and the transformed cells were plated onto LB Amp plates which contained A0 pg/ml X-gal. Blue-colored lacZ gene placed under the regulation of the Pichia pastoris PH01 An expression cassette comprised of the E. coli promoter element (SEQ ID NO:2) was contained in pAPB10l. pg of uncut pAPB101 were transformed into GS11S spheroplasts and histidine prototrophs were selected. 12 His+ colonies were chosen, the strains grown in liquid high phosphate (HP) and LP media, and assayed for B-galactosidase activity. Positive transformant strains were identified on the basis of B-galactosidase expression after growth in LP media and a lack of B-galactosidase after growth in HP media. B~ga1actosidase was assayed as per J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold Spring Harbor, NY . Blue colonies appeared on the LP-X-gal plates and white colonies appeared on the HP-X-gal plates.

Example XI The following plasmids were constructed for use in other Examples in this application. 1. Construction of Plasmid pAO810 E. coli JM103 cells, and correct phage was identified by isolating DNA the presence of a 950 bp fragment. and was called pPSV101.

One picomole of pPSV10l was subjected to in vitro oligonucleotide-directed, site-specific mutagenesis using 20 pmol of an oligonucleotide of the following sequence: ' CGA GGA ATT ccc ccc GAT ccr TAG ACA T 3' synthetic DNA fragment coding for the Pichia pastoris PHO1 signal sequence (SEQ ID N023) using the following optimized Pichia codons: '-AATTC ATG TTC TCT CCA ATT TTG TCC TTG GAA ATT ATT TTA GCT TTG GCT ACT 3'-G TAC AAG AGA GGT TAA AAC AGG AAC CTT TAA TAA AAT CGA AAC CGA TGA TTG CAA TCT GTC TTC GCT CGA G-3' AAC GTT AGA CAG AAG CGA GCT C TTAA-5' pmoles of an oligonucleotide of the sequence: ' CTAA TTA TTC GAA ACG ATG TTC TCT CCA ATT 3' Correct phage DNA was identified by plaque hybridization with the same oligonucleotide used above. The correct plasmid was called pPSV10A.

Plasmid pAO810 was prepared by digesting 10 pg of pPSV104 with Hindlll, and isolating the 400 bp DNA Fragment from a 1.2% agarose gel. ng of this fragment were ligated with 250 ng of the 7.9 kb fligdlll digestion product of pA0807 (the preparation of which is described hereinbelow) as isolated from a 0.8% agarose gel. The ligation was transformed into E. coli HCl06l cells and ampR colonies were selected.

. Creation of pAO807 ; Preparation of fl-ori DNA fl bacteriophage DNA (50 ug) was digested with 50 units of Bsgl and Dral (according to manufacturer's directions) to release the ‘A58 bp DNA fragment containing the fl origin of replication (ori). The followed by extracting the aqueous layer with an equal volume of CI and finally the digestion mixture was extracted with an equal volume of PCI DNA in the aqueous phase was precipitated by adjusting the NaCl concentration to 0.2M and adding 2.5 volumes of absolute ethanol. The mixture was allowed to stand on ice (h°C) for 10 minutes and the DNA precipitate was collected by centrifugation for 30 minutes at 10,000 xg in a microfuge at 4°C.

The DNA pellet was washed 2 times with 70% aqueous ethanol.

The washed pellet was vacuum dried and dissolved in 25 ul of TE buffer.

This DNA was electrophoresed on 1.5% agarose gel and the gel portion containing the "458 bp fl-ori fragment was excised out and the DNA in the gel was electroeluted onto DE81 (Whatman) paper and eluted from the paper in 1H NaCl. The DNA solution was precipitated as detailed above and the DNA precipitate was dissolved in 25 ul of TE buffer (fl-ori fragment). b; Cloning of fl-ori into 9331 sites of pBR322: pBR322 (2 ug) was partially digested with 2 units 9531 (according to the manufacturer's instructions). The reaction was terminated by PCI extraction followed by precipitation of DNA as detailed in step a above. buffer. sites (nucleotide positions 3232 and 3251) of pBR322. c. Creation of_pA0807: pBRfl-ori (10 ug) was digested for A hours at 37°C with 10 units each of Estl and flggl. The digested DNA was PCI extracted, precipitated and dissolved in 25 ul of TE buffer as detailed in step 1 above. This material was electrophoresed on a 1.2% agarose gel and the gggl - Estl fragment (approximately 0.8 kb) containing the fl-ori was isolated and dissolved in 20 ul of TE buffer as detailed in step a above. About 100 ng of this DNA was mixed with 100 ng of pAO80A that had been digested with Egtl and fidgl and phosphatase-treated. A description of pAO80A is provided in V089/0h320. This mixture was ligated in 20 ul of ligation buffer by incubating for overnight at 1h°C with 1 unit of T4 DNA ligase. The ligation reaction was terminated by heating at 70°C for 10 minutes. This DNA was used to transform E; 99;; strain JMl03 to obtain pAO807.

Example XII Construction of pPSV2l8 oligonucleotide with the following sequence: '-GATCAGATCT-3' Positive clones were ng of pPSV203 were partially digested with a limiting amount of fiindlll and which converts the gamfll site to a gglll site. identified on this basis and the plasmid was called pPSV203. blunt-ended with Klenow. Linear full length plasmid fragments were Correct clones were 1350 bp figlll/flindlll fragment and correct plasmids were called pPSV2l0. gel-purified and self-ligated in a 100 pl volume. identified on the basis of plasmid DNA containing a oligonucleotide with the sequence: '-GATCAGATCT-3' which converted the fiamhl site to a gglll site. The correct plasmid was identified on the basis of a 422 bp gggl/figlll fragment, and called pPSV204.

SEQUENCE LISTING (1) GENERAL INFORMATION: NUMBER OF SEQUENCES: 5 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1994 bp (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N021: GGATCCCTAT TGTTACTTTT GCTTAACATT CCAATATTCT TCAACGGTTA ATTGATTAAC 60 ACTGTAACCT CTGCCCATGT GCTTCATCCA AATCTGGTAA TCTGCTTTCT ATTTCTGCCA 120 AAATAGTTAA TCTATGAGAC ATGTGCCCTC AATTGCGCAG TAGATCGAGT GGAAGTCTTC 180 TTTGCGTAAC ACTCAAAGTA TATCCCTGTT AGTCTTTATT CACCTGTTGC TGCATTGGTG 240 TCAGTTACCA TTATTGTTTC CACTTGGAAA AGCTTGTTTT TTTTTGATAG CACAGAAACG 300 TGGGCTCCGA TAAGCTAAAC TTCAACGAGA ATATAAAAGC TGAAAAGATT CTTGTCAAGA 360 ACTTGTACAA CGACCAATAA GTCTTTCAAG GCATCAGAC ATG TTT TCT CCT ATT CTA 417 Met Phe Ser Pro lle Leu -20 AGT CTC GAA ATT ATT CTC GCT TTG GCT ACT CTC CAA TCA GTC TTT GCG GTT 468 Ser Leu Glu lle lle Leu Ala Leu Ala Thr Leu Gln Ser Val Phe Ala Val -15 -10 -5 1 GAG TTG CAG CAC GTT CTT GGA GTC AAC GAC AGA ccc TAT ccr CAG AGG ACA 519 Glu Leu Gln His Val Leu Gly Val Asn Asp Arg Pro Tyr Pro Gln Arg Thr S 10 15 GAT Asp GGT Gly ATT lle AAT Asn GTG Val GTC Val TCT Ser GAT Asp TAC Tyr GAT Asp GTG Val GAA Glu TCT Ser 105 CAA Gln CAG Gln CAG Gln 55 GGG Gly GTC Val GAC Asp ATA lle 140 GAA Glu TAC Tyr GGA Gly GCT Ala AAA Lys CCT Pro 90 GCC Ala ACC Thr AGA Arg TGG Trp no CAA Gln ACA Thr GCT Ala GCT Ala 125 GTT Val ATT lle GGA Gly CTA Leu GGA Gly TGG Trp TTC Phe GTT Val 160 CTG Leu ATT lle GAA Glu TAC Tyr 110 GAA Glu GAC Asp AGA Arg GCT Ala ATG Met 60 GCT Ala GAG Glu TTT Phe GAA Glu 145 GCT Ala AGA Arg TCT Ser 95 GGT Gly "GGA GCA Ala GAG Glu &5 TAC Tyr TTC Phe GAA Glu ACC Thr 130 AAG Lys GGA Gly GGA Gly CAG Gln 80 TTT CAA ACA Thr ACT Thr AAA Lys TAC Tyr 165 GGC Gly GAA Glu AAA Lys ACT Thr 115 TTT Phe ATT lle AGA Arg 65 GAC Asp AAG Lys TCT Ser GCT Ala GGC CCC Gly Pro GAA Glu TCC Ser 50 TAC CCA Tyr Pro GAT Asp TAT GAT TAC TTC Tyr Asp 100 GGT TTC Gly Phe GAA Glu CGA Arg 135 GTT Val TGG Trp CAA Gln GGA Gly TAC Tyr TGT Cys GCT Ala 85 TAC Tyr TAT Tyr GCT Ala TTT Phe 170 ATC S70 lle ACG Thr ACC Thr GAT 723 Phe TCG 825 Set GAA GGC Gly ATG Met AAA Lys GCT Ala ATT lle 240 AAG Lys ACA Thr CAG Gln 190 CAT His GAC Asp AGA Arg 275 GAA Glu GGA Gly ATC lle AGA Arg 225 TTC Phe GAT Asp ATC lle GCT Ala TAC Ty: 175 TAC Tyr ATT lle TGT Cys 260 GGC Gly GAT Asp 310 ACC Thr AAA Lys 210 GCC Ala GAC Asp GGA Gly TAC Tyr 295 GTT Val GAT Asp ACT Thr CTA Leu GTC TGG Trp GTC Val AGA Arg ATG Met CTA Leu GGC Gly TAC Tyr TAT Tyr 280 GAT Asp GTC Val ACG Thr GAC Asp TCT Ser TGT TAC Tyr GCC Ala TAT Tyr GAA Glu 180 GCT Ala GGC Gly AGA Arg 265 GTC Val CGA Arg GGG Gly 215 GGA Gly TTT Phe GAG Glu GTT Val GCC Ala 300 GTT Val ATT lle TTT Phe GAA Glu 250 GGA Gly ACA Thr TAC Tyr GCT Ala TCA Ser GAC Asp AAC Asn AGA TTT Phe TGT Cys ATT lle ATT lle 235 GGG Gly CAG Gln GAA Glu 185 GCT Ala ACT Thr GTA Val TAC Tyr 270 TTT Phe 320 CAG Glu CAA Gln 220 AGA Arg ACT Thr GAG Glu TAT Tyr AGA Arg GAT Asp GGT Gly 255 GCT Ala GAA Glu 305 AAA Lys AAC Asn 205 GAA Glu GAT Asp GAG Glu TAT Tyr GGA Gly 290 GAT Asp ACC Thr GTT Val GGT Gly 375 GTT Val GGA Gly GAC Asp GAT Asp 325 GAA Glu TCT Ser 410 ATG Met 360 GAA Glu GAC Asp AGA Arg TCA Ser GAC Asp GGA Gly AAA Lys TGT Cys 395 AAA Lys CAA Gln CAG Gln 345 GCA Ala TAC Tyr GAG Glu AAT Asn GTA Val ATT lle AGA Arg TAC Tyr 380 GCA Ala GCT Ala TTC Phe 330 CAA Gln GTT Val GGC Gly TTG Leu 415 ACT Thr TTC Phe CTT Leu 365 TAC Tyr CAG Gln ACC Thr ACT Thr GGA Gly 400 ACC Thr 350 CAG Glu ATC lle TTC Phe GAC Asp CTT Leu GGT Gly AGA Arg CTC Leu TCT GAA Glu CTT Leu TTC Phe TGT Cys GAC Asp 420 TTC Phe AAA Lys TTG Leu GAT TTT Phe TGT Cys GCA Ala TTG Leu 405 TTC Phe TAAAAATGGT AAGGAATGTT TTGCATCAGA TACGAGTTCA GTC Val ACC Thr 355 ACT Thr GTC Val GAA Glu TTC Phe A40 ACA Thr GAA Glu GTT Val TTC Phe 390 TGG Trp GAT Asp 340 GAA Glu TGT Cys 425 GAT Asp lA88 AAACGATTAA 1853 GAAGAGAATG CTCTTTTTTT TGTTTCTATC CAATTGGACT ATTTTCGTTT ATTTTAAATA 1913 GCGTACAACT TTAACTAGAT GATATCITCT TCTTCAAACG ATACCACTTC TCTCATACTA 19 GGTGGAGGTT CAATGGATCC [993 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: bp (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGATCCCTAT ACTGTAACCT AAATAGTTAA TTTGCGTAAC TCAGTTACCA TGGGCTCCGA ACTTGTACAA TGTTACTTTT CTGCCCATGT TCTATGAGAC ACTCAAAGTA TTATTGTTTC TAAGCTAAAC CGACCAATAA GCTTAACATT CCAATATTCT TCAACGGTTA ATTGATTAAC GCTTCATCCA AATCTGGTAA TCTGCTTTCT ATTTCTGCCA ATGTGCCCTC AATTGCGCAG TAGATCGAGT GGAAGTCTTC TATCCCTGTT AGTCTTTATT CACCTGTTGC TGCATTGGTG CACTTGGAAA AGCTTGTTTT TTTTTGATAG CACAGAAACG TTCAACGAGA ATATAAAAGC TGAAAAGATT CTTGTCAAGA GTCTTTCAAG GCATCAGAC 399 (2) INFORMATION FOR SEQ ID NO:3: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 bp (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG TTT TCT CCT ATT CTA 18 Net Phe Ser Pro lle Leu -20 AGT CTG GAA ATT ATT CTC GCT TTG GCT ACT CTC CAA TCA GTC TTT GCG 66 Ser Leu Glu lle Ile Leu Ala Leu Ala Thr Leu Gln Ser Val Phe Ala -15 -10 -5 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1341 bp (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA GTT Val GAG Glu GAT Asp GGT Gly ATT lle AAT Asn 70 GTC Val (xi) SEQUENCE DESCRIPTION: GAT Asp TAC Tyr GAT Asp GTG Val GAA Glu TCT Ser 105 CAG Gln CAG Gln CAG Gln 55 GTC Val GAC Asp CAC His TAC Tyr GGA Gly GCT Ala AAA Lys CCT Pro 90 GCC Ala GTT Val TGG Trp L0 CAA Gln ACA Thr GCT Ala ATT lle GGA Gly CTA Leu GGA TGG Trp GGA Gly CTG Leu ATT lle GAA Glu TAC Tyr 110 GTC Val AGA Arg GCT Ala ATG Met 60 GAG Glu AAC Asn GCT Ala AGA Arg TCT Ser CAA Gln ID N0:4: GAC Asp GAG Glu #5 TAC Tyr TTC Phe GAA Glu AGA Arg GGA Gly GGA Gly CAG Gln 80 GGC Gly GAA Glu AAA Lys TAT Tyr ATT lle AGA Arg 65 CCT Pro GAA Glu TAC Tyr CAG Gln TCC Ser 50 GAT Asp AGG Arg TAC Tyr TGT Cys GCT Ala 85 ACA S4 Thr ATC lle ACG Thr ACC Thr GAT 258 Asp TTT CAA GAC TAT GAT TAC TTC 309 ACA Thr ACT Tht 115 Asp Tyr Asp Tyr Phe AAG GGT TTC TAC TCG 360 Lys Gly Phe Tyr Ser GGG TTA AAC ACC GCT TTC GAT TTT GGT ACC ACT TTG AGA GAA CGA TAT GAA 411 Gly Leu Asn Thr Ala Phe Asp Phe Gly Th: Thr Leu Arg Glu Arg Tyr Glu 125 130 135 CAT TTG ATA AAC AAT AGC GAA GAA GGA AAG AAA CTT TCT GTT TGG GCT GGC 462 His Leu lle Asn Asn Ser Glu Glu Gly Lys Lys Leu Ser Val Trp Ala Gly 140 145 150 TCT CAA GAA AGA GTT GTT GAC AAC GCA AAG TAC TTT GCT CAA GGA TTT ATG 513 Set Gln Glu Arg Val Val Asp Asn Ala Lys Tyr Phe Ala Gln Gly Phe Met 155 160 165 170 AAA TCT AAC TAC ACC GTT ATG GTC GAA GTC GTT GCT CTA GAA GAG GAG AAA 566 Lys Ser Asn Tyr Thr Val Met Val Glu Val Val Ala Leu Glu Glu Glu Lys 175 180 185 TCC CAG GGA CTC AAC TCT CTA ACG GCT CGA ATT TCA TGT CCA AAC TAT AAC 615 Ser Gln Gly Leu Asn Ser Leu Thr Ala Arg lle Ser Cys Pro Asn Tyr Asn 190 195 200 205 AGC CAT ATC TAC AAA GAT GGC GAC TTG GGG AAT GAC ATT GCT CAA AGA GAA 666 Ser His lle Tyr Lys Asp Gly Asp Leu Gly Asn Asp lle Ala Gln Arg Glu 210 215 220 GCT GAC AGA TTG AAC ACT CTT TCT CCA GGA TTT AAC ATT ACT GCA GAT GAT 717 Ala Asp Arg Leu Asn Thr Leu Ser Pro Gly Phe Asn lle Thr Ala Asp Asp 225 230 235 ATT CCA ACA ATT GCC CTA TAC TGT GGC TTT GAA CTA AAT GTA AGA GGT GAG 768 lle Pro Thr lle Ala Leu Tyr Cys Gly Phe Glu Leu Asn Val Arg Gly Glu 240 245 250 255 TCA TCC TTC TGT GAC GTC TTG TCA AGA GAG GCT CTA CTG TAC ACT GCT TAT 819 Ser Ser Phe Cys Asp Val Leu Ser Arg Glu Ala Leu Leu Tyr Thr Ala Tyr 260 265 270 AAG Lys ACA Thr ACC Thr GTT Val GGT Gly 375 GTT Val AGA Arg 275 ACA Thr GAA Glu GAT Asp 325 GAA Glu TCT Ser GAT Asp ATC lle GCT Ala ATG Met 360 GAC Asp AGA Arg GGC Gly GAT Asp 310 GAC Asp GGA Gly AAA Lys TGT Cys 395 GGA Gly TAC Tyr 295 CAA Gln CAG Gln 3&5 TAC Tyr GAG Glu TGG Trp GTC Val AGA Arg GTA Val ATT lle AGA Arg TAC Tyr 380 GCA Ala TAT Tyr 280 TAT Tyr GAT Asp TTC Phe 330 CAA Gln GTT Val GGC Gly TTG Leu TAC Ty: GCC Ala TAT Tyr ACT Thr TTC Phe CTT Leu AGA CCT Pro CAG Gln ACC Thr ACT Thr GGA Gly 400 GAG Glu GTT Val GCC Ala 300 ACC Thr 350 ATC lle TTC Phe GAC Asp GGA Gly ACA Thr TAC Tyr GGT Gly AGA Arg CTC Leu TCT AAC Asn AGA Arg TTT Phe CTT Leu TTC Phe TGT Cys GAC Asp A20 GGG Gly CAG Gln TTC Phe AAA Lys TTG Leu 370 TTT Phe TTT Phe 320 TGT Cys GCA Ala TTG Leu 405 GCG Ala GTG Val ACC Thr 355 GTC Val GAA Glu GAA Glu 305 ACA Thr GAA Glu GTT Vai TTC Phe 390 GGA Gly 290 GAT Asp GAT Asp 340 ATA 11e GAA Glu TAT Tyr TGT Cys A25 GGA GTT CCT AAA AAT GCT TCC TAC CCA CTT GAA CTA TCA TTC TTC TGG GAT 1329 Gly Val Pro Lys Asn Ala Ser Tyr Pro Leu Glu Leu Ser Phe Phe Trp Asp A30 435 4.40 GAC TTC TCA TAA 1361 Asp Leu Ser 4(2) INFORMATION FOR SEQ ID N025: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 137 bp (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AAATGGTAAG GAATGTTTTG CATCAGATAC GAGTTCAAAA CGATTAAGAA GAGAATGCTC 60 TTTTTTTTGT TTCTATCCAA TTGGACTATT TTCGTTTATT TTAAATAGCG TACAACTTTA 120 ACTAGATGAT ATCTTCTTCT TCAAACGATA CCACTTCTCT CATACTAGGT GGAGGTTCAA 180 TGGATCC 187

Claims (19)

1. An isolated DNA fragment comprising the Piip_a_s_to_ri_s_ acid phosphatase gene having the nucleotide sequence set forth in SEQ ID N021.
2. An isolated DNA fragment comprising the Pichia pastoris acid phosphatase 5’ regulatory region having the nucleotide sequence SEQ ID N022.
3. An isolated DNA fragment comprising the Pichia pastoris acid phosphatase signal sequence having the nucleotidepsequence SEQ ID NO:3.
4. The DNA fragment of claims 2 or 3 wherein said DNA fragment is operably- linked to a heterologous DNA sequence which codes for at least one polypeptide.
5. A method for the secretion of a heterologous protein from a yeast cell wherein said secretion is directed by the presence of the signal sequence from the Pichia pastoris acid phosphatase gene (SEQ ID NO:3).
6. The method according to claim 5 wherein said yeast cell is a yeast cell ofthe genus Pichia pastoris.
7. The method according to claim 6 wherein said heterologous protein is tissue plasminogen activator.
8. An isolated DNA fragment comprising the Pichia pastoris acid phosphatase structural gene having the nucleotide sequence SEQ ID NO:4.
9. An isolated DNA fragment comprising the Pichia pastoris acid phosphatase 3’ transcription termination sequence having the nucleotide sequence SEQ ID NOO:5.
10. . A vector comprising the DNA sequence SEQ ID NO:l, NO:2, N023, NOL4 or NO:5.
11. An expression plasmid comprising the DNA coding for the Pichia pastoris PHOI signal sequence (SEQ ID N013) and the DNA coding for the mature tissue plasminogen activator, operably linked the Pichia pastoris alcohol oxidase l (AOXI) 5’ regulatory region.
12. A method for activating the acid phosphatase 5' regulatory region derived from the Pichia pastoris acid phosphatase gene (SEQ ID NO:l) to facilitate ‘the expression of a heterologous DNA sequence, which encodes at least one polypeptide, comprising contacting a suitable yeast host cell which has been transformed with the acid phosphatase 5' regulatory region (SEQ ID N012) or variants thereof due to clonal variation or sequencing errors operably- linked to a heterologous DNA sequence with a medium in which the concentration of phosphate will effect expression.
13. The method according to claim 12 wherein said yeast host cell is pastoris. Pichia
14. The method according to claim 12 wherein said heterologous DNA additionally contains a signal sequence, particularly the acid phosphatase signal sequence ofPichia pastoris (SEQ ID NO:3), operably linked thereto.
15. An isolated DNA fragment Comprising the Pichia pastoris acid phosphatase signal sequence (SEQ ID NO:3) operably linked to a 5’ regulatory region, particularly the acid phosphatase 5’ regulatory region of Pichia pastoris (SEQ ID NO:2) or the Pichia pastoris alcohol oxidase (AOXI) 5' regulatory region.
16. The DNA fragment of claim 15 wherein said DNA fragment is operably linked to a heterologous DNA sequence which encodes for at least one polypeptide.
17. The DNA fragment of claim 4 or 16 wherein said heterologous DNA codes for tissue plasminogen activator.
18. A method of directing integration at the Pichia pastoris PHOI locus utilizing an integrative vector which comprises the 5’ (SEQ ID NO:2) and 3 (SEQ ID N025) flanking sequences of the Pichia pastoris PHOI gene or variants thereof due to clonal variation or sequencing errors. 66
19. 19. A method of identifying Pichia pastoris transformants containing expression vectors as defined in claim 18 integrated at the £0; locus, wherein }_°_ig1i_;_1 iris which has been transformed is plated on low phosphate indicator plates, allowed to grow overnight, and the resulting colonies are screened for a white color. F. R. KELLY & C0,, AGENTS FOR THE APPLICANTS