Enhanced yeast transcription employing hybrid promoter region constructs.

BACKGROUND OF THE INVENTION

Field of the Invention

With the advent of hybrid DNA technology, production of mammalian proteins in microorganisms became a reality. For the first time, mammalian proteins could be synthesized in a unicellular microorganism by introduction of a gene encoding for the mammalian protein under the transcriptional and translational control of regulatory sequences recognized by the microorganism host. The introduction of these foreign constructions into a microorganism host resulted in competition between the regulatory signals of the construct and the regulatory signal endogenous to the host for the host systems involved with expression. The structural gene of interest is usually directed to a product which is nonproductive and may be injurious to the host. Therefore, host cells which can turn off the foreign gene can effectively dominate modified host cells.

Substantial progress has been made in isolating sequences concerned with transcriptional and translational regulation for protein expression. But frequently flanking sequences, as well as distant sequences, may also affect the efficiency and regulation of the expression of the protein. Therefore, as one manipulates these various sequences, removing them from their native environment, and joining them to unnatural sequences, that is sequences other than the wild-type sequence, one can never be certain as to the result.

In order to enhance the economies of producing proteins in microorganisms, there have been substantial efforts directed to improving the efficiency of transcription and translation, maximizing the proportion of total protein directed to production of the desired product, enhancing the viability of the modified host, as well as improving the efficiency with which the modified host may be obtained.

Description of the Prior Art

Guarente <u>et</u><u>al</u>., <u>Proc. Natl. Acad. Sci. USA</u> (1982) <u>79</u>:7410-7414, describes a hybrid promoter region employing the GAL4 regulatory region. Guarente and Ptashne, <u>ibid</u>. (1981) <u>78</u>:2199-2203, report the presence of two domains in a yeast promoter, with a region upstream from the TATA sequence providing an activation site. Kramer <u>et</u><u>al</u>., <u>ibid</u>. (1984) <u>81</u>:367-370, describe the regulated expression of a human interferon gene in yeast employing the yeast acid phosphatase promoter, where expression is induced by phosphate or a temperature shift. Tekamp-Olson <u>et</u><u>al</u>., Cold Spring Harbor Meeting, <u>Molecular Biology of Yeast</u>, 1983, describe the absence of deleterious effects on yeast viability when employing "short" promoters, as distinct from the presence of such effects, when employing an extended promoter region or "long" promoters.

SUMMARY OF THE INVENTION

Novel hybrid promoter regions are provided for use in conjunction with constructs having a structural gene under the transcriptional control of the hybrid promoter region and a terminator region. The hybrid promoter comprises a first segment providing transcriptional enhancement, either constitutive or regulated, and a second segment defining an RNA polymerase binding site and transcriptional initiation site. The hybrid promoter regions lack the deleterious effects associated with a wild-type promoter region in recombinant constructs, which results in reduced transformation efficiencies and lower yeast viability.

BRIEF DESCRIPTION OF THE DRAWINGS

<ul id="ul0001" list-style="none"><li>Figure 1 is a diagrammatic view of plasmids GAP1-6;</li><li>Figure 2 is a diagrammatic view of plasmids Pykl-6; and</li><li>Figure 3 indicates the DNA linker sequence and a flow diagram showing its use in a construct for hSOD.</li></ul>

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel DNA fragments are provided as well as constructions employing the fragments for enhanced expression of structural genes in a yeast host. The constructs employing a hybrid promoter region provide for enhanced efficiencies of transformation and greatly improved viability of the yeast host as contrasted with those employing a wild-type yeast promoter. Concomitant with the improved viability is increased expression of a structural gene, in comparison with the truncated promoter region, and, therefore, greatly enhanced overall yields of expression products.

For the purposes of the subject invention, the "promoter region" is divided into two domains: (1) the structural gene proximal region, which includes the transcription initiation site, the "TATA" sequence capping sequence, as appropriate, and an RNA polymerase binding sequence, which sequence intends a sequence which includes nucleotides upstream from the initiation site for directing the initiation of synthesis of the messenger RNA; and (2) a distal region which provides for regulated or constitutive expression, with enhanced expression as compared to the first domain linked to non-functional yeast DNA.

The hybrid promoters of the subject invention employ the RNA polymerase binding region of a yeast glycolytic enzyme promoter and a region upstream from said polymerase binding region, which is different from the wild-type upstream region of the RNA polymerase binding region and provides for enhanced efficiencies of transcription. This distal region will be derived from either a sequence, usually a yeast sequence, involved in regulation of transcription, or a prokaryotic sequence which provides for enhanced constitutive expression of the desired gene.

Conveniently, cassettes or constructs can be prepared which provide for one or more restriction sites intermediate the promoter region and a related terminator region where the structural gene may be inserted, so as to be under the transcriptional control of the hybrid promoter region. By having one or more restriction sites, one can provide for ease of insertion of the structural gene intermediate the transcription initiation and termination regions. The cassettes which can be prepared comprising the transcriptional initiation and termination region, having appropriate restriction sites for structural gene insertion can be cloned in prokaryotic vectors, so that after insertion of the structural gene, the resulting cassette, including the structural gene, may be cloned, isolated and purified, before introduction into a yeast vector.

The cassette, will for the most part, have the following formula:         -(P.R.(2)-P.R.(1))-R.S.-T.R.- wherein:    P.R.(1) is the promoter region proximal to the structural gene and having the transcription initiation site, the RNA polymerase binding site, and including the TATA box, the CAAT sequence, as well as translational regulatory signals, e.g., capping sequence, as appropriate;    P.R.(2) is the promoter region joined to the 5'-end of P.R.(1) associated with enhancing the efficiency of transcription of the RNA polymerase binding region;    R.S. is a sequence having one or more restriction recognition sites, preferably at least two restriction recognition sites, where the sites may result upon restriction into blunt ends or overhangs;    T.R. intends the termination region, which will include the terminator, which may be a stem and loop structure, and which may be associated with one or more stop codons, a polyadenylation signal sequence, if any, as well as any other transcriptional and translational termination sequences.

P.R.(1) will generally be at least about 150bp, more usually at least about 200bp, usually not more than about 600bp, more usually not more than about 500bp, generally not more than about 450bp and preferably less than about 400bp; the sequence will extend in the downstream direction of transcription to about bp +3, more usually bp -1 and may extend only to bp -20, more usually to bp -10 (the numbering intends that +1 is the first bp of the initiation codon with which the promoter region is associated in the wild-type host, while -1 is the immediately upstream bp and the integers increase in the direction of transcription;    P.R.(1) will be derived from a strong yeast promoter, normally a glycolytic enzyme promoter, such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, alcohol dehydrogenase, phosphoglucoisomerase, triose phosphate isomerase, phosphofructokinase, etc.,    P.R.(2) will provide for an enhancing function of transcription, which enhancing function may provide for constitutive or regulated transcription; regulators will be derived from regions associated with regulation of yeast genes, other than the natural or wild-type gene associated with, the first domain in the wild-type or natural host, such as UDP-galactose epimerase (GAL10), galactokinase (GAL1), acid phosphatase (PHO5), alcohol dehydrogenase I and II (ADH I and II), etc. For yeast regulatory sequences, the domain will usually be at least about 100bp, more usually at least about 200bp, for convenience generally not exceeding about 3kbp, usually not exceeding about 1kbp, desirably not exceeding about 600bp. The regulatory region will generally begin at least about 200bp from the initiation codon, usually at least about 300bp and may begin at 400bp or farther upstream from the initiation codon.

Regulation can be as a result of a change in the chemical or physical environment of the host, such as a change in carbon source, e.g., glucose to galactose or vice versa; a change in concentration of a nutrient, e.g., an inorganic nutrient such as a phosphate; or a change in temperature, e.g., 25°C to 35°C. Constitutive transcription can be achieved employing prokaryotic sequences of at least about 500bp, usually 1kbp or more, for convenience, generally not exceeding about 5kbp; conveniently, the prokaryotic sequence can be obtained from the vector in which the cassette is cloned, illustrative vectors including pBR322, lambda, Charon 4A, pACYC184, pUC5, etc.

R.S. will generally be at least 4bp, more usually at least 6bp, and may be 100bp or more, more usually being not more than about 60bp and may include one or more, usually not more than about 10 restriction sites, where such restriction sites may be illustrated by <u>Eco</u>RI, <u>Bam</u>HI, <u>Sal</u>I, <u>Hin</u>dIII, <u>Alu</u>I, <u>Ava</u>I, <u>Tag</u>I, <u>Hpa</u>I, etc., having at least one unique restriction site for the construct sequences.

T.R. is the termination region which will include the necessary transcriptional and translational signals for termination, such as the polyadenylation site, etc.;    T.R. will generally be at least about 100bp, more usually at least 150bp, and generally less than about 1kbp, usually less than about 600kbp; the termination region may be derived from any convenient yeast sequence, so long as the terminator balances the promoter, conveniently being derived from a glycolytic enzyme terminator, where the terminator may be associated with the same or different enzyme with which the promoter is associated.

Where a cassette is cloned in a bacterial vector, the construction will have the following formula:<maths id="math0001" num=""><img file="EP0480480A2_D0001.tif" /></maths> wherein all the symbols have been defined previously, except for:    Rep (B), which intends a replicon or replication system recognized by a prokaryotic host and may be derived from a plasmid or phage, such as ColE1, and R plasmid, e.g., pRK290, lambda, e.g., Charon 4A, λdv, etc.;    M is a marker which provides for selection of hosts containing the construction, where (B) intends a prokaryotic, e.g., bacterial, host and a intends an integer of from 0 to 3, usually 1 to 2, although additional markers may be present, where the marker allows for selection of the host containing the construct as well as providing for selective pressure on maintaining hosts having the construct; the markers include biocide resistance, such as antibiotic resistance, toxin resistance and heavy metal resistance; providing prototrophy to an auxotrophic host; providing immunity; and the like;    the markers may provide for complementation of an auxotrophic host, e.g., his⁻, ura⁻, trp⁻, leu⁻ genotype, resulting in prototrophy; resistance to metals, such as cup⁺ genotype; resistance to antibiotics, such as amp^r, tc^r, cam^r, str^r, tur^r genotype, etc.;    b is 0 or 1, intending that the construction is either linear or circular, usually circular.

The above construct can be used for insertion of a wide variety of structural genes, both prokaryotic and eukaryotic, both naturally occurring and synthetic, where the genes may include signal leaders for secretion, and the like. The genes may express enzymes, hormones, proteins from pathogens for vaccines, structural proteins, lymphokines, membrane surface proteins, immunoglobulins, blood proteins, or the like. The particular structural gene which is inserted is not critical to this invention and any polypeptide or protein of interest may be prepared employing the constructions of the subject invention. The structural genes will usually be foreign to the yeast host, where foreign intends different from wild-type yeast structural genes and from a source that does not normally exchange genetic information with yeast.

Usually, the structural gene will be at least about 36bp, and not more than about 20kbp, usually not more than about 3000bp, usually not more than about 1500bp. Included in the structural gene may be non-coding flanking regions, the 5'-flanking region normally being quite short, usually less than about 30bp, while the 3'-flanking region may be extended, usually not exceeding about 500bp. Thus, the structural gene fragment will usually include the translational stop codons for proper termination of amino acid chain extension.

When the structural gene has been inserted into the cassette which is joined to a yeast replication system, normally including one or more markers recognized by yeast, the resulting construct will have the following formula: <chemistry id="chem0001" num="0001"><img file="EP0480480A2_D0002.tif" /></chemistry> wherein all of the symbols have been defined previously except for:    gene, which intends the structural gene, having its initiation codon and stop codons as appropriate; and    Y, which intends that the symbol is related to yeast.

Convenient yeast replication systems include the 2µm plasmid replication system, combination of CEN3 and ARS1 or ARS3, or the like. The replication systems may be high or low copy number, depending on the effect of the construct on the viability of the host. While the indicated replication systems are those which have found common employment, any replication system useful in yeast may be employed which provides for efficient replication and maintenance. Often the structural gene will be inserted into an appropriate shuttle vector capable of replication and selection in either a yeast or bacterial host, where the resulting construction will have the following formula: <chemistry id="chem0002" num="0002"><img file="EP0480480A2_D0003.tif" /></chemistry> where all symbols have been defined previously. Also, it is, of course, understood that the cassette without an inserted structural gene but containing the restriction enzyme recognition sequence, R.S., may be propagated in yeast or contained within a shuttle vector, where the construction will have the following respective formulae: <chemistry id="chem0003" num="0003"><img file="EP0480480A2_D0004.tif" /></chemistry><chemistry id="chem0004" num="0004"><img file="EP0480480A2_D0005.tif" /></chemistry> where all symbols have been defined previously.

The various fragments which form the cassette and final constructions may be joined together in accordance with conventional ways. In many cases, genes have been isolated and restriction mapped, as well as sequenced. To that extent, one can select the sequence of interest by restriction of the gene, employing further manipulation as necessary such as resection with <u>Bal</u>31, <u>in</u><u>vitro</u> mutagenesis, primer repair, or the like, to provide a fragment of a desired size, including the desired sequence, and having the appropriate termini. Linkers and adapters can be used for joining sequences, as well as replacing lost sequences, where the restriction site is internal to the region of interest. The various fragments which are isolated, may be purified by electrophoresis, electroeluted, ligated to other sequences, cloned, reisolated and further manipulated.

The use of regulatory sequences for controlling transcription of the structural gene of interest allows for growing the host cells to high density with no or low levels of expression of the structural gene, and then inducing expression by changing the environmental conditions, e.g., nutrient, temperature, etc.

For example, with the GAL4 regulatory region, the yeast cells could be grown in rich media with a glycerol-lactic acid combination to high density, e.g., mid or late log phase, followed by switching the carbon source to galactose. For PHO5 regulation one could grow the cells at high phosphate, about 1 to 10mM, and then decrease the phosphate concentration to about 0.1 to 0.5mM. For temperature sensitivity, one could grow the cells at 25° to 37°C and then change the temperature as appropriate by about 5° to 20°C. The host cells would have the regulatory system associated with the regulatory region employed.

Various techniques will be exemplified in the Experimental section of this application, which techniques can be used as paradigmatic for constructions employing fragments from sources other than those exemplified. Of particular interest, as evidenced by the Experimental section, will be the use of the glyceraldehyde-3-phosphate dehydrogenase promoter region for the RNA polymerase binding site in conjunction with regulator sequences, such as those associated with GAL4, PHO5, ADHII or the like. In referring to the GAL4 regulatory region or associated sequence, the region intends the sequence associated with regulation of other galactose metabolism genes, e.g., GAL1 and GAL10, which are under the regulatory control of such sequence in conjunction with the expression product of the GAL4 gene. The PHO5 and ADHII sequences refer to regions associated with the PHO5 and ADHII genes which provide for transcriptional regulation of the PHO5 and ADHII gene, respectively.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

All DNA manipulations were done according to standard procedures. See <u>Molecular Cloning</u>, T. Maniatis <u>et</u><u>al</u>., Cold Spring Harbor Lab., 1982. Enzymes used in cloning were utilized as per the manufacturer's specifications. Enzymes were obtained either from New England Biolabs or Bethesda Research Laboratories. Procedures with these enzymes employed the supplier's directions. Yeast were transformed and grown using a variety of media including selective medium (yeast nitrogen base without leucine); YEPD medium, containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose, and others as appropriate and/or detailed below. In the case of plating medium contained 2% (w/v) agar and for transformation 3% top agar. Hepatitis B surface antigen was determined after lysis of yeast by glass bead agitation and clarification by centrifugation, using the AusriaII assay (Abbott Laboratories). Protein is determined by the Coomassie dye binding method.

Construction of GAL regulator containing plasmids

.

Plasmid pLGSD5 is prepared as described in Guarente <u>et</u><u>al</u>., (1982) <u>supra</u>. The plasmid was manipulated as follows: After restriction with <u>Xho</u>I, the overhangs were filled in with the Klenow fragment of DNA polymerase I ("Klenow fragment"), ligated with <u>Eco</u>RI linkers (GGAATTCC) and then completely digested with <u>Eco</u>RI and <u>Sau</u>3A to provide a 370bp fragment which was isolated by gel electrophoresis and included the intergenic sequence between GAL1 and GAL10 genes of yeast, and provides for the GAL4 regulation sequence of the GAL1 and GAL10 genes.

This fragment was inserted into pBR322 which had been completely digested with <u>Eco</u>RI and <u>Bam</u>HI, followed by treatment with alkaline phosphatase to prevent oligomerization. The resulting plasmid pBRGAL4 was treated in two different ways.

In the first procedure pBRGAL4 was completely digested with <u>Sau</u>3A, the overhangs filled in with the Klenow fragment, and the resulting blunt-ended fragment ligated with <u>Sal</u>I linkers (CGTCGACG), followed by digestion with <u>Sal</u>I and <u>Xho</u>I. The resulting 370bp fragment was isolated by gel electrophoresis. This fragment has the original 370bp yeast GAL4 regulator sequence with <u>Xho</u>I and <u>Sal</u>I termini.

The second fragment was obtained by complete digestion of pBRGAL4 with <u>Xho</u>I and <u>Sal</u>I to provide a <u>Xho</u>I-<u>Sal</u>I fragment which included the 370bp yeast GAL4 regulator sequence as well as about 280bp of pBR322, the GAL4 sequence extending from <u>Sau</u>3A to <u>Sal</u>I.

The two fragments were then cloned in the plasmid plot5. plot5 was prepared by inserting the 40bp polylinker fragment of the following sequence<img file="EP0480480A2_D0006.tif" /> into pBR322 as an <u>Eco</u>RI-<u>Pvu</u>II substitution followed by insertion of the <u>trp</u>-<u>lac</u> promoter (Russell and Bennett, <u>Gene</u> (1982) <u>20</u>:231-245) into the <u>Pvu</u>II site with transcription oriented toward the polylinker sequence. plot5 was completely digested with <u>Sal</u>I, followed by treatment with alkaline phosphatase and the 370bp and 650bp fragments independently inserted into plot5 to provide plasmids plot5GAL4/370 and plot5GAL4/650, respectively. Each of the plasmids was then completely digested with <u>Bam</u>HI and <u>Sal</u>I to reproduce the individual fragments extended by 6bp of the polylinker fragment. These fragments were then ligated into pC1/1, which had been completely digested with <u>Bam</u>HI and <u>Sal</u>I followed by treatment with alkaline phosphatase to prevent recircularization. Plasmid pC1/1 is a derivative of pJDB219 (Beggs, <u>Nature</u> (1978) <u>275</u>:104) in which the region corresponding to bacterial plasmid pMB9 in pJDB219 has been replaced by pBR322 in pC1/1. The resulting plasmids were designated pC1/1GAL4/370 and pC1/1GAL4/650, respectively. The <u>Bam</u>HI-<u>Sal</u>I fragment is located in the pBR322 portion of the vector pC1/1.

The next construction develops a hybrid promoter for expression of the Hepatitis B surface antigen (HBsAg or sAg), employing the RNA polymerase binding region of GAPDH. The previously prepared plasmid pHBS56/16-3, a yeast shuttle vector containing the alcohol dehydrogenase 1 (ADH1) promoter, the HBsAg gene and ADH terminator as a <u>Sph</u>I fragment, was digested with <u>Sph</u>I and the ends modified with <u>Bam</u> linkers. The Bam linkers have the sequence CGGATCCG.

pHBS56/16-3 was prepared as follows: A <u>Taq</u>I-<u>Hpa</u>I fragment obtained from the HBsAg coding region which included 26bp of the pre-sAg region, 681bp of the sAg region and 128bp of the 3'-untranslated region, was linked with <u>Eco</u>RI linkers and cloned at the <u>Eco</u>RI site in pBR322. The <u>Eco</u>RI linkers have the sequence GGAATTCC. The plasmid pHBS5 was thus obtained.

After digesting pHBS5 with <u>Eco</u>RI, the digest was resected with <u>Bal</u>31 and religated with <u>Eco</u>RI linkers (GGAATTCC). After digestion with <u>Eco</u>RI the material of about 800bp was isolated from a polyacrylamide gel. This isolate was then recloned into pBR322 which had been digested with <u>Eco</u>RI and treated with alkaline phosphatase. Where the resection was to the sequence CATGG, which included the methionine codon, the <u>Eco</u>RI linker created an <u>Nco</u>I site. The plasmids were screened for the presence of an <u>Nco</u>I site and one of the plasmids chosen for further manipulation. This plasmid, designated pHBS5-3, was restricted with <u>Eco</u>RI, the <u>Eco</u>RI fragment made blunt-ended with Klenow fragment and dNTPs, and the blunt-ended fragment was then restricted with <u>Xba</u>I to provide an about 100bp fragment having an <u>Xba</u>I overhang and blunt end at the former <u>Eco</u>RI site.

pHBS5 was then digested with <u>Cla</u>I, made blunt-ended with the Klenow fragment and dNTPs, digested with <u>Xba</u>I, followed by alkaline phosphatase treatment. The 100bp fragment was then inserted into the vector to provide the plasmid pHBS6. Upon sequencing of the blunt-ended ligation site, it was found that an adenosine had been lost, so as to lose the <u>Eco</u>RI site, where the sequence was now ATCGATTCCCATGG. The <u>Cla</u>I and <u>Nco</u>I sites were retained. The loss of the A resulted in pHBS6 having a single <u>Eco</u>RI site.

pHBS5-3 was digested with <u>Eco</u>RI and the resulting <u>Eco</u>RI fragment having the sAg fragment isolated by gel electrophoresis and inserted into the vector pHBS16 (Valenzuela <u>et</u><u>al</u>., <u>Nature</u> (1982) <u>298</u>:347-350). This plasmid has the ADH1 promoter and the sAg gene in an <u>Eco</u>RI fragment in a plasmid containing the 2µm origin, a <u>Trp</u>I gene and pBR322. The plasmid was digested with <u>Eco</u>RI, treated with alkaline phosphatase to prevent recircularization, and the <u>Eco</u>RI fragment from pHBS5-3 inserted to provide pHBS16-3, where the sAg gene isolated as a <u>Taq</u>I-<u>Hpa</u>I fragment had been modified by <u>Bal</u>31 resection. The plasmid pHBS16-3 was digested with <u>Sph</u>I and <u>Xba</u>I to provide a fragment which had the ADH promoter at the <u>Sph</u> terminus and the 5'-end of the sAg gene.

pHBS56 was then digested with <u>Sph</u>I. pHBS56 was prepared from pC1/1 by digestion with <u>Sph</u>I, which deletes a portion of the plasmid spanning the 2µm-pBR322 joint. The active portion of the ADH1 promoter region is contained within the <u>Sph</u>I-<u>Hin</u>dIII fragment of approximately 300bp (Bennetzen <u>et</u><u>al</u>., <u>J. Biol. Chem.</u> (1982) <u>257</u>:301). The <u>Sph</u>I site in the ADH promoter begins at position -413 and the yeast terminator sequence is contained within a <u>Hin</u>dIII-<u>Sph</u>I fragment of about 330bp. In each case the <u>Sph</u>I site is distal to the coding region. A 1500bp ADH1 promoter fragment terminating at position -9 (Hitzeman <u>et</u><u>al</u>., <u>Nature</u> (1981) <u>293</u>:717) and an approximately 450bp terminator unit from nucleotides 913 to 1368 in the ADH gene nucleotide sequence were joined at a <u>Hin</u>dIII site between the fragments and cloned into the <u>Bam</u>HI site of the vector YEp13 (Broach and Hicks, <u>Gene</u> (1979) <u>8</u>:121) to provide pADH5.

The HBsAg-DNA segment of pHBS5 was excised by <u>Eco</u>RI digestion, blunt-ended with the Klenow fragment and joined at both ends with <u>Hin</u>dIII linkers, CAAGCTTG. After digestion with <u>Hin</u>dIII, the HBsAg fragment was inserted into the <u>Hin</u>dIII site of the plasmid pADH5 which had been digested at the <u>Hin</u>dIII site intermediate the ADH1 promoter and terminator sequence. A plasmid with the HBsAg gene in the correct orientation as determined by restriction analysis was designated pHBS22. The cassette was included between two <u>Sph</u>I restriction sites. pHBS22 was digested with <u>Sph</u>I to obtain a fragment of about 1500bp and inserted into <u>Sph</u>I digested pC1/1 to provide pHBS56 which was cloned in <u>E</u>. <u>coli</u> HB101.

pHBS56 was digested with <u>Sph</u>I and <u>Xba</u>I to provide a 1.1kb fragment having the ADH terminator region and the 3'-portion of the sAg gene with the <u>Sph</u>I site proximal to the terminator region. The 1.1kb <u>Sph</u>I-<u>Xba</u>I fragment was joined to the <u>Sph</u>I-<u>Xba</u>I fragment from pHBS16-3, which resulted in providing the complete sAg gene in the correct orientation between the ADH promoter and terminator. This <u>Sph</u>I-<u>Sph</u>I fragment was then ligated to <u>Sph</u>I digested pHBS56, replacing the cassette of pHBS56 to provide the plasmid pHBS56/16-3 with the resected sAg coding region fragment. The cassette was then excised from pHBS56/16-3 by digestion with <u>Sph</u>I, followed by chewing back the overhangs with the Klenow fragment in the presence of dNTPs, then ligated with <u>Bam</u>HI linkers, followed by digestion with <u>Bam</u>HI to provide a 1.6kb fragment which was isolated by gel electrophoresis. The fragment included the ADH promoter region, the sAg gene and ADH terminator region, as described above. This fragment was inserted into the <u>Bam</u>HI site of pBR322 to provide pPGT16-3 which was digested with <u>Bam</u>HI and <u>Xba</u>I and the resulting 1.1kb fragment gel isolated, where the <u>Xba</u>I-<u>Bam</u>HI fragment had the 3' portion of the sAg gene and the ADH terminator region.

pHBS6 was digested with <u>Xba</u>I and <u>Nco</u>I and the 94bp fragment gel isolated to provide the 5'-portion of the sAg gene. A synthetic adapter was prepared of the formula<img file="EP0480480A2_D0007.tif" /> having <u>Taq</u>I and <u>Nco</u>I termini and providing the -25 to -1 nucleotides of the GAPDH (GAP49) promoter and the initiation codon of the sAg gene. This synthetic fragment, the <u>Nco</u>I-<u>Xba</u>I fragment, and the <u>Xba</u>I-<u>Bam</u>HI fragment were ligated simultaneously, followed by digestion with <u>Taq</u>I and <u>Bam</u>HI. The resulting fragment was then substituted into pBR322 linearized with <u>Cla</u>I and <u>Bam</u>HI, followed by treatment with alkaline phosphatase. The resulting plasmid, which contains the -1 to -25bp of the GAPDH promoter region, the sAg gene, and the ADH terminator, where the <u>Nco</u>I restriction site is lost was called pHBS6LGAPsAgtADH.

pGAP1, a plasmid prepared by insertion of a <u>Hin</u>dIII fragment containing the GAPDH gene GAP49 (Holland and Holland, <u>J. Biol. Chem.</u> (1979) <u>254</u>:5466-5474) inserted in the <u>Hin</u>dIII site of pBR322, was digested with <u>Hin</u>fI and a 500bp promoter containing fragment isolated. The fragment was resected with <u>Bal</u>31 to remove about 50 or 90bp, followed by ligation with <u>Hin</u>dIII linkers and digestion with <u>Hin</u>dIII. pBR322 was digested with <u>Hin</u>dIII, followed by treatment with alkaline phosphatase and the about 450 or 410bp fragment inserted to provide pGAP128 and pGAP396, respectively.

pGAP128 was digested with <u>Hin</u>dIII, the fragment made blunt-ended with the Klenow fragment and dNTPs and the resulting 450bp fragment isolated by gel electrophoresis. This fragment was inserted into <u>Sma</u>I digested plot5, which had been treated with alkaline phosphatase, to provide plasmid plot5pGAP128, which contained about -400 to +27bp of the GAPDH promoter and coding region. Plasmid plot5pGAP396 was prepared from pGAP396 in an identical manner and thus differs from plasmid plot5pGAP128 in having about 15-30 fewer bp at each terminus of the GAPDH promoter region (about -385 to -3).

Plasmids GAP1-GAP4 were then prepared in the following manner. Plasmid plot5pGAP128 was digested with <u>Taq</u>I and <u>Bam</u>HI to provide an about 390bp <u>Taq</u>I-<u>Bam</u>HI fragment which included the -26 to about -400bp of the GAPDH promoter region and a portion of the <u>Hin</u>dIII and plot5 polylinker. pHBS6LGAPsAgtADH plasmid was also digested with <u>Taq</u>I and <u>Bam</u>HI and a 1.1kb <u>Taq</u>I-<u>Bam</u>HI fragment containing the 3'-terminus of the GAPDH promoter region, the sAg gene and the ADH terminator region was gel isolated and ligated to the other <u>Taq</u>I-<u>Bam</u>HI fragment to provide a <u>Bam</u>HI-<u>Bam</u>HI fragment which included approximately 400bp of the GAPDH promoter region, the sAg gene in proper orientation for transcriptional regulation by the GAPDH promoter, followed by the ADH terminator region. This fragment was ligated into pBR322 which had been digested with <u>Bam</u>HI and treated with alkaline phosphatase to provide plasmid pPGT80. This <u>Bam</u>HI cassette could now be isolated and inserted into plasmid pC1/1, at the <u>Bam</u>HI site in the pBR322 portion of pC1/1, where in plasmid GAP1 the ADH terminator region is proximal to the <u>amp</u>^<u>r</u> gene with the pBR322 portion divided into an approximately 4kb sequence including the <u>amp</u>^r gene and a 375bp region separating the cassette from the 2µm sequences. In GAP2, the promoter is adjacent to the long pBR322 sequence with transcription in the same direction as the <u>amp</u>^r gene. The same cassette was inserted into <u>Bam</u>HI-digested pC1/1GAL4/650 to obtain plasmids GAP3 and GAP4, where GAP3 has the GAPDH promoter distal from the GAL4 regulator region and the long pBR322 sequence and GAP4 has the GAPDH promoter adjacent to the GAL4 regulator region, which is adjacent to the long pBR322 sequence.

Plasmids GAP5 and GAP6 were isolated as follows. Plasmid plot5pGAP396 was digested with <u>Sal</u>I and <u>Taq</u>I and a fragment containing 9bp of the plot5 polylinker sequence and the GAPDH promoter sequence extending from about -385 to -26bp was isolated. An approximately 130bp <u>Taq</u>I-<u>Xba</u>I fragment including -25 to -1bp of the GAPDH promoter and +1 to +93bp of the sAg gene was obtained from pHBS6LGAPsAgtADH. A 1.1kb <u>Xba</u>I-<u>Sal</u>I fragment containing the 3'-portion of the sAg gene and the ADH terminator as well as 6bp of plot5 polylinker sequence was obtained from plasmid plot5sAgtADH (described below---Pyruvate Kinase Promoter). These three fragments were ligated, digested with <u>Sal</u>I and then cloned into <u>Sal</u>I-digested pC1/1GAL4/370. GAP5 has the GAPDH promoter region adjacent to the GAL4 regulator region, which is proximal to the short pBR322 sequence, and GAP6 has the GAPDH promoter region distal from the GAL4 regulator region and proximal to the long pBR322 sequence (see Fig. 1).

Pyruvate kinase promoter

.

Plasmid pHBS6Pyk containing the sAg gene under the transcriptional regulatory control of the Pyk promoter was obtained by cloning a 4.4kb insert of yeast genomic DNA in pBR322 containing the Pyk gene and 911 nucleotides of 5'-untranslated region, and digestion of this plasmid pPyk9.1.1 with <u>Xba</u>I. After making the ends blunted-ended, the linear fragment was digested with <u>Bam</u>HI providing a 912bp <u>Bam</u>HI-blunt fragment containing the Pyk promoter and 8 bases from the Pyk coding region. This fragment was inserted into the plasmid pHBS6, which had been digested with <u>Nco</u>I, blunt-ended and digested with <u>Bam</u>HI. The plasmid pHBS6Pyk was totally digested with <u>Eco</u>RI, to obtain a fragment including the sAg gene and a portion of the Pyk promoter region. The fragment was made blunt-ended with the Klenow fragment and dNTPs, followed by ligation to <u>Bam</u>HI linkers, digested with <u>Xba</u>I, which is internal to the sAg gene, the <u>Xba</u>I terminus made blunt-ended with the Klenow fragment and dNTPs, followed by digestion with <u>Bam</u>HI, to provide a 580bp <u>Bam</u>HI-blunt-ended (<u>Xba</u>I) fragment. The plasmid plot5 was digested with <u>Eco</u>RI, made blunt-ended, digested with <u>Bam</u>HI and treated with alkaline phosphatase and the two fragments joined to provide plasmids plot5PyksAg51 and plot5PyksAg.57. The two differ in that the <u>Bam</u>HI site of the latter was not regenerated during cloning, possibly as a consequence of minimal nuclease contamination (digestion).

plot5 was treated as previously described (<u>Eco</u>RI digestion, blunt-ended, <u>Bam</u>HI digestion and treatment with alkaline phosphatase) and joined to a 1.1kb fragment obtained by digestion of pPGT16-3 with <u>Xba</u>I, followed by blunt ending, followed by digestion with <u>Bam</u>HI and gel isolation. This fragment was introduced into plot5 to provide the plasmid plot5sAgtADH. Again the <u>Bam</u>HI site in this plasmid was not regenerated, presumably due to digestion by contaminating nuclease.

Plasmids Pyk1 and Pyk2 were prepared as follows. Plasmid plot5PyksAg51 was digested with <u>Bam</u>HI, then with <u>Xba</u>I, and an approximately 580bp fragment containing about 480bp of Pyk promoter and 93bp of the 5'-end of the sAg gene was gel isolated. A 1.1kb <u>Xba</u>I-<u>Sal</u>I fragment containing the 3'-portion of the sAg gene, the ADH terminator and about 6bp of the plot5 polylinker was isolated from plot5AgtADH. These two fragments were ligated, digested with <u>Bam</u>HI and <u>Sal</u>I and then cloned into plasmid pC1/1, which had been cleaved with <u>Bam</u>HI and <u>Sal</u>I and treated with alkaline phosphatase, to yield plasmid Pyk1. Plasmid Pyk2 was prepared similarly but the 580bp <u>Sal</u>I-<u>Xba</u>I, Pyk promoter/HBsAg gene 5'-end fusion fragment was isolated from plot5PyksAg.57 and included about 6bp of plot5 polylinker sequence upstream from the promoter region. Also the 1.1kb <u>Xba</u>I-<u>Bam</u>HI fragment containing the 3'-part of the HBsAg gene and the ADH terminator was derived from plasmid pPGT16-3.

Plasmids Pyk3-Pyk6 were prepared as follows. Plasmid plot5PyksAg51 was digested with <u>Bam</u>HI, then with <u>Xba</u>I and the about 580bp fragment containing the Pyk promoter and the 5'-part of the HBsAg gene isolated as above. The 1.1kb <u>Bam</u>HI-<u>Xba</u>I fragment, containing the 3'-portion of the HBsAg gene and ADH terminator, was recovered from pPGT16-3, also as above, and the two fragments ligated, digested with <u>Bam</u>HI and inserted with different orientations into the <u>Bam</u>HI site of pC1/1GAL4/650 (Pyk3, Pyk4). Plasmids Pyk5 and Pyk6 were prepared similarly except that the <u>Sal</u>I-<u>Xba</u>I fragment containing the Pyk promoter and 5'-end of the sAg gene was isolated from plot5PyksAg.57 and the <u>Xba</u>I-<u>Sal</u>I sAg gene 3'-portion/ADH terminator fusion fragment was derived from plot5sAgtADH and thus both fragments included approximately 6bp of plot5 polylinker sequence. The cassette so formed was then cloned into the <u>Sal</u>I site of pC1/1GAL4/370 in opposite orientations.

The six plasmids designated Pyk1-6 (see Fig. 2) are distinguished by Pyk1 having the promoter region proximal to the short pBR322 sequence; Pyk2 having the promoter region proximal to the long pBR322 sequence; Pyk3 having the promoter region proximal to the short pBR322 sequence and distal from the GAL4 sequence; while Pyk4 has the promoter region proximal to the GAL4 region, which in turn is proximal to the long pBR322 sequence; Pyk5 has the promoter region proximal to the GAL4 region which is proximal to the short pBR322 sequence; while Pyk6 has a promoter region distal from the GAL4 region and proximal to the long pBR322 sequence.

These plasmids described above were transformed into <u>S</u>. <u>carlsbergensis</u> strain 2150-2-3 (available from Lee Hartwell, University of Washington) under conventional conditions (Hinnen <u>et</u><u>al</u>., <u>Proc. Natl. Acad. Sci. USA</u> (1978) <u>75</u>:1929-1933). Cultures of 50-150ml were grown to mid or late log phase in rich media (YEP) under neutral conditions (3% glycerol, 2% lactic acid), and then inducing conditions (+2% galactose), or repressing conditions (+2% glucose) for the final 1-2 generations. After lysis with glass beads and clarification of the supernatants by centrifugation, HBsAg expression was determined as described above. The results for the 12 plasmids are set forth in the following Table 1. <tables id="tabl0001" num="0001"><img file="EP0480480A2_D0008.tif" /></tables>

Construction of pPGAP

.

A yeast expression vector was prepared called pPGAP having a polyrestriction site linker between the GAPDH terminator and short promoter region. Plasmid plot5pGAP128 was digested with <u>Bam</u>HI and <u>Taq</u>I to yield an approximately 390bp <u>Bam</u>HI-<u>Taq</u>I fragment having the -400 to -26bp of the GAPDH promoter. The <u>Bam</u>HI-<u>Taq</u>I fragment was ligated to a synthetic fragment having the following sequence:<img file="EP0480480A2_D0009.tif" /> to provide a <u>Bam</u>HI-<u>Sal</u>I fragment, which was digested with <u>Bam</u>HI and <u>Sal</u>I and used to replace the <u>Bam</u>HI-<u>Sal</u>I fragment of <u>Bam</u>HI-<u>Sal</u>I digested pBR322 treated with alkaline phosphatase. After ligation, the plasmid pGAPNRS was obtained which was digested with <u>Bam</u>HI and <u>Sal</u>I to provide a 400bp <u>Bam</u>HI-<u>Sal</u>I fragment which was gel isolated. This fragment was ligated to an about 900bp <u>Sal</u>I-<u>Bam</u>HI fragment containing the GAPDH terminator region and a short segment of 3' coding region and the resulting 1.4kb <u>Bam</u>HI-<u>Bam</u>HI fragment digested with <u>Bam</u>HI. The <u>Sal</u>I-<u>Bam</u>HI GAPDH terminator fragment was obtained by <u>Sal</u>I and <u>Bam</u>HI digestion of pGAP2, a plasmid prepared by insertion of an about 3.3kb <u>Bam</u>HI fragment containing the GAPDH gene GAP49 (Holland and Holland, <u>supra</u>) into the <u>Bam</u>HI site of pBR322. Plasmids pGAP2 and pGAP1 were obtained as follows: A yeast gene library was prepared by inserting fragments obtained after partial digestion of total yeast DNA with restriction endonuclease <u>Sau</u>3A in lambda-phage Charon 28 (Blattner <u>et</u><u>al</u>., <u>Science</u> (1977) <u>196</u>:161-169). The phage library was screened with DNA complementary to the yeast GAPDH mRNA and the yeast GAPDH gene from one of these clones was subcloned as either an about 3.3kb <u>Bam</u>HI fragment in the <u>Bam</u>HI site of pBR322 (pGAP-2) or as an about 2.1kb <u>Hin</u>dIII fragment in the <u>Hin</u>dIII site of pBR322 (pGAP-1).

pBR322 was digested with <u>Eco</u>RI and <u>Sal</u>I, the termini blunt-ended and ligated to <u>Bam</u>HI linkers, followed by <u>Bam</u>HI digestion and the <u>Bam</u>HI-<u>Bam</u>HI 3.8kb fragment gel isolated, recircularized by self-ligation, cloned and designated pBRΔR1-Sal. The 1.4kb <u>Bam</u>HI-<u>Bam</u>HI fragment was inserted into the <u>Bam</u>HI-digested, alkaline phosphatase treated pBRΔR1-Sal vector to provide the plasmid pPGAP of about 5.3kb with the orientation in the opposite direction of the <u>amp</u>^r.

The plasmid phSOD was prepared as follows:

Molecular cloning of hSOD cDNA

.

Total RNA was prepared from an adult human liver by the guanidinium thiocyanate/lithium chloride method (Cathala <u>et</u><u>al</u>., <u>DNA</u> (1983) <u>2</u>:329-335). polyA RNA was used to synthesize double-stranded cDNA (Maniatis <u>et</u><u>al</u>., <u>Molecular</u><u>Cloning</u>, 213-242, Cold Spring Harbor, 1982) and this was passed over a Sepharose CL4B column to enrich for cDNAs of greater than 350bp (Fiddes and Goodman, <u>Nature</u> (1979) <u>281</u>:351-356). The cDNA was inserted at the <u>Pst</u>I site of p<u>lot</u>4, a pBR322 derivative having the following sequence replacing the <u>Pst</u>I-<u>Eco</u>RI site.<img file="EP0480480A2_D0010.tif" /> The cDNA insertion employed the oligo-dG:dC tailing method (Maniatis <u>et</u><u>al</u>., <u>supra</u>). <u>E</u>. <u>coli</u> strain D1210 was transformed with this mixture and transformants selected on L-agar containing 10µg/ml tetracycline (Kushner, S.R. (1978) In: <u>Genetic</u><u>Engineering</u>, eds. Boyer, H.B. and Nicosia, S., (Elsevier/North Holland, Amsterdam) p. 17). Plasmid DNA constituting a liver cDNA library was prepared (Maniatis <u>et</u><u>al</u>., <u>Molecular</u><u>Cloning</u>, pp. 86-94, Cold Spring Harbor 1982) directly from approximately 62,000 recombinant colonies plated at a density of approximately 3,000 colonies per 9cm diameter Petri dish.

Isolation of r-hSOD clones

.

Strain D1210 was retransformed with the liver cDNA library and about 40,000 clones were grown on nine 14cm diameter Petri dishes. After transfer of the colonies to nitrocellulose paper and chloramphenicol amplification of plasmid DNA, the cells were lysed and the filters prepared for hybridization (Ish-Horowicz and Burke, <u>Nucleic</u><u>Acids</u><u>Research</u> (1981) <u>9</u>:2989-2998). Oligonucleotide probes were employed for screening by hybridization, with the probes consisting of enzymatically-radiolabeled, chemically-synthesized DNA molecules complementary to the mRNA encoding amino acid residues 19 to 24 of the protein (Jabusch <u>et</u><u>al</u>., <u>supra</u>.; Barra <u>et</u><u>al</u>., <u>supra</u>.); the mixture had the following sequences:<img file="EP0480480A2_D0011.tif" /> where all of the indicated possibilities for encoding the peptide sequence were prepared (32-fold degenerate).

The probes were labeled with ³²P to a specific activity of 1-3x10⁸cpm/µg and Millipore (0.45µm) filtered before use. Filters were prehybridized for 6hrs at 30°C in 4x SSC, 2x Denhardts's solution, 40mM sodium phosphate, pH 7.5, 300µg/ml sonicated salmon testes DNA. Hybridization was for 20hrs at 30°C in the same solution containing 2x10⁶cpm/ml hSOD DNA probe (residues 19-24). Filters were washed in 4x SSC, once for 15min at r.t. and twice for 15min at 30°C, blotted dry and autoradiographed with an intensifying screen for 24hrs at -70°C.

Areas on the master plates that corresponded to duplicate positive signals were picked into L-broth and plasmid DNA prepared by the miniscreen procedure (Maniatis <u>et</u><u>al</u>., <u>Molecular</u><u>Cloning</u>, 178, 368-369, Cold Spring Harbor 1982). This DNA was cut with <u>Pst</u>I and subjected to Southern blot analysis (Southern, <u>J</u>. <u>Mol</u>. <u>Biol</u>. (1975) <u>98</u>:503-517) hybridizing initially with the previous labeled probes (amino acid residues 19-24) and then with additional radiolabeled probes derived from amino acid residues 109-114 and having the following sequences (all possible variations, 72-fold degenerate) present as a mixture:<img file="EP0480480A2_D0012.tif" /> One plasmid pool (pSOD1) contained a cDNA insert of 520bp that hybridized with both probes and after colony purification, plasmid DNA was prepared from this clone and sequenced by the method of Maxam and Gilbert (<u>Proc. Natl. Acad. Sci. USA</u> (1977) <u>74</u>:560-564). The hSOD cDNA clone pSOD1 constitutes the coding region for amino acids 10-153 of hSOD, a single translational stop codon and a 3' untranslated region. Therefore, in the expression vector construct, the base sequence of the region encoding amino acids 1-9 is derived from the published amino acid sequence of hSOD (Jabusch <u>et</u><u>al</u>., <u>supra</u>; Barra <u>et</u><u>al</u>., <u>supra</u>) and synthesized chemically as a part of the variable linker segment (see discussion relating to Fig. 3).

Construction of plot5 derivatives containing r-hSOD

.

The synthetic DNA molecules F(26), C(16), B(31), D(11), E(13) and 4(24) shown in Fig. 3, were synthesized by the phosphoramidite method.

The single strand 4(24) was prepared by using all four bases, at each site where X is indicated. Furthermore, silica was withdrawn from the synthesis of the 24mer, such that single-stranded 21mers, 22mers, and 23mers are obtained in addition to the 24mers. After removal from the silica support, the four mixtures are combined in appropriate proportions to provide for equimolar amounts of each of the possible single strands. This mixture was treated as a single product in the subsequent steps.

Molecules F(26), C(16), B(31) and D(11) were mixed together in equimolar amounts and 10µg phosphorylated using T4 polynucleotide kinase. After phenol-ether extraction, the additional non-phosphorylated synthetic DNA molecules 4(24) and E(13) were added, such that all fragments were equimolar. The equimolar mixture contained 13µg of DNA in 133µl of 0.3x kinase buffer.

After annealing by cooling at a uniform rate from 70°C to 20°C over 60min, the single strands were ligated together with T4 ligase in 200µl ligation mix at 14°C for 4hrs, phenol-chloroform extracted, ethanol precipitated and the 5'-ends of 4(24) and E(13) phosphorylated using T4 polynucleotide kinase (Maniatis <u>et</u><u>al</u>., <u>supra</u>). Preparative polyacrylamide gel electrophoresis was used to isolate the completely ligated 53bp material having 5'- and 3'-overhangs.

The above purified fragment mixture was then ligated to the 460bp <u>Taq</u>I-<u>Pst</u>I segment of the hSOD cDNA as shown in Fig. 3. This segment was itself constructed by isolating the 454bp <u>Taq</u>I-<u>Alu</u>I hSOD fragment, making it flush-ended using Klenow and inserting it into p<u>lot</u>5 between its <u>Eco</u>RI and <u>Sal</u>I sites which had been similarly made flush-ended. After preparation of plasmid DNA from this recombinant, the 460bp <u>Taq</u>I-<u>Pst</u>I hSOD fragment was isolated by preparative polyacrylamide gel electrophoresis. After extraction and precipitation, the 515bp fragment resulting from the joining of the synthetic fragment to the 460bp <u>Taq</u>I-<u>Pst</u>I hSOD fragment was blunt-ended (525-528bp) and then digested with <u>Sal</u>I and the resulting 519-522bp hSOD fragment isolated by polyacrylamide gel electrophoresis. This fragment was then inserted into p<u>lot</u>5 which had been digested with <u>Pvu</u>II and <u>Sal</u>I and then treated with alkaline phosphatase. The resulting plasmids were used to transform strain D1210. Recombinants obtained after transformation of strain D1210 were selected on L-agar containing 100µg/ml ampicillin to give a set of clones, which were screened for an <u>Nco</u>I site. One was selected and designated phSOD.

Construction of a yeast vector for SOD expression

.

The plasmid phSOD was digested with <u>Nco</u>I and <u>Sal</u>I and a 550bp fragment obtained, which included 1 nucleotide untranslated at the 5'-terminus and the entire coding region for hSOD. pPGAP was digested with <u>Nco</u>I and <u>Sal</u>I followed by treatment with alkaline phosphatase and the <u>Sal</u>I-<u>Nco</u>I fragment substituted for the <u>Nco</u>I-<u>Sal</u>I fragment in pPGAP to provide pPGAPSOD. <u>Bam</u>HI digestion of pPGAPSOD resulted in a 2kb fragment which was gel isolated and inserted into the <u>Bam</u>HI site of pC1/1 and pC1/1 GAL4/370. These plasmids were transformed into yeast strain 2150-2-3 as described previously, with the results of expression set forth in the following Table 2. <tables id="tabl0002" num="0002"><table frame="all"><title>Table 2</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Expression of Human SOD in Yeast Strain 2150</entry></row><row><entry namest="col1" nameend="col1" align="center">Plasmid</entry><entry namest="col2" nameend="col2" align="center">Carbon Source</entry><entry namest="col3" nameend="col3" align="center">SOD² µg/mg protein</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="center">pC1/1</entry><entry namest="col2" nameend="col2" align="left">g, L¹</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="center">pC1/1GAPSOD</entry><entry namest="col2" nameend="col2" align="left">g, L</entry><entry namest="col3" nameend="col3" align="char" char=".">148</entry></row><row><entry namest="col1" nameend="col1" align="center">pC1/1GALGAPSOD</entry><entry namest="col2" nameend="col2" align="left">g, L</entry><entry namest="col3" nameend="col3" align="char" char=".">0.4</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="left">gal</entry><entry namest="col3" nameend="col3" align="char" char=".">68</entry></row></tbody></tgroup><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col3" align="justify">¹ All cultures grown in Minus Leucine media with 2% lactic acid, 3% glycerol with or without 2% galactose to late log or early stationary phase.</entry></row><row><entry namest="col1" nameend="col3" align="justify">² Determined by RIA.</entry></row></tbody></tgroup></table></tables> hSOD levels were measured using a standard radioimmunoassay with iodinated authentic hSOD as standard. Constitutive synthesis from the GAP promoter leads to very high levels of hSOD production, of the order of 10-30% of the total cell protein. The induction with galactose works almost as well, yielding about 7% of the cell protein as hSOD.

Construction of pYASI1

This yeast expression plasmid contains the hSOD gene fused to the amino terminus of the human proinsulin gene, with a methionine codon at the junction between both genes. The fusion gene is under control of the hybrid inducible ADH2-GAP (yeast alcohol dehydrogenase 2) promoter and the GAP terminator. An about 3kbp <u>Bam</u>HI expression cassette was constructed.

The ADH2 portion of the promoter was constructed by cutting a plasmid containing the wild type ADH2 gene (plasmid pADR2, see Beier and Young, Nature (1982) <u>300</u>:724-728), a pBR322 derivative, which contains the yeast centromere gene from chromosome 3 (CEN3) (U.S. Patent No. 4,464,472), the <u>Trp</u>1 gene and the yeast ADHII gene with the restriction enzyme <u>Eco</u>R5, which cuts at a position +66 relative to the ATG start codon, as well as in two other sites in pADR2, outside of the ADH2 region in the <u>CEN</u>3 and <u>Trp</u>1 gene. The resulting mixture of a vector fragment and two smaller fragments was resected with <u>Bal</u>31 exonuclease to remove about 300bp. Synthetic <u>Xho</u>I linkers were ligated onto the <u>Bal</u>31 treated DNA. The resulting DNA linker vector fragment was separated from the linkers by column chromatography, cut with the restriction enzyme <u>Xho</u>I, religated and used to transform <u>E</u>. <u>coli</u> to ampicillin resistance. The positions of the <u>Xho</u>I linker additions were determined by DNA sequencing. One plasmid which contained an <u>Xho</u>I linker located within the 5' non-transcribed region of the ADH2 gene (position -232 from ATG) was cut with the restriction enzyme <u>Xho</u>I, treated with nuclease S1, and subsequently treated with the restriction enzyme <u>Eco</u>RI to create a linear vector molecule having one blunt end at the site of the <u>Xho</u>I linker and an <u>Eco</u>RI end at the pBR322 proximal terminus.

The GAP portion of the promoter was constructed by cutting plasmid pPGAP (supra) with the enzymes <u>Bam</u>HI and <u>Eco</u>RI, followed by the isolation of the 0.4Kbp DNA fragment. The purified fragment was cut with the enzyme <u>Alu</u>I to create a blunt end near the <u>Bam</u>HI site.

Plasmid pJS014 was constructed by the ligation of the <u>Alu</u>I-<u>Eco</u>RI GAP promoter fragment to the ADH2 fragment present on the linear vector described above.

Plasmid pJS104 was digested with <u>Bam</u>HI (which cuts upstream of the ADH2 region) and with <u>Nco</u>I (which cuts downstream of the GAP region). The about 1.3Kbp fragment containing the ADH2-GAP promoter was gel purified and ligated to an about 1.7Kbp fragment containing the hSOD-proinsulin fusion DNA sequences and GAP terminator present in pYSI1. This 3Kbp expression cassette was cloned into <u>Bam</u>HI digested and phosphatase treated pC1/1 to yield pYASI1.

Plasmid pYSI1 was constructed as follows. Three fragments were employed which involve a 454bp <u>Nco</u>I-<u>Sau</u>3A isolated from phSOD, where the fragment includes the entire coding sequence for human superoxide dismutase (hSOD) with the exception of the last three 3'- codons; a 51bp <u>Sau</u>3A-<u>Hin</u>dIII synthetic adapter, which codes for the last three codons of hSOD, methionine, and the first 14 codons of proinsulin; and a 231bp <u>Hin</u>dIII-<u>Sal</u>I fragment, isolated from pINS5 (a pBR322-derived vector which contains a proinsulin coding sequence chemically synthesized according to the amino acid sequence reported by Bell <u>et</u><u>al</u>., <u>Nature</u> (1979) <u>282</u>:525-527), which encodes proinsulin excepting the first 14 amino acids. These fragments were ligated together and introduced into the plasmid pPGAP, which had been previously digested with <u>Nco</u>I and <u>Sal</u>I and alkaline phosphatase treated. The resulting plasmid pSI1 was digested with <u>Bam</u>HI to provide an expression cassette which was cloned into plasmid pC1/1 to yield pYSI1.

Cloning of alpha-1-antitrypsin

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A cDNA library was made from 10µg of polyA⁺ RNA isolated from a part of a human liver. This library was prepared by oligo-dT priming of the first cDNA strand and self-priming of the second cDNA strand. The ds cDNA was size fractionated on a Sepharose CL4B column and those molecules greater than 300bp isolated. This fraction was treated with nuclease S1 and tailed with dCTP, using terminal transferase. The tailed cDNA was annealed to pBR322 which had been digested with <u>Pst</u>I and tailed with dGTP. Transformation of <u>E</u>. <u>coli</u> HB101 yielded 60,000 colonies, where greater than 90% of the clones were recombinant.

Two synthetic oligonucleotide probes were used to isolate the alpha-1-antitrypsin (α₁-AT) cDNA, the first probe corresponding to amino acid residues 344-350 near the C-terminus of the protein was used to probe 5,000 colonies and the second probe, corresponding to amino acid residues -23 to -17 (+1 being the first nucleotide of the first codon of the mature α₁-AT) of the signal peptide, was used to probe 25,000 colonies. The probe sequences were taken from the partial nucleotide sequence described by Kurachi <u>et</u><u>al</u>., <u>Proc. Natl. Acad. Sci. USA</u> (1981) <u>78</u>:6826; Leicht <u>et</u><u>al</u>., <u>Nature</u> (1982) <u>297</u>:655). Approximately 3% of the colonies hybridized to the C-terminal probe and four hybridized to the N-terminal probe. The four N-terminal clones and 12 C-terminal clones were isolated and subjected to restriction analysis. From these, three overlapping clones which cover the entire cDNA were subjected to further study and were used to construct the full length cDNA clone.

The entire sequence of a composite full length cDNA derived from the three plasmids is as follows:<img file="EP0480480A2_D0013.tif" />

LEGEND

Nucleotide and predicted amino acid sequences of α₁-AT cDNA. The reactive center met-ser at positions 358-359 is boxed. Subscripts to amino acids in parentheses identify differences between the subject protein sequence and those derived from (a) protein sequencing (Carrell <u>et</u><u>al</u>., 1982), (b) the cDNA of Woo et al., [see Carrell <u>et</u><u>al</u>., 1982]), and (c) the cDNA of Bollen <u>et</u><u>al</u>., 1983. The synthetic DNA molecules used in the construction of the <u>Bam</u>HI to <u>Sal</u>I fragment encoding the mature protein are shown as are the cDNA restriction sites used in this construction.

The above sequence was determined using the dideoxy sequencing method of Sanger <u>et</u><u>al</u>., <u>Proc. Natl. Acad. Sci. USA</u> (1977) <u>74</u>:5463, in the M13 vectors of Messing <u>et</u><u>al</u>., <u>Nucleic Acids Res.</u> (1981) <u>9</u>:309. The differences at the nucleotide and amino acid level from the published cDNA sequences are shown.

Construction of the full length clone for expression of yeast began with three fragments isolated from cDNA clones: 1) a 630bp <u>Bam</u>HI-<u>Bst</u>EII fragment; 2) a 450bp <u>Bst</u>EII-<u>Ava</u>I fragment; and 3) an 85bp <u>Ava</u>I-<u>Hin</u>fI fragment. A synthetic adapter was employed having the following sequence:<img file="EP0480480A2_D0014.tif" /> Approximately two pmoles of fragments 1 and 2 were ligated together and after removal of the ligase, digested with <u>Bam</u>HI and <u>Ava</u>I. Fragment 3 and the synthetic adapter were ligated and digested with <u>Ava</u>I and <u>Sal</u>I and the two resulting fragment mixtures were ligated followed by digestion with <u>Bam</u>HI and <u>Sal</u>I. Fragments migrating upon electrophoresis in the region of about 1000-1400bp were isolated and cloned by substitution into <u>Bam</u>HI and <u>Sal</u>I digested and alkaline phosphatase treated pBR322. The resulting plasmid is referred to as pATi.

Plasmid pPGAP was digested with <u>Nco</u>I, followed by blunt-ending, followed by <u>Sal</u>I digestion and treatment with alkaline phosphatase. The <u>Nco</u>I-<u>Sal</u>I fragment was substituted with an approximately 1250bp blunt-ended (<u>Bam</u>HI)-<u>Sal</u>I fragment obtained from plasmid pATi, by <u>Bam</u>HI digestion, blunt ending, and <u>Sal</u>I digestion. This was inserted into the pPGAP vector to produce the plasmid pGAPATi, a 6.6kb plasmid, which was digested with <u>Nco</u>I and <u>Bam</u>HI and a 2.3kb <u>Nco</u>I-<u>Bam</u>HI fragment obtained having the α₁-AT gene and the GAPDH terminator and approximately 400bp <u>Bam</u>HI-<u>Nco</u>I fragment obtained having the GAPDH promoter. These fragments were ligated together and inserted into the <u>Bam</u>HI site of pC1/1. The plasmids pC1/1GAPATi8 and pC1/1GAPATi9 were obtained with the orientation of expression clockwise in the former and counterclockwise in the latter, with <u>amp</u>^r being in the counterclockwise direction. These plasmids were transformed in <u>S</u>. <u>cerevisiae</u> AB103 (A.T.C.C. No. 20658, deposited January 5, 1983) by standard methods, selecting for leucine prototrophy and grown as described above. Yeast extracts were prepared by lysis with glass beads and the α₁-AT activity determined by inhibition of human leukocyte elastase.

Assays contained in 1ml:0.1-0.2 human leukocyte elastase (HLE); 0.1mM MeO-Suc-Ala-Ala-Pro-Val-<u>p</u>-nitroanilide (Beatty <u>et</u><u>al</u>., <u>J. Biol. Chem.</u> (1980) <u>255</u>:3931); 50mM Tris, pH 8, 0.5M NaCl, and the indicated amounts of yeast extract or human α₁-AT. Assays were initiated by the addition of elastase, incubated at 28°C for 15min, terminated by the addition of 100µl of 8N acetic acid and the absorbance at 410nm determined. Typical results are shown in the following Table 3. <tables id="tabl0003" num="0003"><img file="EP0480480A2_D0015.tif" /></tables> The above data demonstrate that plasmids having the orientation of the expression cassette in the counterclockwise orientation, the promoter proximal to the long sequence of pBR322, make 10-20 times more α₁-AT than the same cassette in the other orientation.

Yeast strain AB110

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Yeast strain 2150-2-3 was crossed with a yeast strain AB103 transformant containing pC1/1GAPATi9. The diploids were sporulated and the tetrads dissected. Strains were maintained on leucine selective plates in order to ensure maintenance of the plasmid, since the parents are auxotrophs. A series of colonies were screened for their genotype with respect to a number of markers. The most vigorous strains were selected and cultures grown on leucine selective media. The best strain was designated AB110 (pC1/1GAPATi9), gave 6-7.5% of the total cell protein as α₁-AT as shown in the above Table 3. The strain AB110 has the following genotype: Matα, ura3-52, leu2-04 or both leu2-3 and leu2-112, pep4-3, his4-580 (cir°).

Phosphate induction

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Plasmid pPGT80 was digested with <u>Bam</u>HI, the ends blunt-ended, followed by digestion with <u>Xba</u>I and the 500bp fragment containing the GAPDH promoter and 5'-end of the sAg gene isolated.

The PHO5 gene was isolated from a yeast genomic library employing an oligonucleotide probe 5'-GGCACTCACACGTGGGACTAG-3' derived from the published partial sequence (Meyhack <u>et</u><u>al</u>., <u>The EMBO Journal</u> (1982) <u>1</u>:675-680). A subfragment of this clone containing 550bp of the 5'-untranslated region and approximately 80bp of coding sequence was subcloned as a <u>Bam</u>HI-<u>Sal</u>I substitution in pBR322 to provide pPHO5. This fragment has the sequence 5'-ATGTTTAAA-3', encoding the first three amino acids, the second and third codons specifying an <u>Aha</u>III site. The plasmid pHBS6 was digested with <u>Nco</u>I, blunt-ended, followed by digestion with <u>Bam</u>HI and treatment with alkaline phosphatase. The PHO5 promoter region was obtained by digesting the pPHO5 plasmid with <u>Aha</u>III, resecting the resulting fragment with <u>Bal</u>31 for a short time, followed by digestion with <u>Bam</u>HI and isolation of a 500-550bp <u>Bam</u>HI blunt-ended fragment. This fragment was employed for substitution of the <u>Nco</u>I-<u>Bam</u>HI fragment from pHBS6 and was screened for regeneration of the <u>Nco</u>I restriction site to provide plasmid pHBS6PHO5/1.

Plasmid pHBS6PHO5/1 was digested with <u>Bst</u>EII which cleaves at position -175 in the PHO5 promoter. This molecule was blunt-ended, digested with <u>Sal</u>I and the 650bp fragment having the 5'-portion of the promoter domain, containing 275bp of pBR322 and 375bp of the PHO5 promoter region isolated. This fragment was ligated with the blunt-ended (<u>Bam</u>HI)-<u>Xba</u>I fragment obtained from digestion of pPGT80 with <u>Bam</u>HI, blunt ending, followed by <u>Xba</u>I digestion. After digesting the ligated fragment with <u>Sal</u>I and <u>Xba</u>I, the resulting fragment was then substituted into pPGT16-3 which had been digested with <u>Sal</u>I and <u>Xba</u>I and treated with alkaline phosphatase. The resulting plasmid pPHO5PGT80 had a cassette comprising the PHO5 regulatory region, the GAPDH promoter, the sAg gene and the ADH terminator. This cassette was excised from the plasmid by <u>Bam</u>HI digestion, whereby a 1.8kb <u>Bam</u>HI-<u>Bam</u>HI fragment was gel isolated and ligated into the <u>Bam</u>HI site of <u>Bam</u>HI digested and alkaline phosphatase treated pC1/1 to provide plasmids PHO5GAP1 and PHO5GAP2 where the PHO5 was distal and proximal to the long pBR322 sequence, respectively.

The two plasmids were transformed into yeast strain 2150-2-3 as described above and grown in rich media as described above for 8 to 10 generations in either high (7mM) or low (0.2mM) phosphate. Samples were harvested in late log phase and HBsAg determined as described previously. The results are shown below in Table 4. <tables id="tabl0004" num="0004"><img file="EP0480480A2_D0016.tif" /></tables>

From the above results, it is evident that effective regulation with phosphate is obtained, with one orientation being superior to the other.

It is evident from the above results, that highly efficient expression can be obtained, either constitutive or regulated, by providing for truncated promoter regions of yeast glycolytic enzyme gene promoters, employing the 3' domain proximal to the coding region of the gene in conjunction with a 5'-portion or second domain of the promoter region of a yeast gene subject to inducible regulation by a nutrient, e.g., carbon source or phosphate, temperature, or other externally controllable source or condition. Alternatively, the second domain may be replaced by prokaryotic sequences of at least about 1kb or greater, which provide for constitutive enhancement in the absence of the second domain of the promoter region. Thus, a wide variety of genes exogenous to yeast may be expressed in high yield in high percentages of the total protein of the yeast host.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.