AU614959B2 - Expression system - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1952 COMPLETE SPECIFICATION Form

(ORIGINAL)

FOR OFFICE USE Short Title: Int. Cl: Application Number: Lodged: os:Complete Specification-Lodged: o a, Accepted: Lapsed: o Published: Priority: o o Related Art: o o COMPLETE-AFTER-PROVISIONAL PI 1788 °o0 0 0 0 0 TO BE COMPLETED BY APPLICANT o o-'°ame of Applicant: Address of Applicant: Actual Inventor: Address for Service: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION LIMESTONE AVENUE CAMPBELL, 2601 AUSTRALIAN CAPITAL TERRITORY

AUSTRALIA

Ian Geoffrey MACREADIE Paul Richard VAUGHAN Ahmed Abdullah AZAD CLEMENT HACK CO., 601 St. Kilda Road, Melbourne, Victoria 3004, Australia.

Complete Specification for the invention entitled: EXPRESSION SYSTEM The following statement is a full description of this invention including the best method of performing it known to me:r- 1C -i-;^nClc-TinCS~"e~

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eat F I S 0I I S 000 0 SEXPRESSION SYSTEM This invention relates to novel expression systems suitable for use in yeasts and E. coli, and to the production of desired proteins using said system.

Background and Prior Art Biotechnology probably has its beginnings with the first exploitation of the unicellular eukaryote, Saccharomyces cerevisiae, for baking and brewing. Today traditional uses continue but this budding yeast now tinds new roles in the new biotechnology industry where it can be made to produce valuable polypeptides such as interferons and antigens for vaccines.

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3 Many years of intensive studies in yeast genetics and molecular biology have contributed to making S. cerevisiae one of the best understood eukaryotes and ideal for the expression of foreign genes. Most expression systems designed to date utilize a constitutive yeast promoter which means that foreign genes are expressed throughout the growth cycle provided that their product is not deleterious or lethal to the yeast cell. The use of a convenient inducible expression system, therefore, can offer considerable advantages. In the 10 work now presented here, such a system, utilizing the CUP1 0 00 o'gene, is developed for use in yeast and in addition, o" significant constitutive expression is found in E. coli. CUP1 °,"',codes for yeast copper-metallothionein, a cysteine-rich 0 0 o o metallothionein-like protein.

0 0 Metallothionein-like proteins and oo0 'metallothionein-producing microorganisms have been suggested to be useful for chelation of transition metals, such as 0 ,metals found in industrial waste streams and in mining and mineral processing fluids. It has also been suggested that they may be useful in the treatment of metal poisoning, for example by passing a patient's plasma through columns having a metallothionein bound to the column support (Australian Patent Application No. 31637/84; European Patent Application EP-A2-78154).

The CUP1 gene in Saccharomyces cerevisiae cells confers a resistance level to copper that is proportional to CUPl copy number (Fogel and Welch, 1982) and, most importantly, the expression of the gene is tightly copper-dependent; when resistant cells are transferred from copper-depleted medium to medium with copper ions, CUP1 mRNA switches from being undetectable to being probably the most abundant mRNA. (Butt et al, 1984a, b; Karin et al, 1984; Etcheverry et al, 1986).

This facet of CUP1 expression has been previously exploited for the expression of foreign genes in yeast.

Expression vectors containing the yeast gene coding for metallothionein for transformation into a suitable host -4organism, and an isolated structural gene and gene expression units coding for yeast copper metallothionein have been disclosed (EP-A2-78154; EP-A3-96491; Australian Patent Application No. 31637/84). Each of these applications discloses expression systems of specific sequence. Butt et al (1984b) first reported the successful utilization of a BamHI-RsaI fragment, containing the CUP1 promoter, to direct the 50-fold inducible expression of the E. coli galactokinase (galk) gene: termination signals downstream of galk were 10 provided by the yeast CYCl gene. The precise region directing 0 00 o the copper-inducible expression, recently identified by Thiele 0o0 and Hamer (1986), was found to come primarily from two tandem 0o°o, copies of closely related sequences. The greatest induction o0 0 So1 ratios, however, were found when sequences from the BamHI 1iS, (Sau3AI) to the RsaI site were present.

000000 0 A second use of the CUP1 promoter was demonstrated by Etcheverry et al (1986), who utilised the same 430bp 000., Sau3AI-RsaI CUP1 fragment. In addition, however, they added o o0 on to the RsaI-cut end a synthetic linker which extended the o o 0 S'0 native 5' sequence by 18 nucleotides, including the (CAAT) 4 o o sequence; the subsequent bases generated XbaI and EcoRI 0oo 0 recognition sites which allowed for the expression of EcoRI or XbaI fragments containing their own ATG start codon. Such a e- construct allowed for inducible high levels of expression of the normally highly expressed yeast gene phosphoglycerate kinase (PGK). Replacement of the yeast PGK gene with the gene for human serum albumin (HSA), however, resulted in a marked reduction in inducible expression such that HSA comprised 1% of total yeast protein. Using these constructs, induction ratios of 6-fold were measured by Etcheverry et al (1986), Swhile deletions retaining 300 nucleotides or less upstream of the start codon led to the loss of induction, resulting in constitutive levels of expression for the CUP1 promoter.

These latter results are in direct contrast to those of Thiele Hamer (1986).

5 A recent study showed that maximal expression of a foreign viral gene, the gene of HBcAg in S. cerevisiae, took place when all non-translated 5' and 3' sequences flanking the viral sequences were yeast derived (Kniskern et al 1986).

We reasoned that the use of the 5' portion of the CUP1 sequence alone should be sub-optimal, so we sought to make a versatile construct, employing the complete native CUP1 sequence, which would allow for the expression of genes without their own start codon.

10 We have now found that by retaining CUP1 0 00- S 'transcription and translation regulatory signals upstream and 02 downstream of the gene it is possible to make in-frame gene o°o' fusions which translate a few codons from the start of the o CUP1 sequence but then translate codons from the foreign 1 5, sequence until reaching its own stop codon or a stop codon 0° 'derived from CUP1. Such a fusion masks the foreign sequence as much as possible and is in keeping with the criteria o00 ,required for maximal expression of heterologous genes. As a further refinement of the system, unique restriction sites 0 a were engineered at the extreme 5' and 3' ends such that the So- entire modified expression construction can be excised and moved as a unit which we have term a cassette, into a variety of yeast shuttle vectors, including those allowing for high, _0,medium and low copy number as well as those designed for 27 integration into the host genome.

Summary of the Invention According to one aspect of the present invention there is provided a gene expression system comprising the CUP1 promoter sequence, and the CUP1 coding sequence, transcriptional terminators, and unique restriction sites at the extreme 5' and 3' ends.

Preferably the expression system comprises a multiple cloning site provided by further unique restriction endonuclease cleavage sites. More preferably the multiple restriction endonuclease cleavage sites are situated soon after the start codon of the CUP1 sequence.

1 -6- 6 The restriction endonuclease sites in the multiple cloning site may be present in either orientation.

According to a second aspect of the invention there is provided an expression system for a desired protein comprising the above-defined gene expression system and the DNA sequence encoding said desired protein. Preferably said DNA sequence encoding said desired protein is inserted into the CUP1 sequence. The protein expression system according to the invention allows for the expression of foreign genes in 10 the absence of their own start codon.

0 00 Signal sequences enabling the desired protein to be secreted into the medium may optionally be incorporated into 0, the expression system according to the invention.

o According to a third aspect of the invention there is provided a method of construction of a gene expression o° system, comprising the steps of: cloning a portion of the CUP1 locus of S, Saccharomyces cerevisiae into restriction endonuclease cleavage sites in the replicative form of a bacteriophage; inserting an oligonucleotide encoding part of .0 the copper-inducible regulatory region of CUP1; creating a new restriction endonuclease cleavage site within the CUP1 gene; and cloning the DNA sequence thus produced into a Shost plasmid.

Preferably the restriction endonuclease cleavage site created in step above is an EcoRI site. Still more preferably said restriction endonuclease cleavage site is near to and after the start of the CUP1 coding sequence. Most preferably an A to C substitution is effected in the third base of the fifth codon of the CUP1 gene.

The EcoRI site enables further unique restriction endonuclease cleavage sites to be inserted into the sequence.

According to a fourth aspect of the invention, there is provided a method of producing a desired protein, comprising the steps of 7 cloning the DNA sequence encoding the desired protein into a host plasmid bearing the above-defined gene expression system; transforming the host plasmid into a susceptible microorganism; cultivating the microorganism under conditions suitable therefor; and isolating the desired protein.

Preferably the host microorganism is selected from S. cerevisiae, Schizosaccharomyces pombe and E. coli.

o The desired protein may optionally then be treated 0 to remove N-terminal methionine or formylmethionine, for °,'*,example using methionine aminopeptidase.

00 SDetailed Description of the Invention 015 The invention will now be further described by way of reference only to the following non-limiting examples, and S to the drawings, in which: S t Figure 1 is a simplified schematic representation of Sthe strategy for construction of the copper-inducible S2,4 expression system according to the invention, in which M13mpl8/CUP1, pUAS36, pMCE5, pMCPR and pVYC30 represent recombinants in the M13 host, and pYEUC19, pYEUC33, pYEUC89, ctand pYEUC94 represent recombinants in the yeast shuttle vector; Figure 2 shows the map of plasmid pFL44 together with the multiple cloning site (MCS) sequence between the Hind III and EcoRI sites; Figure 3 shows the nucleotide sequence of the 0.7 kb HindIII EcoRI fragment of M13mpl8/CUPl. Restriction endonuclease cleavage sites are listed below the DNA sequence; the CUP1 translation product is listed above the sequence.

The arrow indicates a nucleotide destined for mutagenesis, and the asterisk indicates the CUP1 stop codon; numbering is from the start of the CUP1 coding sequence; 8 Figure 4 shows the nucleotide sequence of the copper-inducible expression cassette according to one embodiment of the invention. The translation product of the cassette is shown above the nucleotide sequence, and the multiple cloning site in the cassette is shown; Figure 5 shows the structure of pYELC5, a high copy number vector. All restriction sites shown are unique to this plasmid; and Figure 6 shows the sequence of an alternative embodiment of the expression cassette according to the invention, in which the restriction sites at the multiple cloning site are in the reverse orientation.

o 00 0 0 MATERIALS AND METHODS 0.'0 Materials o 0 °o0 Tryptone, agar, yeast extract and peptone were from Soo Difco. Phytagar was purchased from Gibco. Calf intestinal Doo 0 ,:alkaline phosphatase, Klenow enzyme, and 0 S 5-bromo-4-chloro-3-indolyl- -D-galactopyranoside (X-gal) were from Boehringer Mannheim. O-nitrophenyl- -D-galactopyranoside o °iQ° (ONPG) was from Sigma.

0 o o The restriction endonucleases XbaI, KpnI, SacI, EcoRI, HindIII, BamHI, PstI, SphI, PvuII and SalI, as well as o °°T4 polynucleotide kinase and mung bean nuclease were from Pharmacia and were used according to the manufacturer's directions. Ligations employed T4 DNA ligase (BRL) in the suoplied buffer.

The M13 vectors Ml3mpl8 and M13mpl9 and the universal M13 17-mer sequencing primer were purchased from Amersham. All other oligonucleotides were synthesized using an Applied Biosystems synthesizer.

Other reagents were analytical grade.

Cloning Cloning methods were basically as described by Maniatis et al (1982). E. coli host strains were TG1, JM101 and JM109 for M13 cloning and MC1061 and MC1066 (from Malcolm 9- Casabadan, The University of Chicago) for cloning of shuttle vectors. Strains were grown in LB medium with ampicillin when required.

Yeast strains FLa (a ura3 CUP1 s and FL (a ura3 CUP1 s (from F. Lacroute, CNRS, Strasbourg, France), BZ31-1-7Ba (a ura3 CUP1 s ade8 trpl arg4) (Fogel and Welch, 1982), 6657-4D (a his3 leu2 CUP1 s (from P. Nagley, Monash University, Clayton, Australia), DBY747 (a his3 ura3 leu2 trpl CUP1

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(from D. Botstein, Massachusetts Institute of Technology, Cambridge, Maryland), FYHl (a leu2 trpl his3 CUP1R), FYH6 leu2 trpl his3 ura3 CUP1

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(from D.

Finkelstein, University of Texas Health Science Centre, o o Dallas, Texas) MY2 (a leu2 trpl his3 URA3 CUP1 R/ leu2 trpl 0 0 his3 URA3 CUP1R), a diploid obtained in our laboratory by a i5 cross between FYH1 and FYH6, Schizosaccharomyces pombe strains o .0'38399 (h leul) (Yeast Genetic Stock Centre, Berke ley, and SP-ULA (h leul ura4 ade6) (from P. Nurse, Oxford S,,,,University, were used in yeast transformations (Hinnen et al., 1978; Ito et al., 1983). Medium was generally 20 solidified with agar, but minimal medium containing copper o 0o oosulphate was solidified with 2% Phytagar.

DNA sequencing o Nucleotide sequencing of M13 clones was by the dideoxy-chain termination method (Sanger et al., 1977).

-galactosidase assays -galactosidase activity in E. coli was monitored in the lac host strain MC1066 (Casabadan et al (1983).

L-Broth+Ampicillin plates, containing approximately 20 ml of A solidified medium received 50pl 2% X-gal in dimethyl formamide; on such L-Broth+Ampicillin+X-gal plates colonies expressing -galactosidase were clearly blue, while lac colonies were the usual pale yellow colour.

For assays of -galactosidase in yeast, cells were first grown in minimal synthetic medium and were induced with copper sulphate, added to a final concentration of 0.025mM to 10 ImM. Following four hours of induction, cells were assayed for -galactosidase according to Casabadan et al (1983), or according to Guarente (1983) using ONPG and normalizing the OD420 to OD600 of the culture.

Immunological Methods Methods described by Azad et al (1986) were used for immunological detection of antigens of Infectious Bursal Disease Virus.

STRATEGY FOR THE CONSTRUCTION The strategy enabling the construction of the o oo copper-inducible expression cassette is shown in Figure 1 as a 0 0 osimplified schematic representation. The actual details of the construction are as follows. The 0.8 kb XbaI-KpnI °o .'restriction fragment containing most of the CUP1 locus from °oA: Saccharomyces cerevisiae was obtained from Brian Cheetham ,,,,,'(Department of Biochemistry, Monash University). In order to o analyze its sequence and check functionality, it was cloned into the XbaI-KpnI sites of the replicative form DNAs of the °o°,°bacteriophages Ml3mpl8 and Ml3mpl9 (Yanisch-Perron et al, 2'0.1985) and the yeast shuttle vector pFL44, (from F. Lacroute) shown in Figure 2, which contains the Ml3mpl8 multiple cloning 0 0 .°sites. The URA3 gene allows selection of the plasmid in ura3 yeast (and in pyrF E. coli). Replication in yeast is from the 2pm fragment. In E. coli, the origin of replication and 4.1ampicillin resistance gene are from pUCl9. The bacteriophages enabled rapid Ml3-dideoxy DNA sequencing (Sanger et al, 1977) to be carried out on each DNA strand of the fragment. Each carries the same sequence, which we designate the pUC19 multiple cloning sites (MCS), between the EcoRI and HindIII sites, but the orientation of the sequence is reversed in one phage compared to the other. This allows for M13 DNA sequencing to take place on both strands of the cloned XbaI-KpnI fragment. Important for this study was the fact that the MCS contained KpnI and XbaI sites. The DNA sequence analysis between the HindIII and EcoRI sites in these 11 recombinants, designated MI3mpl8/CUP1 and M13mpl9/CUPl respectively, is shown in Figure 3, where the cloned segment runs between the XbaI and KpnI sites. This sequence is more than 99% homologous to CUP1 sequences, reported by Karin et al (1984) and Butt et al (1984b). The only differences shown in Table I occur within the last 100 nucleotides.

TABLE 1 Sequence Comparisons Between CUP1 Genes Nucleotide Karin et al (1984) Butt et al 1984b Present Position(s) Invention o 00 390 C G C 0o00 414 C C G 0o0 442 A G A 445-460 16 A's 16 A's 15 A's 0 0 Numbering is as shown in Figure 3 o o0 The shuttle vector pFL44 is one of many shuttle 0' '.°vectors that could be used to maintain the XbaI-KpnI CUP1 fragment in yeast. It was chosen here simply because it oo 0 contained the MCS. Other plasmids, e.g. pYEL2 derived from pMH158, would also perform suitably, as will be appreciated by those skilled in the art. The pFL44 recombinant plasmid, t*6 which we designated pYEUC19 (yeast episomal URA3, CUPl), was isolated from E. coli and then transformed into yeast.

Comparisons of yeast transformed with pFL44 and yeast S' transformed with pYEUC19 clearly showed that copper resistance was conferred upon the yeast by pYEUC19. Although this recombinant plasmid confers copper resistance on copper-susceptible yeast strains, it lacks a sequence considered to form part of the upstream activation sequence (UAS) or copper inducible regulatory region (Thiele and Hamer, 1986). This missing sequence was replaced by digesting the r i 2 12 recombinant plasmid M13mpl8/CUP1 shown in Figure 1 with SphI and XbaI. The gap left in the plasmid was repaired with the oligonucleotides TCTTTTGCTGGCATTTCTT 3'OH and 3'OH GTACAGAAAACGACCGTAAAGAAGATC OH which annealed together and ligated into gapped plasmid. The presence of 5' hydroxyls on the oligonucleotides ensured that no end-to-end joining could occur, so that only one copy could insert into a gap. This construct, detected by hybridization with the second oligonucleotide, and given the name pUAS36, restored a portion of one of the CUP1 upstream activation o o. sequences, which had been missing, and simultaneously removed some unwanted restriction endonuclease cleavage sites. The DNA sequence of the pUAS36 HindIII-EcoRI fragment was °o ,'analysed, verifying the nucleotide sequence substitution.

S".Furthermore, when the fragment was cloned into pFL44 (to make the plasmid which we have called pYEUC33) and transformed into 0 t yeast, copper resistance was conferred to the yeast in a manner very similar to resistance conferred by the pYEUCl9 o o* recombinant.

o oo obo The next stage in the construction involved engineering a convenient restriction endonuclease cleavage Sosite near to and after the start of the CUP1 coding sequence.

A sequence 5' GAATAA, including the fourth and fifth codons of CUP1 as shown in figure 2 being just one nucleotide different NV0 from an EcoRI site, was chosen as the target for mutagenesis.

The oligonucleotides TGTTCAGCGAATTCATTAACTTCCAAA 3' and the M13 universal primer 17-mer (Amersham), which hybridizes upstream of the first primer oligonucleotide, were annealed to the single-stranded DNA of pUAS36, which contains the modified CUP1 EcoRI-HindIII fragment in Ml3mpl8. Second strand DNA synthesis in vitro primed from the above primers and in the presence of the four dNTPs, DNA polymerase I and T4 DNA ligase essentially as described by Colleaux et al (1986) produced a heteroduplex molecule. This heteroduplex molecule 13 replicated in E. coli to produce homoduplexes of the pUAS36 type and mutant versions of pUAS36 having a new EcoRI site.

These plasmids were purified in plaques and differentiated by hybridization.

Restriction digests and DNA sequence analysis confirmed that in the mutant phage containing the resultant plasmid, which we called pMCES, an A to C substitution in the third base of the fifth codon of the CUP1 gene (marked by the arrow in Figure 2) had created an EcoRI site at the position.

The 0.7 kb HindIII-SacI fragment of pMCE was cloned into the pFL44 HindIII-SacI sites, forming a plasmid which we named pYEUC89. Transformation of this plasmid into yeast showed o that copper resistance was still conferred, even though a leucine was now substituted by a phenylalanine.

The new EcoRI site now conscituted a position for 'the placement of multiple cloning sites within CUP1 coding «e Osequences. Complementary oligonucleotides a 5'OH AATTGGATCCGCAGCTCTCGACTGCAG 3'OH and 3'OH CCTAGGCGTCGACAGCTGACGTCTTAA .o*were synthesized and annealed to make an adaptor with EcoRI ,\o".site-compatible ends 5' AATT overhanging ends). The adaptor was then ligated into plasmid pMCE that had been S""*cleaved with EcoRI. The ligation of the adaptor restores an EcoRI site at one end only and recombinants containing the adaptor in the forward orientation shown above were obtained.

4:Recombinants with the adaptor in the reverse orientation were also detected in the initial hybridization screen with the labelled oligonucleotide shown on the upper strand above.

These were distinguished in HindIII EcoRI digests where a parental 0.27 kb fragment was replaced by a 0.30 kb fragment plasmid pMCPR in Figure Such recombinants had simultaneously lost the downstream 0.5 kh EcoRI fragment.

This was replaced in the plasmid pMCPR following EcoRI digestion and alkaline phosphatase treatment of the cut plasmid pMCPR to prevent religation on itself; thus religation in the presence of the 0.5 kb EcoRI fragment led to efficient 14 insertion of this fragment. Recombinants with inserts in the forward orientation were distinguished by strand specific plaque hybridizations with the oligonucleotide AAGTCATGCTGCAGTGGGAAATGAAA and by digests with HindIII Sacd.

The resulting construction, which we have designated contains the copper inducible expression cassette.

The versatility of the construct is due in part to the placement of unique restriction sites, as can be seen by examination of the sequence, which is shown in Figure 4. For expression of the reading frames of foreign genes, fragments may be cloned into any of the greater than five unique restriction endonuclease cleavage sites in the CUP1 coding region. Additional fill-in or exonuclease treatments can be used to create blunt ends in all possible reading frames, enabling any restriction fragment to be cloned in a desired phase. The ends of the cassette contain unique HindIII, KpnI 4'and SacI restriction sites which are useful in mobilizing the cassette.

The cassette can be cloned into yeast, resulting in copper-inducible expression, or into Schizosaccharomyces pombe a 4* 0 o or E. coli, resulting in constitutive expression, and thus o ,"°enabling convenient selection of organisms bearing the cassette.

Example 1 Construction of the expression vector according to I tthe strategy set out above was monitored using agarose gel electrophoresis of restriction fragments.

EcoRI-HindIII fragments of pYEUC19 recombinant plasmids were stained and probed with the 27-mer from the 30 adaptor. Sequence analysis of one of these fragments, denoted pYEUC33, confirmed the desired olignucleotide replacement of the upstream activation sequence (UAS). The presence of the second UAS conferred an increased level of copper resistance to yeast.

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15 Electrophoretic analysis of EcoRI fragments of pUAS36 mutagenized plasmids showed that a novel 0.5kb EcoRI fragment was present in three of the plasmids. Sequence analysis showed the single introduction of a C for an A residue at the position arrowed in Figure 3.

Analysis of EcoRI-HindIII fragments of recombinant plasmids containing the CUP1 polylinker stained and probed with the 27-mer upper strand of the adaptor showed that four lanes contained hybridising EcoRI-HindIII fragments 0.03kb larger than the 0.27kb fragment seen in the first lane, indicating the insertion of the linker in the forward orientation: the downstream EcoRI fragment had simultaneously a been lost in each case.

Example 2 'o155 As stated above, the ends of the cassette contain S'unique HindIII, KpnI and SacI restriction sites which are useful in mobilizing the cassette. As an example, we excised 0 the cassette as a HindIII SacI fragment of pVYC30 and ligated it into the HindIII SacI sites of pFL44, creating an 0 2p 0 E. coli/yeast expression vector, which we called pYEUC94. The .cassette in pYEUC94 confers copper resistance in yeast, even 0 00 though an additional nine amino acids, from the polylinker, 0 00 o o 0 are encoded by the modified CUP1 gene. The nonapeptide was fused into a pre-sequence which is not observed in the mature metallothionein. We have not determined the fate of the v pre-sequence in pYEUC94 yeast tranformants; however, it is of interest that the transformants were copper resistant. This could mean either that the addition to the pre-sequence does not affect processing at the normal site, or that processing is altered but the altered metallothionein is still functional in conferring copper resistance. The fact that copper resistance is conferred by this modified CUP1 gene is significant; thus disruptions to the gene caused, for example, by the cloning of a foreign restriction fragment into the CUP1 cassette, would probably result in the loss of copper -16resistance in yeast. The cassette therefore can act as a phenotypic marker, capable of discriminating recombinant from non-recombinant.

Example 3 Expression from the cassette was tested by fusing only the protein coding sequences from E. coli p-galactosidase the protein coding sequences lacking an initiation sequence) into the above construct. A general purpose cassette constructed for such gene fusions was provided by Malcolm Casabadan. This cassette, obtained from plasmid MC1871 (Casabadan et al 1983) as a PstI fragment, was inserted S in-frame into the PstI site of the pYEUC94, forming a plasmid which we named pMG4. A lac E. coli strain, MC1061, was transformed with this DNA and transformants were selected on solidified L-Broth+Ampicillin+X-gal plates. The production of dark blue colonies in the experiment indicated that the correct fusions had occurred and that CUP1 sequences were directing efficient expression in E. coli; restriction endonuclease digests further confirmed the desired *2 'construction. This expression result is highly important since it implies bimodal expression, thus ensuring that constructs destined for yeast can be screened prior to yeast .°'transformation.

In yeast strains FLa and FL< the plasmid pMG4 directs A-galactosidase production upon copper induction.

Example 4 cDNA sequences from Infectious Bursal Disease Virus i of chickens (IBDV) have also been successfully expressed from a the CUP1 expression cassette. A 1.35 kb XhoII fragment from a construct known as pEX.PO (Hudson et al, 1986) was isolated.

This fragment contains 0.4 kb of lacZ sequence followed by 1 kb of sequence encoding the host protective antigen of IBDV.

Again insertion in the forward orientation of this XhoII fragment into the BamHI site of pYEUC94 creates an in-frame fusion. Immunological analysis of E. coli transformants 17 containing this plasmid, which we called pMC941, showed constitutive production of the IBDV antigen. All monoclonal antibodies known to react with the antigen have given positive results with the transformants.

Yeast transformed with pMC941 showed production of IBDV antigen only upon copper induction.

Example 5 Effect of plasmid copy number on expression level Plasmid pMG4 was also transformed into cupl s yeast strains FLa and FL Such transformants were copper sensitive, but a low frequency of revertants which were copper resistant and had lost the (-galactosidase insert were i observed (data not shown).

In Table 2 it can be seen that the level of S,/-galactosidase activity was increased 9-10 fold over the basal level (negligible) in these two strains when induced by C CuSO This is a little better than the 6-fold induction ratio reported for HSA (Etcheverry et al., 1986) but not as good as the 50-fold induction ratio reported for galK (Butt et al., 1984b). A number of factors may be contributing to the in expression levels.

i11 I 18 TABLE 2 A-GALACTOSIDASE ACTIVITIES OF E. coli AND S. cerevisiae TRANSFORMED WITH pMG4 CURA3, CUP1-lacZ) OR (LEU2, CUPl-lacZ) -Galactosidase Activitya E.coli b S. cerevisiae C Strain MC1066 FLa FL 6657-4D DBY747 1euB pyrF ura3 CUPl 5 s ura3 CUPl 5 s leu2 CUPi R leu2 ura3 No DNA 2.0 2.0 2.1 3.8 3.8 00 -f 5 pMG4 78.2 2.7 2.1 NT 3.3 0aj 0 no copper 0 0 6pMG4 NT 25.5 21.0 NT 32.8 0 copper 0 a 80.2 NT NT 3.8 3.8 no copper NT NT NT 174.0 184.0 Ratio 39.1 9.4 10.0 -8.4 0 pMG4/pFL44 Ratio 40.1 45.8 55.8 a. Cells were grown to an 0D600 of 0.6. 1 ml aliquots were then assayed for 10 mins for -galactosidase activity J)according to Guarente (1983) using ONPG as substrate. Units are calculated as OD 420 nm x 1000/OD 600 nm x t~min) x V(ml).

Each value is the average of at least 3 determinations.

b. E. coli strain MC1066 required no induction with CuSO4. leuB and pyrF are complemented by yeast (the LEU2 and URA3 genes respectively).

I I I II Y 19 c. Yeast strains were induced for 4 hours with 1000pM and 1000pM CuSO4 respectively for strains FLa, FLA 6657-4D and DBY747.

NT not tested.

One obvious factor which may effect expression levels is plasmid copy number. We have noticed that the PFL44-based vectors are maintained at low copy number, so the CUP1 cassette was transferred into a high copy number vector, pYEL2, engineered from pMH158 (Heuterspreute et al., 1985).

pYEL2 was derived from the T4 polishing and ligation of the large 6.6 kb PstI-XhoI fragment of pMH158. The Hind III-SacI CUP1 cassette was then replaced into the 1.3 kb HindIII-SacI fragment (containing the Tet gene) of pYEL2 making a plasmid which we have called pYELC5, the structure of which is shown 5 in Figure 5. In pYELC5 the BamHI, PvuII, Sall and PstI sites ,within the CUP1 cassette are unique, allowing for greater t cloning versatility than in pYEUC94, which has two PvuII sites. Copy number is noticeably higher, as judged by plasmid stability and the visibility of the plasmid in a total yeast DNA isolation (data not shown).

o R In the leu2, ura3, CUP1 strain, DBY 747, we were So,"able to directly compare the effect of copy number on expression from the CUP1 cassette. It is apparent from Table 2 that in this strain the pYELC5-based construct containing a the 4-galactosidase cassette (denoted pYELC5-G) gives expression levels that are about six times higher than pMG4, the pYEUC94-derived construct. These levels of expression are at least 50-fold over any basal level of CUP1 expression.

Note, however that our measurements of basal levels are not significantly different from background levels of ONPG breakdown.

Strain differences also can have a large effect on expression levels. While the leu2 CUP1 R strain 6657-4B is very similar to DBY747 in terms of CUP1 expression levels, another strain FYHl a leu2 trpl his3 CUP1 produces a level of 630 units. The diploidization of this strain with FYH6, an 0( r~--rscr; 1 1, 20 leu2 trpl his3 ura3 sister spore, results in an expression level of 820 units. In these instances the induction ratios approximate 200-fold or more.

Example 6 Constructs with the multiple cloning sites in the reverse order As a further means of increasing the versatility of the CUP1 cassette we have made another construct having the multiple cloning sites in the reverse orientation. The most significant benefit of this is that the BamHI, SalI and PstI sites now fall within a different reading frame, allowing for S the ligation of sticky-ended fragments in a shifted reading o0 0 O frame. The sequence of this alternative form of the expression cassette according to the invention is shown in oFigure 6.

156. In order to make the alternative cassette, the EcoRI ,site of pFL44 was removed by mung bean nuclease treatment of the EcoRI digested plasmid. This modified plasmid, pFL44-17, was then used as a recipient for the HindIII-SacI fragment pYEUC89 to make a plasmid pYEUC91 that differs from pYEUC89 by 0 6 the presence of a unique EcoRI site in the metallothionein sequence. EcoRI digestion followed by ligation with the o.polylinker led to constructs having the polylinker inserted in both orientations. A construct pYEUC114 has the polylinker in the reverse orientation and its CUP1 cassette was also been 0m moved into pYEL2 making a LEU2 based vector named pYELC7.

Such vectors are also suitable for expression work, as judged by /-galactosidase assays (data not shown).

Other host plasmids may be used, and in some of these the construct with multiple cloning sites in the reverse order has an additional AATTC sequence added at the 5' end, so that it commences in an EcoRI site.

Ir 21 Example 7 Construction of plasmids using the expression cassette according to the invention.

A variety of plasmids was constructed using the expression cassette as described above, and a variety of host plasmids, with the restriction sites at the multiple cloning site in either orientation. These are summarized in Table 3.

TABLE 3 Plasmids bearing the CUP1 cassette Host plasmid a v 00s pFL44 pFL44-17 pYEL2 0 Multiple cloning site orientation BamH EcoRl EcoRl BamHl pYEUC94 pYEUC113 pYELC5 pYELC51* pYELC511** pYELCo pYEUC1l4 pYELC7 0 a I o2Q Sa IpYELC51 is derived from pYELC5 by changing a HindIII a site to a KpnI site.

pYELC51 is derived from pYELC51 by changing a StuI site to an EcoRl site.

pYELC< is derived from pYELC511 by inserting a PstI to SalT site from the yeast o mating factor gene (MFO<1) into the PstlI XhoI sites of pYELC511.

The protein coding sequences of E.coli /-galactosidase were fused into pYEUC94, pYELC5 and pYELC7 to form plasmids designated respectively pMG4, pYELC5-G, and pYELC7-G.

22 Example 8 Transformation of host cells using plasmids according to the invention.

In order to transform yeast (either Saccharomyces cerevisiae or Schizosaccharomyces pombe) with either pYELC7, or pYELC5-G it was found that it was necessary to modify the conventional lithium acetate method (Ito et. al., 1983). The following modifications were made: Cells were incubated in 100 or 200 mm lithium acetate at 300C for 2 hours instead of 1 hour.

200 ml of cells instead of 100 ml cells were added to the DNA (10 ug).

Cells and DNA were incubated at 300C for 1 hour instead of 30 minutes.

Polyethylene glycol treatment was for 2 hours o1S instead of 1 hour.

0 00 o 0 Heat shock at 42°C was for 5 minutes instead of 2 .minutes.

SUsing this modified method for pYELC5, pYELC7, and and the conventional method for other plasmids, the Oo2Q,, -galactosidase activity of Saccharomyces cerevisiae, o oSchizosaccharmomyces pombe and E.coli was estimated as in Example 2 above. Results obtained using plasmids pYELC5-G and °pMG4 are presented in Table 4. Host strains are Saccharomyces cerevisiae unless otherwise stated. Induction was by addition of CuSO 4 for periods and at concentrations indicated.

R

For Saccharomyces cerevisiae, CUP1 R strains were induced for 4 hours with 500-1000 pM CuSO 4 CUP1 s strains were induced for 4 hours with 25 pM or 75 pM CuSO 4 (Fla and Flo ura3 respectively).

'I -23 TABLE 4 (-GALACTOSIDASE ACTIVITY OF HOST CELLS TRANSFORMED WITH pYELC5-G OR pMG4 /3-galactosidase Host strain Plasmid Activity (units) 6657-4D (a leu2 CUP R) DBY747 (a leu2 ura3 CUPi FYH6 leu2 trpl his3 ura3 CUPi 0 FYHi (a leu2 trpl his3

R

CUPi 4 X5mY2 (FYHi x FYH6) (phenotype as in Materials and Methods) FLa~a ura3 cupi FLo((c( ura3 cupis a',E.coli MC1066(leuB pyrF) Sch. pombe 38399 (h leul) Sch. pombe SP-ULA *~(leul ura4 ade6 ura4) pYELC5-G pYEL5-G pMG4 pYELC5-G pMG 4 pYELC5-G pYELC5-G 174 184 152 170 820 pMG 4 pMG 4 pYELC5-G pYELC5-G pYELC5-G 21 210 190 pMG4 4 Activity in E.coli was constitutive. LeuB and pyrF are complemented by LEU2 (pYELC5-G) and URA3 CpMGF) respectively.

Activity in Schizosaccharomyces pombe constitutive; leul and ura4 are complemented by respectively.

was also LEU2 and URA3 i 24 Thus we have constructed a series of expression vectors and cassettes that make use of the entire CUP1 gene of yeast. These cassettes are designed to accept gene fusions such that the elements for the control of expression are still those of CUP1.

The cassettes incorporate 9 codons into the CUP1 pre-sequence such that the reading frame is kept open. The additional sequences introduce a number of unique restriction sites for the cloning of DNA fragments. While the additional sequence does not disrupt the copper resistance encoded by the CUPl cassette, recombinants with inserts into the cassette should generally have lost copper resistance. This can be a oparticularly useful asset for one-step cloning into yeast.

The copper mediated expression of the CUP1 cassette o l''makes it especially suitable for the production of foreign oproteins in yeast. The expression of proteins which would be 4 harmful or inhibitory to yeast can be left until a desired Sgrowth stage has been reached. Expression can then simply be induced by the addition of copper.

o2"' The CUP1 cassette structure makes it suitable for a o o wide variety of yeast plasmids. As we have shown, expression in different plasmids and in different hosts varies widely so o ."portability of the expression cassette is a valued asset.

Four additional aspects of the utility off the system relate to the fusions that can be engineered: 1. The CUP1 cassette can provide a protective new N-terminus to the foreign protein that we are expressing.

2. We can insert yeast signal/secretion signals 5' to the multiple cloning sites. These could be made to secrete recombinant protein into the yeast growth medium, or to direct the recombinant protein through the endoplasmic reticulum and Golgi apparatus for possible glycosylation.

Alternatively a less hydrophobic highly charged N-terminal sequence could be used to keep a recombinant protein in the cytoplasm and away from protein modifications.

3. It is possible to engineer constructs where the foreign gene sequences are fused in-frame at both ends to the CUPI cassette sequences. In such a case it may be possible to produce a protein with metal-binding properties. This may considerably aid the isolation of recombinant proteins. As a further development of this for protein purification protease cleavage sites could be inserted between the foreign protein sequence and the metallothionein sequence. Glutathione S-transferase sequences could also be used in place of metallothionein sequences for protein purification, as would be readily appreciated by those skilled in the art.

It may be advantageous to delete the 3' coding region of CUP1, from the BamHl site to the stop codon, after 0t completing the construction of the cassette, in order to oo 1 shorten the transcript; it is known that shorter mRNA Smolecules are more readily expressed, and are possibly more ,stable.

Since the expression system according to the invention is able to act in Saccharomyces cerevisiae and in .Schizosaccharomyces pombe, it would be expected to be active ,also in other industrially important yeast genera, such as Kluyveromyces.

26

REFERENCES

1. Azad, Fahey, Barrett, Erny, K.M., and Hudson, P.J. (1986) Expression in Escherichia coli of cDNA fragments encoding the gene for the host protective antigen of infectious bursal disease virus, Virology 149 190-198.

2. Butt, Sternberg, Herd, J. and Crooke, S.T.

(1984a) Cloning and expression of a yeast copper metallothionein gene. Gene 27: 23-33.

3. Butt, Sternberg, Gorman, Clarke, Hammer, Rosenberg, and Crooke, S.T. (1984b) S Copper metallothionein of yeast: Structure of the gene and regulation of expression. Proc. Natl. Acad. Sci. USA 81: S 3332-3336.

Casadaban, Martinez-Arias, Shapira, S.K.

and Chou, J. (1983) -galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast Meth.

Enzymol. 100: 293-308.

Colleaux, d'Auriol, Betermier, M., Cottarel, Jacquier, Galibert, F. and Dujon, B. (1986) Universal code equivalent of a yeast mitochondrial intron Sreading frame is expressed into E. coli as a specific double strand endonuclease Cell 44: 521-533.

7. Etcheverry, Forrester, Hitzeman, R. (1986) Regulation of the chelatin promoter during the expression of j t human serum albumin or yeast phosphoglycerate kinase in yeast.

Biotechnology 4: 726-730.

8. Fogel, S. and Welch J.W. (1982) Tandem gene amplification mediates copper resistance in yeat'. Proc.

Natl. Acad. Sci. USA. 79: 5342-5346.

27 9. Guarente, L. (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Meth.

Enzymol. 101 181-191.

Fogel, Welch, Cathala, and Karin, M.

(1983) Gene amplification in yeast: CUP1 copy number regulates copper resistance Current Genetics 7: 347-355.

11. Hamer, Thiele, and Lemontt, J.E. (1985) Function and autoregulation of yeast copperthionein. Science 228: 685-690.

0 -C S. 12. Heuterspreute, Oberto, Ha-Thi, V. and o. Davison, J. (1985) Vectors with restriction site banks III.

'Escherichia coli Saccharomyces cerevisiae shuttle vectors.

Gene 34 363-366.

13. Hinnen, Hicks, J.B. and Fink, G.R. (1978) Transformation of yeast. Proc. Natl. Acad. Sci. USA 1929-1933.

14. Hudson, McKern, Power, B.E. and Azad, A.A. (1986) Genomic structure of the large RNA segment of infectious bursal disease virus. Nucl. Acids Res. 14: 5001-5012.

Ito, Fukuda, Murata, Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations, J. Bact. 153: 163-168.

16. Karin, Najarian, Haslinger, Valenzuela, Welch, J. and Fogel, S. (1984) Primary structure and transcription of an amplified gene locus: the CUP1 locus of yeast. Proc. Natl. Acad. Sci. USA 81: 337-341.

28 17. Kniskern, Hagopian, Montgomery, D.L., Burke, Dunn, Hofmann, Miller, W.J. and Ellis, R.W. (1986) Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae.

Gene 46: 135-141.

18. Maniatis, Fritsch, and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

19. Sanger, Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors Proc. Natl. Acad.

Sci. USA 74: 5463-5468.

0' 20. Sherman. Fink, G.R. and Hicks, J.B. (1983) Methods in Yeast Genetics. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

21. Thiele, D.J. and Hamer, D.H. (1986) Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene. Mol. Cell Biol. 6 1158-1163.

22. Yanisch-Perron, Vieira, J. and Messing, J.

(1985) Improved M13 phage cloning vectors and host strains: .menucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33: 103-119.

It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to hereinabove.

Claims (21)

1. A gene expression system comprising: the complete native DNA sequence encoding the CUP1 gene of Saccharomyces cerevisiae, including all upstream regulatory sequences and transcriptional terminators thereof, a multiple cloning site provided by unique restriction endonuclease cleavage sites downstream from the CUP1 start codon, and unique restriction endonuclease cleavage sites at the extreme 3' and 5' ends of the system whereby the whole S, expression system can be moved as a cassette.

2. A gene expression system according to Claim 1, which o: is expressible in a yeast shuttle vector.

3. A gene expression system according to Claim 1 or Claim 2, in which the unique restricton endonuclease cleavage sites in the multiple cloning site are oriented in the reverse direction.

4. A gene expresson system according to Claim 1 or Claim 2 or Claim 3, which is copper-inducible in cupl s or R CUP1 strains of Saccharomyces cerevisiae. A gene expression system according to Claim 1, Claim 0 Claim 3 or Claim 4, in which the CUP1 sequence copper-binding region is fused in correct reading frame.

6. A gene expression system according to any one Claims 2 to 5, in which the yeast shuttle vector is selected from vectors allowing high, medium or low copy number when expressed in a host, or adapted to integrate into a host genome.

7. A gene expression system according to any one of the preceding claims in which the transcriptional terminators are derived from CUP1 or from a fused 4 DNA sequence.

8. An expression system for a desired protein comprising: 30 the complete native DNA sequence encoding the yeast CUP1 gene of Saccharomyces cerevisiae, including all upstream regulatory sequences and transcriptional terminators thereof, a multiple cloning site provided by unique restriction endonuclease cleavage sites downstream from the CUP1 start codon, unique restriction endonuclease cleavage sites at the extreme 3' and 5' ends of the system whereby the whole expression system can be moved as a cassette, and a DNA sequence encoding the desired protein.

9. An expression system according to Claim 8, which is o °expressible in a yeast shuttle vector. An expression system according to Claim 8 or Claim Oo 9, in which the unique restriction endonuclease cleavage sites o in the multiple cloning site are oriented in the reverse 0° 'direction. g4n. expression system according to Claim 8, Claim 9 or Claim 10, which is copper-inducible.

12. Ae expression system according to Claim 8, Claim 9, Claim 10 or Claim 11, in which the CUP1 sequence copper-binding region is fused in correct reading frame. .13. An expression system according to any one of Claims 8 to 12 which enables expression of the DNA sequence for the desired protein in the absence of the start codon of said DNA ,,sequence. I qec4. An expression system according to any one of Claims 8 to 13 in which the transcriptional terminators are derived from the CUP1 sequence or from the DNA sequence encoding the desired protein. i 15. An expression system according to any one of Claims 8 to 14, additionally comprising signal sequences enabling the desired protein to be secreted into the medium.

16. An expression system according to any one of Claims 8 to 15, which encodes sequences which provide a protective new N-terminus to the desired protein. L U_; 7 31

17. An expression system according to any one of claims 8 to 14, which comprises yeast signal sequences 5' to the multiple unique restriction endonuclease cleavage sites which direct the desired protein, when expressed in a host cell, to sites where post-translational glycosylation occurs.

18. An expression system according to any one of Claims 8 to 14 which encodes sequences which provide a highly-charged N-terminal sequence to the desired protein, whereby the desired protein when expressed in a host cell is retained in the cytoplasm.

19. An expression system according to any one of Claims 8 to 18, wherein DNA sequences encoding the desired protein are fused in-frame at both ends of the cassette. Method of construction of a gene expression system according to Claim 1 or Claim 8, comprising the steps of: cloning at least a portion of the CUP 1 locus of Saccharomyces cerevisiae into restriction endonuclease cleavage sites in a replicative form of a bacteriophage; inserting an oligonucleotide encoding part of the copper-inducible regulatory region of CUP 1; creating a new restriction endonuclease cleavage site within the CUP 1 gene; and cloning the DNA sequence thus produced into a I. I Vi I i I I 1 P1 I host plasmid.

21. Method according to Claim 20 in which the new 'i restriction endonuclease cleavage site is an EcoRI site.

22. Method according to Claim 20 or Claim 21 in which said restriction endonuclease cleavage site is near to and Safter the start of the CUP 1 coding sequence.

23. Method according to Claim 20 or Claim 21, in which in step an A to C substitution is effected in the list nucleotide arrowed in Figure 3.

24. Method of producing a desired protein, comprising the steps of cloning the DNA sequence encoding the desired protein into a host plasmid bearing a gene expression system according to Claim 1 or Claim 8; I 32 transforming the host plasmid into a susceptible microorganism; cultivating the microorganism under conditions suitable therefor; and isolating the desired protein. Method according to Claim 4, in which the host microorganism is selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Escherischia coli.

26. A copper inducible gene expression cassette having DNA sequence as shown in Figure 4 or in Figure 6.

27. A vector comprising an expression cassette according to Claim 26.

28. A plasmid selected from the group consisting of pUAS36, pVYC30, pYEUC33, pYEUC89, pYEUC91, pYEUC94, pYEUC113, pYEUC114, pMCE5, pYELC5, pYELC7, pYELC51, pYELC511, pYELC pYELC7-G, and pMG4 Os here\o oe escr'\\ze

29. Host cells transformed by a plasmid according to Claim 28. o co 00? 4 I Cy 4 4, 4 4 Ja DATED this 6th day of May 1988 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION By its Patent Attorneys CLEMENT HACK CO. Fellows Institute of Patent Attorneys of Australia YEAST CUPI LOCUS COPPER INDUCIEBLE EXPRESSION CASSETTE o Co 00 000 0 0 CO 0 00 0 00 0 000000 O 0 00 0 0 00 O 00 0 0 00 0 CO O 0 0 000 0 -S insertion of second upstream activator sequence replacement of CUPi 3' end Ecog Eco RI digestion /-pIy I In ke r insertion -ff I. 0 00 oo c, 0 0 9 C 0 0 0 0 0 0 0 0 0 0 a0 co 00 0 0. 0 00 0 0. 0 C 0 ,0 00 2 0 0 0 0 1 2 3 4 PVX1i T,,R WET k E TI* ATG ACC AIG ATl AO1G (1 2 3 A 5 6 7 8 9 10 11 12 13 it Is '6 17 PrV we 6ri Nsi 414 CY'1 4(9 W. "N *e 9kU aw wQ vs.f P'D w CCJ. AC3C 110 CAT 0CC TCC AGG TCG ACT CIA GAG GM-k CCC CGG GTA crG ArC ACC I x~I 5 6 ASN SEA AAT TCA 7 t L EU A-LA CTG GC PvuI pol~linker 0 PUCIG. H c i Ct Amp r Ori origin 2/m rn rmen kb Ciaj -7-2- Hind T9f FmaqmenI b/w 51l11 [inkexs hffm.- J AAGCTTGCATGCCTGCAGGTCGACTCTAGAAGCAAAAAGAGCGATGCGTGT TTTCCGCTGAACCGTTCCAGCAAA HindlIll SphI PstI Satl XbaI AAAGACTACCAACGCAATATGGATTGTCAGAATCATATAAAAGAGAAGCAAATAACTCCTTGTCTTGTATCAATT GCATTATAATATCTTCTTGTTAGTGCAATATCATATAGAAGTCATCGAAATAGATATTAAGAAAAACAAACTGTA M FS EL I N F Q NEG HE CQ CAATCAATCAATCAATCATCACATAAAATGTTCAGCGAAT TAATTAACTTCCAAAATGAAGGTCATGAGTGCCAA A C QCG SC K NN EQC Q KS C S CP T GCN S D TGCCAATGTGGTAGCTGCAAAAATAATGAACAATGCCAAAAATCATGTAGCTGCCCAACGGGGTGTAACAGCGAC D KC P CG, N K S E E T K KS C C S G K GACAAATGCCCCTGCGGTAACAAGTCTGAAGAAACCAAGAAGTCATGCTGCTCTGGGAAATGAAACGAATAGTCT TTAATATATTCATCTAACTATTTGCTGTTTTTAATTTTTAAAAGGAGAAGGAAGTTTAATCGACGATTCTACTCA GTTTGAGTACACTTATGTATTTTGTTTAGATACTTTGTTAATTTATAGGTATACGTTAATAATTAAGAAAAGGAA ATAAAGTATCTCCATATGTCGCCCCAAGAATAAAATATTATTACCAAATTCTAGTTTGCCTAACTTAGAACTCTG TATAGAATCCCCAGATTTCGAATAAAAAAAAAAAAAAAGCTATTCATGGTACCGAGCTCGAATTC KpnI SacI EcoRI 0 oo o 00 00 00 00 0 00 0. 000 0 00 00 00 0 0 AAGCTTGCATGTCTTTTGCTGGCATTTCTTCTAGAAGCAAAAAGAGCGATGCGTCTTTTCCGCTGAACCGTTCCA HindIll Xbal GCAAAAAAGACTACCAACGCAATATGGATTGTCAGAATCATATAAAAGAGAAGCAAATAACTCCTTGTCT7 GTAT CAATTGCATTATAATATCTTCTTGTTAGTGCAATATCATATAGAAGTCATCGAAATAGATATTAAGAAAAACAAA M F S E L DP LS TAE F CT GTACAAT CAATCAAT CAAT CAT CACATAAAAT GT TCAGCGAAT TGGAT CCGCAGCT GT CGACT GCAGAATT CA E@mNI PvuU:Sa1I PstI EcoRI I N F Q N E G H E C Q COQ CG S C K N N EQ0 C QK TTAACTTCCAAAATGAAGGTCATGAGTGCCAATGCCAATGTGGTAGCTGCAAAAATAATGAACAATGCCAAAAAT Sc CS C P T G C N S D D K CP COG N K SE E TK K CATGTAGCTGCCCAACGGGGTGTAACAGCGACGACAAATGCCCCTGCGGTAACAAGTCTGAAGAAACCAAGAAGT S C CS GK CATGCTGCTCTGGGAAATGAAACGAATAGTCTTTAATATATTCATCTAACTATTTGCTGTTTTTAATTTTTAAAA GGAGAAGGAAGTTTAATCGACGATTCTACTCAGTTTGAGTACACTTATGTATTTTGTTTAGATACTTTGTTAATT TATAGGTATACGTTAATAATTAAGAAAAGGAAATAAAGTATCTCCATAT GTCGCCCCAAGAATAAAATATTATTA CCAAAT T CTAGTTTGCCTAACTTAGAACTCT GTATAGAAT CCCCAGATT TCGAATAAAAAAAAAAAAAAAGCTAT T CAT GGTA CCGAGCT CGAAT TC KpnI Sadl EcoRI pYELC HpaI1 6.2 kb z jI~C AAGCTTGCATGTCTTTTGCTGGCATTTCTTCTAGAAGCAAAAAGAGCGATGCGTCTTTTCCGCTGAACCGTTCCA HindIll XbaI GCAAAAAAGACTACCAACGCAATATGGATTGTCAGAATCATATAAAAGAGAAGCAAATAACTCCTTGTCTTGTAT CAATTGCATTATAATATCTTCTTGTTAGTGCAATATCATATAGAAGTCATCGAAATAGATATTAAGAAAAACAAA M F SE F CS RQ LR IQ F CTGTACAATCAATCAATCAATCATCACATAAAATGTTCAGCGAATTCTGCAGTCGACAGCTGCGGATCCAATTCA EcoRI PstI SaLI PvuII EBarHI I N FQ0 N E G H E C Q C Q C G SC K NN E QCQ K TTAACTTCCAAAATGAAGGTCATGAGTGCCAATGCCAATGTGGTAGCTGCAAAAATAATGAACAATGCCAAAAAT S CS C PT G C NSD D KC P C G NK S EE T K K CAT GTAG CT GC CCAACG GGG T GTAACAG CGACGACAAAT GC CCCT GCG GTAACAAG TCT GAAGAAACCAAGAAGT S C CS GK* CATGCTGCTCTGGGAAATGAAACGAATAGTCTTTAATATATTCATCTAACTATTTGCTGTTTTTAATTTTTAAAA GGAGAAGGAAGTTTAATCGACGATTCTACTCAGTTTGAGTACACTTATGTATTTTGTTTAGATACTTTGTTAATT TATAGGTATACGTTAATAATTAAGAAAAGGAAATAAAGTATCTCCATATGTCGCCCCAAGAATAAAATATTATTA CCAAATTCTAGTTTGCCTAACTTAGAACTCTGTATAGAATCCCCAGATTTCGAATAAAAAAAAAAAAAAAGCTAT T CAT GGCTAC CGAGCT CO KpnI Sacl