JPH0775551B2 - Stable DNA constructs for expression of alpha-1-antitrypsin - Google Patents

Description

BACKGROUND OF THE INVENTION The use of microorganisms for the production of useful polypeptide products utilizing recombinant DNA technology is well established in the industry. Foreign gene material can be introduced into the culture of the microorganism and the desired product can be synthesized from the foreign gene given the appropriate intracellular and extracellular conditions. Such genetic material is generally introduced into the microorganism in the form of a plasmid, which is an autonomously replicating extrachromosomal element. It was necessary to grow these cells under special conditions to ensure maintenance of the plasmid in the transformed cell culture. Under the absence of such conditions, a plasmid that would be inherently unstable would not be maintained,
The population of cells will return to the untransformed state.

Increased plasmid stability and increased copy number are important to the biotech industry as a means of maintaining high levels of plasmid-encoded protein production at all times. Previously reported attempts to increase plasmid stability do not appear optimal for industrial use. Introduction of yeast centromeres into ARS-containing plasmids with increased stability has been shown to significantly reduce plasmid copy number (clarke and carbon,
Nature, 287 : 504-509,1980, and Stinchcomb et al., Journal of Molecular Biology, 158: 157-179,1.
982). Linear centromere yeast plasmids also show an inverse relationship between stability and copy number (Murry and Szostak, Neitia, 305 : 189-193,198).
3).

Plasmids typically contain a genetic sequence known as a selectable marker, which encodes a host cell's antibiotic resistance or supplemental nutritional requirements. To select for the presence of such a plasmid, the transformed cells are
It must be grown in a special culture medium containing selective agents or depleted of certain nutrients. These culture medium requirements are expensive and prohibit optimal cell growth rates during large scale fermentation processes. Many such plasmids have been reported in the literature. These containing antibiotic resistance genes include pBR322 (Bolivar et al., Gene
e) 2 : 95-113, 1977) and its derivatives such as pUC vector (Vieira and Messing,
Gene, 19 : 259-268,1982) (which is resistant to ampicillin) and pBR325 (Prntki et al., Gene, 14 : 289,1981) (which is resistant to ampicillin, tetracycline and chloramphenicol). It has a gene). Yeast vector YEp13 (Broach et al., Gene, 8 : 121-133,1979) (which carries the LEU2 gene), and YRp7 '(Stinkcom et al., Natia, 282 ) are plasmids that complement host auxotrophy. :
39,1979), which carries the TRP1 gene.

Alpha-1-antitrypsin is a protease inhibitor whose primary function is to inhibit the broad spectrum protease elastase.
Mammalian lung tissue is particularly vulnerable to attack by elastase, thus deficiency or inactivation of α-1-antitrypsin can result in loss of lung tissue elasticity and thus emphysema. α
The loss or reduction of -1-antitrypsin activity is the result of the oxidation of α-1-antitrypsin by environmental pollutants, including tobacco smoke. Deficiency of α-1-antitrypsin can result from one of several genetic defects. Gadek, James E. and RD Crystal, "alpha-1-antitrypsin deficiency",
Metabolic basis of genetic diseases, Stanbury, JB
Et al., McGraw-Hill, New York (1982), pp1450
-1467; and Garroll et al., Neitia, 2988 , 3
29-334 (1982).

Therefore, a DNA structure containing a DNA sequence encoding α-1-antitrypsin and, as a selectable marker, a gene sequence whose product is essential for the growth or normal growth of host cells on complex culture media. It is an object of the invention to provide

Another object of the present invention is to provide a transformant strain of a microorganism containing a plasmid which can be selected by growth on a complex medium and can express α-1-antitrypsin.

Still another object of the present invention is to provide a microbial strain which has a defect in essential function and which can serve as a host for a DNA structure having a gene sequence which can complement these defective essential functions and express α-1-antitrypsin. Is to provide.

Another object of the present invention is a method for producing α-1-antitrypsin in a transformed microorganism, wherein α-1-antitrypsin comprises a DNA structure containing a gene sequence as a selectable marker that complements a defect of an essential gene in a host microorganism. It is to provide a method which is the product of a gene contained on an object.

Other objects of the invention will be apparent to those skilled in the art.

SUMMARY OF THE INVENTION According to the present invention there is provided a DNA construct capable of expressing α-1-antitrypsin and maintained in high copy number without the need for special selective culture media and a suitable host cell. Growth in such conditions results in faster growth, greater cell density and reduced production costs.

The present invention further relates to α-1-in host cells deficient in the functions required for normal cell growth in complex media.
It is an object of the invention to provide a method of making antitrypsin, which comprises the step of transforming a host cell with a DNA molecule containing a gene which complements this defect and a sequence encoding α-1-antitrypsin.

As used herein, the term DNA construct means any DNA molecule modified by humans so that the nucleotide sequence in the molecule is not the same as the naturally-occurring sequence. DNA
The term construct also includes clones of DNA molecules so modified. The term expression vector is defined as a DNA construct containing an autonomous site for replication, a transcription initiation site and at least one structural gene encoding the protein to be expressed in the host organism. Expression vectors will usually also include appropriate control regions, such as promoters and terminators, which control the expression of the protein in the host organism. The expression vector according to the present invention will also include a selectable marker containing the essential genes described herein.

The word plasmid has its commonly used meaning,
That is, it means DNA that is autonomously replicated and is normally closed loop.

In the accompanying drawings: Figure 1 shows the structure of plasmid pB4.

FIG. 2 shows the structure of plasmid pB5.

FIG. 3 shows the structure of plasmid pB15L.

FIG. 4 is co-transformed with plasmids pB5 and pB15L
Figure 7 shows a Southern blot of DNA from S. cerevisiae strain A2.7.C. This blot was probed with a 2.5 kb Bam HI-Hind III fragment from the 5'flanking region of CDC4 to test for disruption of the genomic CDC4 locus. Lane a contains DNA from cells transformed with pB5 alone; lane b contains untransformed cells: lanes ch contain cotransformants. Arrows indicate genomic fragments that hybridize to the probe.

Figure 5 shows S. pombe POT1 and S. cerevisiae TPI.
The sequence of one gene is shown along with each deduced protein sequence. The entire S. pombe TPI protein sequence is given.
The S. cerevisiae protein sequence is shown only where it differs from the S. pombe sequence. The methionine at position 1 in the S. cerevisiae protein sequence is not present in the native protein.

FIG. 6 shows the structure of plasmid pCPOT.

FIG. 7 shows the structure of plasmid pFATPOT.

FIG. 8 shows the structure of plasmid pTPI-LEU2.

DETAILED DESCRIPTION The present invention is based in part on the discovery that essential genes can be used as selectable markers on DNA constructs such as plasmids capable of expressing α-1-antitrypsin. The term essential gene is defined as any gene that encodes a function required for cell growth or normal growth in complex culture media. The complex medium contains nutrients
Products whose composition is not well defined, eg crude cell extract,
It is a culture medium derived from meat extract, fruit juice, serum, protein hydrolyzate and the like. Therefore, in order to select the desired transformants according to the present invention, the selective growth medium is simply a conventional complex growth nutrient, which is a relatively expensive antibiotic, metalloantagonist, or lethal to non-transformed host cells. It is not a particular culture medium that contains other agents or lacks one or more specific nutrients required for a non-transformed host. The essential genes are cell division,
It includes, but is not limited to, genes required for membrane biosynthesis, cell wall biosynthesis, organelle biosynthesis, protein synthesis, carbon source utilization, RNA transcription and DNA replication.

In order to use the essential gene as a selectable marker on a DNA construct such as a plasmid, it is essential to provide a suitable mutant host cell line. Rothstei
n) One-step gene disruption method (Meth.in Enzymology 101 :
202-210, 1983) or the co-transformation procedure described herein can be used to construct a suitable host strain having a deletion of the appropriate essential gene in the genome. Such deletion mutants grow when the mutation is complemented by the function encoded by the genetic material carried on the plasmid. Deletions in the essential gene (s) of the host's genome preferably include major segments of the coding and / or flanking regions. If mutations in essential genes are made in a manner that is achieved by point mutations only, the host cell suddenly reverts to a wild species by a mutation or recombinational repair mechanism, which may result in selectivity that may be achieved by the use of plasmid-carrying genes. Can be reduced or eliminated.

Essential genes are often present in multiple copies (eg histone or ribosomal RNA genes) and / or in multiple related forms called gene families (eg different hexokinase genes, or different DNA polymerase genes). In such cases, these overlapping functions can be mutated one after the other to create multiple defective host cells for a given essential function. However, by using high copy number plasmids to increase the activity of the gene, a single essential gene on the plasmid may compensate for multiple host cell defects. High copy number plasmids are desirable because increased copy number of the cloned foreign gene results in increased production of the protein product encoded by this gene.

Selection of a form-transformant containing a high copy number of a plasmid having an essential gene is carried out by reducing the expression level of each plasmid-carrying essential gene and / or reducing the activity of the gene product encoded by the plasmid-carrying selection marker. Can be achieved. One approach is to mutate an essential gene so that the transcription and / or translation rate of the gene is reduced or the gene product is altered to have a lower specific activity. Another way to reduce the expression level of essential genes used as selectable markers is to use genes from another organism to compensate for the defect in the host cell. Such foreign genes can be naturally defective for expression in host cells. Because the signal for transcription and / or translation will be suboptimal in another species. Alternatively, the gene product may have reduced activity or stability. Because it is in a heterogeneous cellular environment.

There is a wide range of functions required for cell growth or normal growth in complex media. Defects or deletions in the required genes are lethal, slow cell division, stop cell division, stop DNA, RNA or protein synthesis, stop membrane synthesis,
It can result in arrest of cell wall synthesis, arrest of organelle synthesis, defects in sucrose metabolism, etc. Examples of the essential genes include yeast Saccharomyces cerevisiae CDC (cell division cycle) genes (Pringle and Hartwell), "Saccharomyces cerevisiae cell cycle", edited by Strathern and others. Molecular biology, 57
-142, Cold Spring Harbor (Cold Spr
ing Harbor), 1981), S. cerevisiae and Escherichia coli genes encoding the functions of the glycolytic pathway, and S. cerevisiae SEC (Novick and Shekman (Sch.
ekman), Procedures of National Academy of Sciences USA (Proc.Nat.Acad.Sic.USA)
76: 1856-1862, 1979, and Novic et al., Cell 2
1: 205-215, 1980) and INO (Culberts
on) and Henry, Genetics
s) 80: 23-40, 1975) gene.

One preferred class of host cells with essential gene defects is
It contains a defect in the CDC gene known as the cdc mutation, which results in stage-specific arrest of the cell division cycle. Many cdc mutations cause complete blockage of cell cycle essential events by affecting the synthesis or function of specific CDC gene products. Such mutations
It can be identified by the effect on the event that can be observed biochemically or morphologically. Many known cdc mutations are environmentally conditionally lethal (ie, temperature sensitive) mutations that result in the termination of normal growth of mutant cells grown under limiting conditions. However, the main defect resulting from the cdc mutation need not be the defect itself in step-specific function. For example, a continuously synthesized gene product may have a stage-specific function; a defect in the yeast glycolytic gene PYK1 (for the enzyme pyruvate kinase) is a cell division cycle mutation cdc19 (Kawasaki, PhD, Washington University, 1979).
This mutation results in cell cycle arrest in the G1 phase of cells incubated in the typical yeast complex medium YEPD (1% yeast extract, 2% bactopeptone, and 2% dextrose). That is, regardless of whether the cdc mutation results in a deficiency in stage-specific function or it causes a suppressive or inactivating mutation of the gene product with a stage-specific function, the effect of the deficiency can be monitored. .

Pringle and Hartwell (see above) have some 51CD
Describes the function of the C gene. For use in practicing the present invention, such genes can be isolated from the gene library by compensation in strains carrying the desired mutation. Gene libraries can be constructed by known procedures (eg, Nasmyth and Reed,
Proc. Natl. Acad. Sci. USA 77: 2119-2123, 1980: and Nasmith and Tatchell, cels 19: 753-764,198.
0). Strains with the desired cdc mutations are made as described herein or are available from publicly available depository institutions such as the ATCC and Berkeley East Stock Centers.

A second preferred class of essential genes, which encode products involved in the glycolytic pathway, include genes encoding metabolic enzymes and regulatory functions. Examples of glycolytic pathway genes identified in S. cerevisiae are glycolytic regulatory genes
GCR1 and Enzymes Triose Phosphate Isomerase, Hexokinase 1, Hexonase 2, Hoffhoglucose Isomerase, Phosphoglycerate Kinase, Phosphofructokinase, Enolase, Fructose 1,6-bisphosphate Dehydrogenase, and Glyceraldehyde 3-phosphate Dehydrogen It is a gene that encodes Nase. As mentioned above, the pyruvate kinase gene was identified and described by Kawasaki. Contains the yeast phosphoglycerate kinase gene,
Plasmids with regulatory signals are Hitzmann (Hitz
eman) et al. (J. Biol. Chem.) 225: 12073-12080,1 (Journal of Biology and Chemistry).
980). Isolation and sequencing of yeast triose phosphate was performed by Alber and Kawasaki (Journal of Molecular and Applied Genetics) (J.Mol.Appl.Genet.) 1: 419-
434,1982) and Kawasaki and Fraenkel (Biochem.Biophy.
s.Res.Comm.) 108: 1107-1112, 1982).

A particularly preferred glycolytic gene is TPI1, which catalyzes the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone-3-phosphate, and thus is an enzyme essential for glycolysis and glyconeogenesis, the yeast triose phosphate isomerase. Code In S. cerevisiae, the first locus TPI1 encodes this function. Cells with mutations in TPI1 do not grow on glucose and only slightly on other carbon sources.

The S. cerevisiae TPI1 gene was isolated by compensation for the tpi1 mutation (Alber and Kawasaki (supra), and Kawasaki and Frenkel (supra)). The triose phosphate isomerase gene (POT1) from the fission yeast Schizosaccharomyces hombe was isolated by compensation for the same S. cerevisiae mutation and sequenced as shown in FIG. The sequence of the S. pombe gene designated POT1 showed that the S. pombe TPI protein is homologous to the S. cerevisiae TPI protein.

Usually, the essential gene used in the DNA construct (plasmid) will be a wild-type gene from the host species, but in some cases it is preferable to use an essential gene that is foreign to the host cell. Because foreign genes are naturally defective, they are selectable for high plasmid copy numbers. As an example of such an exogenous essential gene used, one of the examples here shows that the S. pombe POT1 gene is effectively used as a selectable marker in S. cerevisiae hosts.

DNA according to the invention containing an essential gene as selectable marker
The construct will be transformed into a mutant host cell that is defective in the function of the essential gene. Appropriately mutated host cells must be made or are readily available from public depository institutions. Mutagenesis of wild-type cells to obtain the appropriate mutants can be accomplished by conventional methods. For example, wild-type cells are treated with a conventional mutagenizing agent such as ethane methyl sulfonate and transformed with a plasmid containing the essential gene to identify colonies for which compensation has occurred. Alternatively, the genome is disrupted to create specific mutations (Rosstin, supra).

The stability of the plasmid containing the essential gene in the host cell is
It may depend on the absence of homologous essential gene sequences in the host cell. Genetic defects in the host ensure that the plasmid is maintained. This is because host cell growth will not occur or will be severely limited by the lack of essential gene function. In addition, the integrity of the plasmid itself will depend on the absence of homology between the plasmid-carrying essential gene and the corresponding locus in the host genome. Because, recombination between each plasmid and the genomic locus will cure both mutation and plasmid cells. That is, it is preferred that the mutation in the host cell genome that inactivates the essential genomic gene is essentially natural, ie the deletion consists of a DNA sequence in the coding region and / or flanking region of a chromosomal gene. . Once this is achieved, cure of recombinational genomic mutations is less likely.

The flanks of the invention are unexpectedly stable when maintained in a suitable mutant host cell. The preferred host cell is yeast; however, other eukaryotic cells as well as prokaryotic cells may be used. In the case of yeast cells, the stability of the flanks according to the invention appears to even exceed that of yeast plasmids containing centromeres. The circular centromere plasmid is
It is among the most stable plasmids ever reported for yeast, but suffers from an extremely low copy number (Krake and Carbon, supra and Stinkcome et al., 1982,
Above). Linear centromere yeast plasmids are unstable or low copy number dependent, depending on plasmid length (Harley and Suzostak, supra). Thus, achieving improved stability of plasmids carrying essential genes is an unexpected advantage.

The POT1 and CDC4 genes are two examples of the use of essential genes as selectable markers on expression vectors. These two genes belong to a broad class of genes required for cell growth on complex culture media. The use of other essential genes allows for plasmid selection in plant or animal tissue culture strains containing complex growth conditions and, in the extreme, in cells that are nourished from blood, serum, or sap from live animals or plants. Allows maintenance of the plasmid at

The data obtained from the experiments with the plasmids described herein show that human α-1-antitrypsin (AT) production is similar to that of the conventional auxotrophic selection marker LEU2 by the use of the S. pombe POT1 gene as a selection marker. It is shown that the AT production is doubled as compared with the AT production obtained by the plasmid. These results indicate that POT1-containing plasmids are functionally larger in copy number than the non-POT1 plasmids from which they were derived.

The techniques used to make the DNA constructs according to the invention, and in particular the plasmids, include the conventional methods. An essential gene used in a DNA construct is separated from the library using a labeled DNA probe if the structure of the gene is known, or a segment of the DNA library is attached to a common vector, This vector is identified by transforming into mutant cells defective in certain essential genes and looking for compensated strains. Once a suitable DNA fragment containing the essential gene has been identified, it encodes the structural protein that will be expressed.

Bound to a vector containing the DNA sequence. The essential gene is
It can be used with its own promoter and other controls required for expression in the host organism. Alternatively, a heterologous promoter can be used to increase or decrease expression of essential genes. The method of ligation of DNA fragments is well described to perform and is well within the skill of the art.

After preparation of the DNA construct, it is transformed into the host organism under transformation conditions. Methods for transforming prokaryotic and eukaryotic cells, including tissue culture cells, are known in the literature.

As mentioned above, the host organism must be deficient in essential function for the selection of essential genes on the plasmid. Mutant host strains are available from conventional depository institutions or can be made by conventional means from wild type by mutagenizing and screening mutants with the appropriate mutations.

Transformed hosts can then be selected by growth on conventional complex media. In the case of yeast, conventional culture media such as YEPD (1% yeast extract, 2% bactopeptin and 2% dextrose) can be used. It will be appreciated that the selectable marker comprising the essential gene according to the invention can be used as a marker whenever it is suitable for the DNA construction, and thus the construct comprising the essential gene selectable marker according to the invention has many uses. . The example below
It is shown for illustration of such applications and not for limitation.

Standard molecular biology techniques were used unless otherwise indicated. Enzymes were obtained from the Bethesda Institute, New England Biolabs, and Boehrnger Mannheim Biochemicals, as directed by the manufacturer or Maniatis et al. (Molecular Cloning: A Laboratory Manual). , Cold Spring Harbor Laboratory, 1982). E. coli cultures were transformed by the calcium chloride method as disclosed in Maniatis et al. (Supra). The yeast culture is Beggs.
Was transformed using the method described above with modifications as described herein.

Example 1 Construction of S. cerevisiae DCD4 Gene A Stable CDC4-Containing Plasmids as Selectable Markers The yeast genomic library consisted of partial digestion of yeast DNA with San3A, size selection by sucrose gradient, and yeast vector predigested with BamHI. Constructed by insertion of selected fragments into YRp7 (Nasmyth)
Reed, Proc.Natl.Acad.Sci.USA.77: 2119-
2123, 1980). A recombinant plasmid containing the CDC4 gene,
Yeast strain GEB5 (MATA cdc4-4 leu2 trpl lys1 ura
l) and GEB7 (MATa cdc4-3 leu2 trpl lysl) were isolated by transformation with the library. These strains were strains known to be transformed with high frequency from strains A364Acdc4-3 and A364Acdc4-4 (Hartwell et al., Genetics 74: 267-286,1975) (K79 [MATαlou2 trp
1) (Nasmith et al., Nichia 289: 244-250,1981: Tatchell et al., Cell 27: 25-35,1981) and subsequent high transformants (K79 and K80 [MATa lou2 trp1 lys1])
On a genetic background (lou2 trp1) desired by backcrossing to
It was induced by obtaining the c4-3 and cdc4-4 mutations. Tryptophan prototrophy and limiting temperature (37 °)
Selection of transformants for their ability to grow in E. coli identified one such plasmid (designated pJY35) that was found to map to the integrated CDC4 locus in the genome.
Spontaneous plasmid integrants were identified based on their selective growth advantage. This growth advantage is detrimental to cell growth when present in high copy number (ie when the plasmid was not integrated into the host genome) due to the presence of the CDC4 linked gene on the original plasmid. is there. In the integrant, the TRP1 plasmid marker was found to be genetically linked to SUP11,
It is bound to CDC4 on chromosome VI (Mortimer (Mo
rtimer) and Shild, “Saccharomyces cerevisiae genetic map”, edited by Strasan et al., Yeast Saccharomyces cerevisiae Life cycle and molecular biology of inheritance, 641-651, Cold Spring Harbor 198.
1). The cdc4-3 compensation region was purified as a 6.4 kb BamHI fragment and was pre-cleaved with BamHI using the T4 DNA ligase, Becotor YRp (Struhl et al., Proh.
c.Natl.Acad.Sci.USA76: 1035-1039,1979). This construct is known as pJY51 and is shown in FIG.

In FIG. 1, the CDC4 coding region was purified from the flanking genomic DNA sequence by the following method. Plasmid pJY
51 was cleaved with Hind III and a 3.6 kb fragment containing the CDC4 region was subcloned in the bacterial plasmid pBR322. This construct was completely digested with Bam HI and
A CDC4 containing fragment of approximately 2.3 kb was purified by partial digestion with nc II.

The end of the Hinc II fragment had a linker sequence (sequence: 5'C
CGGATCCGG3 ', obtained from collaborative research)
Bam HI for the addition of and subsequent removal of excess linker
It was converted to the Bam HI end by digestion with. The resulting fragment containing approximately 1.9 kb of the CDC4 gene was plasmid pJY.
It was inserted into the Bam HI site of YRp7 to make 70. This plasmid appeared to catch the cdc4-3 mutation described above. This 1.9 kb fragment lacks a small portion of both the 5'- and 3'-coding regions of the CDC4 gene, which surprisingly compensates for the temperature-sensitive defect. Probably the transcription and translation of the CDC4 sequence is controlled by sequences located in the pBR322 region of the plasmid, allowing production of a functional gene product.

Plasmid pJY70 is an E for removing the yeast TRP1 and ARS1 sequences.
cBR cleaved and religated to pBR322 and CDC4
A hybrid plasmid containing the sequence was given. This plasmid is known as pJY71 and is shown in FIG.

The 1.9 kb yeast sequence was purified from pJY71 as a Bam HI-Hind III fragment. This fragment is Bam HI
And ligated into linearized pBR322 by digestion with HindIII to create plasmid pB4. This is shown in FIG.

The CDC4 region was reisolated from pB4 for insertion into a high copy number yeast vector. Such a vector would contain the origin of replication of the yeast 2μ plasmid, and one or more restriction enzyme cleavage sites that serve as cloning sites for the foreign gene of interest. Preferably such a site will be a unique site on the plasmid. A preferred vector is MW5, which contains the yeast 2μ plasmid replication origin and unique EcoRI and BamHI cloning sites. In FIG. 2, the plasmid MW5 is the plasmid YRp7 ′ (Stinkcome et al., Nichia 282: 39-43, 19).
79) was derived by cleavage of one of the two EcoRI sites per average molecule by partial cleavage with EcoRI. The resulting unpaired ends of the linear molecule were filled in using DNA polymerase and the resulting blunt ends were religated using T4 DNA ligase. The resulting plasmids carrying an EcoRI site adjacent to the ARS1 sequence were then selected. The ARS1 sequence was removed by digestion with PstI and EcoRI and contained the plasmid YEp13 (broach (Broac
h) et al., Gene 8: 121-133, 1979) PstI-EcoR.
Replaced by I fragment. The resulting plasmid, called MW5, is shown in FIG.

The CDC4 region was reisolated from pB4 for insertion into a high copy number yeast vector. Such a vector would contain the origin of replication of the yeast 2 plasmid and one or more restriction enzyme cleavage sites that serve as cloning sites for the foreign gene of interest. Preferably such a site will be a unique site on the plasmid. A preferred vector is MW5, which contains the yeast 2 rasmid replication origin and unique EcoRI and BamHI cloning sites. In FIG. 2, the plasmid MW5 is the plasmid YR.
7 '(Stinkcome et al., Neacia 282: 39-43, 1979)
From EcoRI, two EcoEs per average molecule due to partial cleavage with EcoRI.
It was induced by cleaving one of the RI sites. The resulting unpaired ends of the linear molecule were filled in using DNA polymerase and the resulting blunt ends were religated using T4 DNA ligase. The resulting plasmids harboring the E | RI site adjacent to the ARS1 sequence were then selected. ARS1 sequence is Pst1
And plasmid YEp13 (broach) containing the origin of replication of the yeast 2μ DNA, which was removed by digestion with E‖RI.
, Gene 8: 121-133, 1979) with the PstRI fragment. The resulting plasmid, called MW5, is shown in FIG.

To construct the final CDC4-containing stable plasmid, MW
5 was cleaved with EcoRI and BamHI. The CDC4 fragment is
The plasmid pB4 was purified by digesting the plasmid with BamHI and EcoRI. The two fragments were ligated using T4 DNA ligase and the chimeric molecule so produced was transformed into the E. coli strain RRI (Nasmis and Reed, supra) and selected for ampicillin resistant, tetracycline sensitive colonies. Plasmid pB5 isolated from one such colony (shown in Figure 2)
Contains the enzyme 2μ replication origin, pBR322 plasmid sequence, selectable marker TRP1, 19 kb of yeast CDC4 coding sequence, and a unique EcoR cloning site.

B. Construction of plasmids for disruption of the host CDC4 gene The stability of the CDC4-containing plasmid according to the invention in transformed hosts depends on the loss of the functional CDC4 gene in the host. It is further desirable that there is no homology between the host genome and the CDC4 containing stable plasmid to eliminate recombination between the plasmid and the stained DNA. To obtain an enzyme strain with the appropriately removed CDC4 locus, a yeast host containing the wild-type CDC4 gene was transformed with a linearized plasmid fragment containing the disrupted "CDC4 gene" (Rosstein, supra). Can be done. Linearized plasmid fragments are the preferred transforming agents because the free ends of the fragments will enhance recombination within the CDC region. Such a plasmid fragment will have a complete CDC4 flanking region at its end to facilitate recombination with the complete genomic CDC4 locus. The genetic material inserted between the CDC4 flanking regions of the plasmid flanking is a tabular feature (selectable marker such as TRP1 or LEU2) that can be selected in the transformed host.
Would code. The disrupting plasmid will also preferably lack a replicating yeast origin to select for integration of the disrupted CDC4 selectable marker sequence into the host genome. Following transformation with the linearized plasmid, genetic recombination results in the replacement of the disrupted sequence instead of the host genomic sequence. Cells in which the CDC4 gene is now removed can then be selected according to the markers used in the disruption.

The method for single-step disruption of the host genome is described by Rosstain (supra). As described above, the disruption of the host strain is achieved by the additional improvement of co-transformation with the complete stable plasmid and the linear plasmid, in addition to achieving the disruption of the host genome, the stable plasmid of the host is also transformed. .

A preferred plasmid for disruption of the host CDC4 locus is pB15
L, shown in FIG. It includes flanking regions of CDC4 and vector pUC13 (Vieira and Messing, Gene 19: 259-268,1982, and Messing, Mesed in Enzymology 101: 20-7.
7,1983) and the yeast LEU2 gene inserted during. When linearized at the junction of the yeast and vector sequences and transformed into a suitable yeast host strain, the plasmid creates a CDC4 deletion in the host genome resulting from the replacement of the LEU2 sequence for the CDC4 region. In the host strain, the leucine auxotrophic disrupted form transformants can then be selected based on leucine prototrophy.

To construct the plasmid pB15L, a 6.4 kb fragment containing the CDC4 gene and its 5'- and 3'-flanking regions was purified from the Bam HI digest of pJY51. This fragment was used in Bam HI to generate plasmid pB14.
It was inserted into the digested pUC13. Much of the CDC4 coding region was removed by digesting pB14 with ClaI and purifying the larger fragment containing pUC13 and the CDC4 flanking sequences. XhoI (Bal II) at the end of the fragment
A 2.8 kb Bgl II LEU2 fragment of YEp13 (Broch et al., Gene 8: 121-133, 1979), modified by the addition of a smart linker (Worthington Diagonostic), was attached to the resulting sticky end. The DNA thus prepared was used to transform E. coli strain RRI.
The sequence complemented the leuB defect in the E. coli host and was selected based on leucine prototrophy. Plasmid pB15L was purified from one such transformed colony.

Plasmid pB15L contains only about 50 base pairs at the 5'end of the CDC4 coding sequence in addition to the 5'and 3'flanking sequences. A comparison of the maps of plasmids pB5 and pB15L shows the lack of homology between their respective CDC4 sequences. Because pB
The junction of the 15L CDC4-LEU2 gene fusion exists in pB5
This is because it is located outside the region of the DCC fragment. This lack of homology excludes recombination between pB5 and the disrupted CDC4 locus in the host cell.

Cotransformation of CS cerevisiae with simultaneous removal of the genomic CDC4 gene and plasmid pB
To introduce 5, Bam HI-cleaved pB15L in yeast cells
Was co-transformed with the complete plasmid pB5. The host strain to be used in transformation is the plasmid pB5
And for the simultaneous selection of genomic CDC4 decay, it must be auxotrophic for tryptophan and leucine. stock
A2 (MATα leu2−2,112 his3−11,15 can1; Suzostak, Method in Enzymology 101: 245−252,1
983) strain GEB7 (see Example 1A) obtained from a cross
2.7.c (MATα cdc4-3 trp1 leu2-2,112 lys1 h
is3-11,15 can1) was used.

In a typical cotransformation experiment, 10 ml of a culture of S. cerevisiae A2,7.c in logarithmic growth phase was treated with about 6 μg of Bam HI digested pB1 5L, 1 μgpB5 and 10 μg calf thymus DNA as a carrier. Transformed. The transformation conditions are as described in Beggs (supra). The cells were plated on medium lacking leucine and tryptophan. They are,
It was grown overnight at 22 ° and transferred to 37 °. About 30 colonies were obtained. Control transformation with pB5 alone and selection for tryptophan prototrophy produced about 1000 transformants.

Six co-transformed colonies were analyzed to verify the disruption of the CDC4 locus and test the stability of the pB5 plasmid. Genomic DNA was isolated from cotransformants by the method of Abraham et al. (Cold Spring Harbor Symposium Quantum Biology 47: 989-998,1893), digested with EcoRI and Bam HI and electrophoresed on an agarose gel. And transferred to nitrocellulose (Southern, J. Mol. Biol. 98: 503-517,
1975). Blot shows C present in pB1 5L but not pB5
2.5 kb Bam HI-Hind from 5'flanking region of DC4
Probed with the III fragment. Figure 4 shows that the probe hybridized to a 6.4 kb fragment of DNA from untransformed cells. (Lane b); this 6.
There is no EcoRI site within the 4 kb Bam HI fragment. Since the LEU2 sequence contains an EcoRI site, disruption of the CDC4 locus results in a reduction in the size of the hybridization zone (indicated by the arrow in Figure 4). This is the case for the transformants shown in lanes c, d, f, g and h. Lane e shows a somewhat different pattern, retaining the genome size band, indicating that no deletion of genomic CDC4 occurred. (The relatively small bands seen in lanes c-h are due to contamination of the gel-purified probe, as shown by the control pattern in lanes a and b.) Six co-transformants were complex media (YEPD. A) Plasmid stability is tested by growing on. Cells
Propagated in liquid YEPD at 25 ° for 30 generations, then YEPD
Plated at 25 ° above and replica plated on 37 ° YEPD, tryptophan depleted and leucine depleted media. The results summarized in Table 1 show that all cotransformants except # 3 were 100% stable for plasmid markers on complex media. (Separation number 3
Is the same co-transformant shown in lane e of FIG. ) Further stability tests were carried out on the two cotransformants, numbers 1 and 2. The test was performed on 663 and 681 colonies, respectively. After 30 generations of growth on YEPD at 30 °, all colonies were prototrophic for tryptophan and leucine.

Cotransformant # 1 was tested for growth rate at 22 ° and found to grow at the same rate as the untransformed A2.7.c control.

Cotransformant # 1 was named BELL1. It is A
It was deposited with the TCC under deposit number 20698.

Example 2 Schizosaccharomyces pombe POT1 gene A. S. pombe POT1 gene as a selectable marker The Saccharomyces cerevisiae TPI1 gene encodes the triose phosphate isomerase protein and tpi1
Obtained by compensating for defects (Kawasaki and Frenkel, supra; Alber and Kawasaki, supra). Surprisingly, the homologous gene from S. pombe has the same S. cerevisiae tp
Isolated by compensating for the i1 mutation. POT1 (pombe
S. pombe named triose phosphate isomerase)
The TPI gene was partially digested with Sau3A and the vector YE
of the genomic S. pombe DNA inserted in p13,
Russell and Hall, (J.Biol.Che
m.258: 143-149, 1983). Preliminary DNA sequence (Maxa (Maxa
m) and Gilbert, Meth, in Enzymology 6
5: 497-559, 1980), showed that the POT1 gene encodes a TPI protein, which is homologous to TPI proteins from other organisms (Alber and Kawasaki, supra). This POT1 DNA sequence is shown in FIG. 5 together with the S. cerevisiae TPI1 DNA sequence and each protein sequence.

The S. pombe POT1 gene is preferred in this example over the S. cerevisiae TPI1 gene as a selectable marker in S. cerevisiae. Foreign genes such as POT1 in S. cerevisiae do not function well in different host cells and may therefore require higher copy numbers to compensate for host cell defects. Also selectable on yeast plasmids
The POT1 gene allows the use of the endogenous TPI1 promoter and TPI1 terminator (a control region showing no homology with POT1) for the expression of industrially important genes on the same beta. Since the flanking regions of POT1 and TPI1 do not show homology, intramolecular recombination and resulting plasmid instability is reduced. Finally, the POT1 gene does not appear to recombine with S. cerevisiae chromosomal DNA. Because it shares little homology with the TPI1 sequence at the DNA level, many of the TPI genes were deleted in the host strain. That is, POT1-containing plasmids can retain the high copy number desirable for enhanced expression of foreign genes of industrial interest in yeast.

The plasmid containing the POT1 gene is S. cerepisiae strain N587-
Compensation for the tpi1 mutation in 2D (Kawasaki and Frenkel, supra) was identified from S. pombe in Russell and Hall (supra).

A restriction map of this plasmid pPOT is shown in FIG. Since pPOT contains the vector YEp13, it is inherently unstable. This is because it lacks the replication function necessary for maintenance of the 2μ plasmid in yeast. Therefore,
The POT1 gene can be mobilized into more competent vectors such as C1 / 1 and related vectors containing the complete 2μ plasmid sequence. Plasmid C1 / 1 was derived from pJDB248 (Beggs, Nichia 275: 104-109,1978) and pBR322 as described in Example 3 herein. It's yeast 2
It contains all of the μ plasmid DNA, the selectable LEU2 gene and the pBR322 sequence.

The POT1 gene was isolated from pPOT as an approximately 3400 base pair BamHI-XbaI restriction fragment and inserted into the corresponding polysonker site of pUC13. The resulting plasmid is pUCP
It is an OT and its partial restriction map is shown in Fig. 6.

The pUCPOT plasmid contains approximately 1 of S. pombe and S. cerevisiae DNA.
Cleaved with Sa1 I to remove 800 base pairs and religated. The obtained pUCPOT-Sa1 plasmid was
It is shown in FIG.

The POT1 gene was inserted into C1 / 1 by the following method. C1 / 1
Since both pUCPOT-Sa1 and pUCPOT-Sa1 have a Bg1 I site in the ampicillin resistance gene and a unique Bam HI at some other position, the PUC1 fragment of pUCPOT-Sa1 is a C1 / 1 p
It can replace part of the BR322 region. C1 / 1 is Bgl I and Ba
Cleavage with mHI released a large fragment of approximately 7700 base pairs containing part of the amp ^r gene, all 2 μDNA, and the LEU2 gene. Similarly, pUCPOT-Sa1 is Ba1 I and Ba1
Cleavage with m HI released a fragment of approximately 3400 base pairs containing another part of the amp ^r gene and the POT1 gene. These two fragments were ligated to form pCPOT, which is the restored selectable amp ^r gene, POT1 gene, LE
It contains the U2 gene, all 2 μDNA, and the bacterial origin of the replication region from pUC13 (the bacterial origin region from pUC13 allows higher plasmid copy numbers than does the origin region of pBR322).

Escherichia coli strain HB101 transformed with pCPOT has accession number 3968
5 was deposited with the ATCC.

The POT1 gene can also be inserted into the C1 / 1 inducible vector with a similar construction. For example, plasmid pFAT5 (7th
The figure) contains an expression unit for the production of human α-1-antitrypsin (AT) inserted in C1 / 1. This expression unit was constructed as described in Example 4 and consists of the TPI1 promoter, ATcDNA sequence, and TPI1 transcription terminator. A restriction map of pFAT5 is shown in Figure 7.

pFAT5 is cleaved by Ba1 I and Bam HI, resulting in AT gene and TP
The fragment containing the I1 terminator (2200 base pairs) was released. Further, as described above, a Ba1 I-Bam HI fragment identical to the C1 / 1Ba1 I-Bam HI fragment is released. However, the fragment from pFAT5 contains an additional 900 base pairs containing the TPI1 promoter. This latter p
The FAT5 piece and the pUCPOT-Sa1 3400hp Bg1 I-Bam HI fragment (described above) were ligated to form the plasmid pFPOT, which has a restriction map as shown in FIG.

The vector pFAT5 is cleaved at the unique Bam HI site and
Allows insertion of the TPI1 terminator fragment from the base pair AT gene and pFAT5. Cloning of the appropriately oriented 2200hb fragment into pFPOT allows expression of human AT in this yeast vector. The appropriately ligated product was named pFATPOT and its restriction map
Give to the figure.

B. Disruption of the host TPI gene The Saccharomyces cerevisiae TPI1 gene was cloned and sequenced (Kawasaki and Frenkel, supra,
And Albert Kawasaki, supra). The plasmid pTPIC10, which contains the structural gene for the TPI protein, is described in Albert and Kawasaki (supra). Bg1 II site is TPI1
Located at DNA position 295 in the coding region, another BglII site is located approximately 1200 base pairs apart in the 5'flanking region. These Bg1 II sites are conventional cloning sites for removing part of the TPI1 gene and for inserting another gene such as the yeast LEU2 gene. Such a construct can be used to create a disruption of the genomic TPI1 locus in a transformed host.

About -1800 in the 5'flanking region of TPI1 is Pst1
It is a part. Therefore, in pTPIC10, the TPI1 gene is
The Pst I site on the 5'side and the Sal I site (te
(in the t ^r gene).

This PstI-SalI fragment containing TPI1 was inserted into pUC13 at the PstI and SalI sites to create pUCTPI. PST
A restriction map of the I-Sal I insert (to pUC13) is shown in FIG.

Next, pUCTPI was cleaved with BglII into the plasmid, and two DN
The A fragment was separated by electrophoresis. The larger plasmid was purified and phosphatase digested to prevent self-ligation. The yeast L is in the Bal II site of this DNA.
The EU2 gene was ligated, which contained the plasmid YEp13 (Brooch et al., Gene 8: 121-13) as a Bal II fragment.
3,1979). The resulting plasmid is pUCTPI-LEU2, which has a partial deletion of TPI1 and an insertion of LEU2. pUCTPI-LEU2 is shown in FIG.

Plasmid pUCTPI-LEU2 contains Pst to linearize DNA.
Cut with 1 and Bam HI. The yeast sequence was then separated from the pUC13 sequence by electrophoresis and gel purification. The yeast DNA portion shown in Figure 8 was used to transform S. cerevisiae strain E2-7B (ATCC No. 20689), which is defective for LEU2, in order to "disrupt" the TPI1 chromosomal gene (Rossten, supra). Used. leu ⁺ transformants were 3% glycerol, 1% lactate (neutralized to pH 7), 1 M sorbitol and leucine-free synthetic (modified Wickelham) medium (Mortimer and Hawthorne, edited by Rose and Harrison, The Yeast, The Yeast). vol.1, 385-460, Academic Press, 1969). Transformants were screened for TPI defects due to their inability to grow on YEP-dextrose. Beginning of 99 screened in one of the transformants tpi ^- nature transformed pair is found. This strain was named E2-7BΔtpi # 29 (hereinafter referred to as Δtpi # 29). Δtpi # 29 grew on YEP-3% glycerol-1% lactate but not on YEP-dextrose. An enzyme assay (clifton et al., Genetics 88: 1-11, 1980) was performed on crude cell extracts and confirmed that Δtpi # 29 lacks detectable levels of triosephosphate isomerase activity. It was

Δtpi # 29 can be crossed to other yeast strains to form diploids that are heterozygous for the tpi ⁻ deletion. Such a diploid, so that other strains defective for trilose phosphate isomerase can be generated,
Can be sporulated. For example, △ tpi # 29 is E8-10A
(Matα leu2) (spore segregant of hybrid E2-7BXGK100 [ATCC20669]) to form the diploid E11. This diploid was sporulated to give rise to the haploid progeny E11-3C. It has the following genotypes: Matαpep4-3tpi
1. E11-3C was backcrossed to Δtpi # 29 to form diploid E18. It is homozygous for the tpi1 deletion. E1
8. Preferred over Δtpi # 29 as a host strain for plasmids as it has no amino acid requirements, has larger cells and grows faster. These tpi ⁻ strains are deficient in the genetic material encoding the glycolytic function and are therefore considered to be irreversible (ie stable) mutants.

Transformation of POT1 gene into CS cerevisiae tpi ^- deleted strains Plasmids pFPOT and pFATPOT were transformed into Δtpi # 29 and related tpi deleted strains. This yeast mutant was grown aerobically at 30 ° in YEP-2% galactose until late log phase overnight. The transformation conditions were as described by Beggs (supra), except that cells were plated with YEP-3% glycerol-1% lactate containing 1M sorbitol instead of YEP-dextrose before being plated on upper agar. Or 1 in YEP-2% galactose
I was allowed to recover at 30 degrees for ~ 2 hours. The top agar and plates contained synthetic, modified Whitcolumn's medium containing 1M sorbitol and 2% dextrose. After 3 days at 30 ° the transformants were visible and detached from the agar due to repute to YEPD. The transformants were then maintained on YEPD or other complex media containing dextrose.

Strain E18 transformed with pFATPOT was named ZYM-3. It has been deposited with the ATCC under deposit number 20699.

Stability of pFPOT and pFATPOT on complex media To study plasmid stability, colonies from single cells were inoculated into tubes containing YEPD and allowed to grow to a total of 10 ⁹ cells (approximately 30 divisions). did. YEP- which allows yeast cells to be sonicated to break up the clumps, diluted appropriately and allowed to grow tpi ^- cells (with or without the plasmid carrying the POT1 gene).
Plated on 2% galactose or YEP-2% glycerol-1% lactate. Colonies generated on YEP-galactose were then replica-plated on YEPD to screen for plasmid deficiency (ie tpi ⁻ cells that had lost the POT1 containing plasmid would not grow on dextrose). The results summarized in Table 2 show that the pFPOT and pFATPOT plasmids are stable in the yeast tpi ^- deficient strain. They are surprisingly much more stable than yeast plasmids containing centromeres. Centromere-bearing plasmids (which have low copy numbers) are among the most stable plasmids reported for yeast, with a general probability of loss of about 1% cells per division in complex media. (See Murray and Suzostak, supra, for a review of centromeric plasmid stability). As shown in Table 2, the POT1 plasmids described here were found to be 1% after 30 rounds of division in complex media in the tpi ^- deleted strain.
Lost with less than probability.

Human α in S. cerevisiae using the D. POT1 plasmid
Expression of -1-antitrypsin To test the use of the POT1 plasmid to enhance expression of heterologous proteins in transformed yeast, plasmids pFATPOT and pFAT5 were added to S. cerevisiae strain Δtpi, respectively.
Used to transform # 29 and E2-7B. Transformed cells were selected on leucine-free medium with dextrose. The culture was grown at 30 ° to an OD600 of 3-4. Cell extracts were prepared and evaluated for AT as described in Example 5.

When made with pFATPOT / Δtpi # 29 it showed 4-6% total soluble protein.

It showed 2-3% total soluble protein when made with pFAT5 / E2-7B.

Although plasmid copy number is difficult to measure accurately and represent the average individual number, empirical observations of gene abundance give an indication of relative plasmid levels, and expression units (promoter, gene of interest, terminator) Indicates that it does not change. Thus pFATPOT appears to be mechanically higher in number than the pFAT5 from which it was derived. Since the two transformants were genetically nearly identical (Δtpi # 29 was derived from E2-7B by plasmid-directed mutagenesis) and were grown under the same conditions, these results are The values for the stable plasmid expression system described herein are shown over the previously described vectors.

Example 3 Preparation of plasmid C1 / 1 C1 / 1 was obtained from plasmid pJDB248 (Beggs, J., Nichia 27).
5, 104-109, 1978). pMB9 sequence is EcoRI
It was removed from pJDB248 by partial digestion with and replaced with pBR322 DNA cut with EcoRI. Figure 6 shows the C1 / 1 restriction map. The C1 / 1 plasmid contains all 2 from the yeast (S. cerevisiae), with pBR322 insertion at the EcoRI site.
Contains μDNA. It also contains the LEU2 gene.

Example 4 Preparation of plasmid pFAT5 The gene encoding the major form of human α-1-antitrypsin (AT) was isolated from a human liver cDNA library by conventional methods using baboon sequences as a DNA hybridization probe ( Kurachi et al., Proc.Natl.Aca
d. Sci. USA 78: 6626-6830, 1980; and Chandra et al., Bio.
chem.Biophys.Res.Comm.103: 751-758, 1981). This library uses human liver cDNA to generate Pst of plasmid pBR322.
It was constructed by inserting it at the I site (Bolivar et al.
Gene 2: 95-113, 1977). The AT gene has 1500 bases (b
p) isolated from the library as a Pst I fragment. This fragment was inserted into the pstI site of pUC13 to make plasmid pUCα1. In pUCα1, the AT sequence was flanked at the 3'end by Xba I and Eco RI sites in the polylinker.

The TPI terminator was purified from plasmid pFG1 (Alber and Kawasaki, supra) as an approximately 700 bp XbaI-EcoRI fragment and inserted into pUCα1 cleaved with XbaI and EcoRI. This structure was then cut with EcoRI,
Oligonucleotide linker (sequence: AATTCATGGAG GT
ACCTCCTAG) is added in the multi-linked copy, T
A Bam HI site is provided at the 3'end of the PI terminator. The resulting plasmid is known as BAT5.

The TPI promoter fragment was obtained from the plasmid pTPIC10 (Alber and Kawasaki, supra). This plasmid is cleaved at the unique Kpn I site and contains the TPI coding region.
It was removed with Bal31 exonuclease and an EcoRI linker (sequence: GGAATTCC) was added to the 3'end of the promoter. Digestion with Bal II and Eco RI gave a TPI promoter fragment with Bal II and Eco RI sticky ends.
This fragment was then digested with Bal II and Eco RI plasmid YRp7 '(Stinkcom et al., Nichia 282: 39-
43, 1979). The resulting plasmid TE32 was transformed with EcoRI to remove a part of the tetracycline resistance gene.
And cleaved with Bam HI. The linearized plasmid is then
It was re-linearized by the EcoRI-Bam HI linker addition described above to make plasmid TEA32. Next TEA32 is Bal I
Cleaved with I and Bam HI, the TPI promoter was purified as a fragment of approximately 900 bp.

In order to construct plasmid pFAT5, plasmid C1 / 1 was cloned into B
It was linearized with am HI and ligated to the 900 bp TPI promoter fragment from TEA32. The resulting construct, known as plasmid F, has a unique BamHI site located at the 3'end of the TPI promoter. This plasmid was digested with Bam HI and contains the AT coding region and TPI terminator 2200b.
The pBam HI fragment was purified from BAT5 and inserted into the Bam HI site. The resulting plasmid, known as pFAT5, is shown in FIG.

Example 5 Evaluation of α-1-antitrypsin As a control, 10 μm of a 100 μg / ml trypsin solution (1 μm)
g), 100 μg of bovine serum albumin (100 μ) and 1 m
M benzoyl arginylyl-p-nitroanilide pH 8.0 buffer solution 0.05 M TRIS (100 μ) was mixed, and 405 nm
The increase in the absorbance was measured with a spectrophotometer over time. The absorbance value of this solution was used as a standard for 100% trypsin activity. All evaluated samples contain the same concentration of substrate and bovine serum albumin.

Example 6 Aspergillus nidulans TPI functional TPI cDNA as a selectable marker was isolated from TPI in S. cerevisiae strain Δtpi29 (McKnight et al., Cell 46: 143-147, 1986).
To complement the deletion, the ability of A. nidulans (A. nidulans)
dulans). 1.15 kb TPI cDNA was added to EcoRI-B
It was inserted into pUC91 as an amHI fragment. This cDNA
Was excised from the plasmid obtained after partial digestion with Sst I, digested to completion with BamHI, and constructed with Sst I-Bglll digested plC19R (Marsh) to construct plasmid pM144.
Et al., Gene 32: 481-486, 1984). The BamHI site was transformed into pM144 by linearizing the plasmid with EcoRV, adding the BamHI linker sequence and religating.
Introduced. The resulting structure was named pM147. TPI
The cDNA was then transformed into pM14 as a Sa11-BamHI fragment.
Separated from 7.

This Sa1l-BamHI A. nidulans TPI was then inserted into C1 / 1 linearized by digestion with the same enzymes. C1 / 1 for which the vector was obtained was named pCTIP.

The plasmid pTIP was then subjected to a stability test in yeast transformation. S. cerevisiae strain GA18-1c (MAT α1cu2-3,
112 ura3 barl Δtpi IILEU2) was transformed with pTIP,
Cultured in glucose medium. This plasmid was shown to complement the TPI deletion in the host and was shown to be present at a high copy number (200 copies per cell) in comparison to the ribosomal DNA band on ethidium bromide stained gels.

These results also indicate that the promoter sequence on the vector is accidentally directed to the expression of the TPI sequence. Further analysis revealed that E. coli l
The acz promoter showed that it was responsive.

Example 7 Cloning of PGI Gene A.PGI as Selectable Marker Yeast DNA library in shuttle vector YEp13 was cloned into Nasmyth and Tatchell (Cell 1).
9: 753-764, 1980). As a plasmid carrying the phosphoglucose isomerase (PGI) gene, co-selection for culturing on glucose and leucine prototrophy (Kawasaki and Fraenlel, Biochem. Bioph.
ys.Res.Comm.108: 1107-1112,1982) using, pgi ^-
It was identified by complementation of a yeast strain and was named pPGI19-7. pPGI
A 19-7 restriction endonuclease map identified a 5.7 kb BamHI-HindIII fragment containing the PGI1 gene.
The PGI1-containing inserts from pPGI19-7, then pUC13
Subcloned into. BamHI and partially Hind III
By supplementing with, by cutting the plasmid and then by isolation on an agarose gel, a 5.3 kb fragment was obtained. The purified fragment was ligated into pUC13 cut with BamHI and HindIII. The identification of this clone is D
Confirmed by NA sequence. The resulting clone is pUCP
I named it GI.

B. Construction of PGI Deletion Strains A plasmid was constructed for use in disrupting the genomic PGI1 locus in yeast host strains. This is pUCPGI's PGI1
The gene contained LEU2 insertion and was named pGD1040.

Plasmid pGD1040 was then digested with BamHI and HindIII and the fragment containing the LEU2 sequence was used to transform S. cerevisiae strain 2-7B. Transformant is 1c
Selected on fructose containing u-synthetic medium. LEU ⁺ transformants were then screened for viability on YEPD plates. The mutant strain was identified in Maitr (Maitr) to confirm that it is defective in PGI.
a) and Lobo (J. Biol. Chem. 246: 475-488, 1
971) and Kawasaki and Fraenkel (ibid), enzyme evaluations were performed on crude cell extracts. The deletion mutant strain showed no enzymatic activity. trpl ^- pgi carry phenotype
The deletion strain was further constructed. Add this to # 51a (MATα 1cu
2 trpI-289 ura3-53 his3-Δ1 Δpgi1 II LEU2).

C. Isolation of PGI cDNA Plasmid pYcDE8 (McKnight et al. EMBO J. 4: 2093-20
99, 1985) and pooled yeast cDNA to McNight and McConorgy (Proc.Natl.Acad.Sci.USA80: 4412-4).
416, 1983) and used for transformation of strain # 51a. The transformants were selected on a synthetic glucose medium containing no tryptophan to obtain one transformant. Plasmid DNA was prepared from this transformant and Ec
Used to transform oli RR1. From the restriction map of this plasmid, called pPHGI cDNA, the 2.0 kb Sst I-Bam III
It was found that the PGI cDNA contained on the fragment corresponded to a part of the 5.4 kb genomic fragment and largely overlapped with the deleted part of pGD1040 that was already used in gene division. This cDNA was subcloned in the M13 phage vector and sequenced by the conventional method. This sequence was used for yeast phosphoryl glucose isomerase (Kempe et al. J. Bio
I. Chem. 249: 4625-4633, 1974).

D. Use of PG1 plasmid as an expression vector Plasmid pPGI cDNA was digested with Sst I and Bam HI to give PGI c
The 2.0 kb fragment containing the DNA was gel purified. This cD
NA was ligated to pZUC13 predigested with Sst I and Bam HI (pZUC13 is the S. cerevisiae chromosome LEU2 gene and pUC1
It contains the origin of replication from the S. cerevisiae 2 μm plasmid inserted in 3. Using plasmid pMT212 described in EP Publication 163,529, pZUC1 described in EP Publication 195,691
Constructed in a similar way to 3. ). The resulting plasmid, designated pA1, is therefore PGI fused to the lacZ promoter.
Contains cDNA, LEU2 sequence, 2 micron origin of replication and ampicillin resistance marker. When pA1 was transformed into strain # 51a, this transformant grew on leu-glucose plates, indicating that the lacZ promoter was functional in yeast cells.

E. Expression of α-antitrypsin using PGI selection A plasmid pA1-B was constructed by inserting an expression unit of the PTI promoter-α1-antitrypsin-encoding sequence-TP1 terminator into pA1. This expression unit was constructed by the following method. Plasmid TEA32
Example 4 was digested with BglII and EcoRI and the 990 bp partial TPI promoter fragment was gel purified.
Plasmid plC19H was cut with BglII and EcoRI and the vector fragment was gel purified. The TPI promoter fragment was then ligated to the linearized plC19H and the mixture was used to transform E. Coli RP1. Plasmid DNA
Was prepared and screened for the presence of the 900 bp BglII-EcoRI fragment. Select the correct plasmid and plCTPI
I named it P.

The plasmid pMVR1 was then constructed. Plasmid pIC7 (M
arsh et al. ibid.) was digested with EcoPI, the ends of the fragment were blunted with DNA polymerase 1 (Klenow fragment) and the linear DNA was recircularized with T4 DNA ligase. E. coli RR1 was transformed with the obtained plasmid. Plasmid DNA was prepared from this transformant and screened for deletion of the Eco R1 site. The plasmid with the correct restriction pattern was named pIC7R1 *.
Plasmid pIC7R1 * was digested with Hind III and Nar I to give 250
Purified with 0 bp fragment. A partial TP1 promoter fragment (approximately 900 bp) is plCTPIP using Nar1 and Sph1
And gel purified.

1750bp containing pFATPOT digested with Sph1 and HindIII and containing partial TPI promoter, ATcDNA and TPI terminator
The fragment was gel purified. This pIC7R1 * fragment, the partial TPI promoter, and the partial TPI promoter-AT-TPI terminator fragment from pFATPOT were ligated at three junctions to produce pMVR1.

Next, an expression vector of α-1-antitrypsin was constructed. The plasmid pMVR1 was digested with Bal II and the 2.3 kb expression unit fragment (TPII promoter-ATcDNA-TPII terminator) was gel purified and inserted into the BamHI site of pA1. The resulting plasmid was named pA1-B.

pA1-B was transformed into S. cerevisiae strain # 51a and transformants were grown in TEPD liquid medium to an OD600 of 1 liter. This transformant expressed α-1-antitrypsin at a total soluble protein of 2.5-4.0%. This level is POT1
Comparable to the level obtained using selection. The copy number of pA1-B was about 100 / cell.

[Brief description of drawings]

FIG. 1 shows the structure of plasmid pB4. FIG. 2 shows the structure of plasmid pB5. FIG. 3 shows the structure of plasmid pB15L. FIG. 4 shows a Southern plot of DNA from S. cerevisiae strain A2.7.c cotransformed with plasmids pB5 and pB1 5L. Figure 5 shows S. pombe POT1 and S. cerevisiae TPI.
The sequence of one gene is shown along with each deduced protein sequence. FIG. 6 shows the structure of plasmid pCPOT. FIG. 7 shows the structure of plasmid pFATPOT. FIG. 8 shows the structure of plasmid pTPI-LEU2.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Glen Kawasaki Washington, USA 98112 Seattle East Sixteen States Avenue 1547 (72) Inventor Leslie Bell, Washington 98102 East Seattle Boyer Avenue 2518

Claims (2)

[Claims] 1. α-1 in yeast host cells having a defect in the function required for normal cell growth in complex media.
-A method for producing antitrypsin, comprising the steps of: (a) transforming the yeast host cell with a DNA molecule containing a gene that complements the defect and a sequence encoding α-1-antitrypsin; (b) step (a) A method comprising the step of culturing cells from from under the conditions in which the gene functions as a selectable marker for transformant cells. 2. The cell from step (a) is cultivated in a growth medium which does not require the inclusion of antibiotics or heavy metals and does not require the depletion of certain nutrients. The method according to claim 1.