# Patent

## Title
Nucleic acids encoding lymphoid CD30 antigen

## Abstract

The invention relates to a novel process for using the yeast ADH II promoter system for the production of heterologous proteins in high yields, for example for preparing hirudins, miniproinsulins, leptins or derivatives thereof.

## Background
The polypeptide hirudin, which was originally isolated from the leech Hirudo medicinalis, is a highly specific thrombin inhibitor possessing a broad therapeutic potential (F. Markwardt, Biomed. Biochim. Acta 44 (1985) 1007-1013). However, it is only possible to prepare the quantities which are required by the recombinant route using transformed microorganisms. In this context, it has been found that the yeast Saccharomyces cerevisiae is suitable as a host organism for producing hirudin which is correctly folded and fully active (EP A1 168 342, EP A1 200 655).

The gene for the alcohol dehydrogenase isoenzyme II (ADH II) is strictly regulated in yeast cells. The product of the ADH II gene is not found when fermentable carbon sources, such as glucose, are added to the fermentation medium. ADH II catalyzes the dehydration of ethanol (C.sub.2 H.sub.5 OH) to acetaldehyde (CH.sub.3 COH). However, conditions for inducing the ADH II promoter can be achieved in simple aerobic batch processes, in shaking flasks or in fermenters, which are started, for example, using a glucose concentration of 4%. When growth takes place on glucose, the so-called "Crabtree" effect results in ethanol being formed initially with the aid of the enzyme ADH I, with the ethanol in turn being used as an additional carbon source once the glucose has been completely consumed. After the glucose has been broken down, the ethanol-degrading enzyme ADH II is induced and the expression of the ADH II gene product begins. If the expression of a gene encoding a heterologous protein is under control of the ADH II promoter, expression of the heterologous protein will begin once the glucose has been metabolized.

As a rule, such batch fermentations do not provide the high yield which is sought for industrial applications. In general, industrial applications require a high yield in a short period of time. What is wanted is the separation of the fermentation into a growth phase and a production phase, as is achieved in classical antibiotic fermentations, for example, by means of a limited fed-batch procedure (also known in the art as feed-batch), in which, after a certain cell density has been established, a carbon source is then fed in a growth-limiting manner in order to form the product (e.g. the secondary metabolite penicillin) at high yield under optimal physiological conditions See Hersbach et al.: The Penicillins: Properties, Biosynthesis and Fermentation, in: Biotechnology of Industrial Antibiotics, pp. 45-140, Ed. E. J. Vandamme, Marcel Dekker, New York, 1984.

Because of the way it is regulated, the ADH II promoter system has been used for the preparation of recombinant proteins in the bakers yeast Saccharomyces cerevisiae. See Price et al.: Methods of Enzymology, Vol. 185, 308-318, 1990, which is hereby incorporated by reference. The fermentation system used by Price depends on the growth of the yeast cells and on the natural elimination of glucose as a carbon source. In the absence of glucose, product formation is switched on and continues until the end of the fermentation. However, the yield of product appears to be less than was hoped.

T.o slashed.ttrup et al. (Biotechnol. Bioeng., 35, 339-348, 1990) describe a modified fermentation procedure for the intracellular production of a human insulin fusion protein, which procedure involves feeding with glucose followed by feeding with ethanol in order to ensure optimal induction of the system. This group used a hybrid promoter, which consisted of the ADH II promoter ligated to the glyceraldehyde-3-phosphate dehydrogenase promoter. These workers succeeded in almost doubling the volume-related product yield (grams protein/liter), as compared with a batch process, by means of continually feeding glucose at a constant rate over the entire fermentation period. A further doubling was finally achieved by means of replacing the glucose feed, from a particular time point onward, with an ethanol feed. It was concluded from this result that even when there is no glucose, or very low glucose concentrations of less than 20 mg/l, in the culture broth, the hybrid promoter is still partially repressed by glucose or a metabolite of glycolysis. Complete induction was only achieved by adding ethanol.

Thus, it would be useful to have a high-yield process for producing heterologous protein in yeast using the ADH II promoter system. As used herein an "ADH II promoter system" means that the expression of a heterologous gene is under the control of the ADH II promoter.

The present application is directed to a biotechnology invention.

It is an object of the invention to provide a process for producing a heterologous protein by fermentation of transformed yeast, said process comprising: wherein said preliminary medium comprises yeast which contain an expression vector comprising a gene encoding said heterologous protein and an ADH II promoter, wherein the expression of said gene is under the control of the yeast ADH II promoter; grams of glucose/liter of culture volume/hour to said main medium, wherein said glucose is added continuously at a constant rate; and

wherein said fermentation is carried out for a time sufficient to produce the desired level of heterologous protein, and wherein said yeast are cultured under aerobic conditions.

It is a further embodiment of the invention to provide such a process where the fermentation is carried out for approximately two days. In other embodiments of the invention, there are provided processes for production of heterologous protein in yeast wherein the glucose concentration is from about 0.7 to about 1.4 grams/liter of culture volume/hour. In a further embodiment, the glucose concentration is from 0.9 to 1.3 grams/liter of culture volume/hour. In still further embodiments, the glucose concentration is from about 0.9 to about 1.3 grams/liter of culture volume/hour, or 1.1 grams/liter of culture volume/hour or about 1.1 grams/liter of culture volume/hour.

In another embodiment of the invention, a process is provided where the compositions of the preliminary culture medium and the main culture medium are equivalent with the exception that the preliminary medium contains 1% glucose. In a further embodiment, the glucose in this preliminary culture medium has been almost completely consumed at the time of the inoculation.

In another embodiment of the invention, the aerobic conditions in a process of the invention are maintained by maintaining an oxygen partial pressure of at least 20% saturation.

In yet another embodiment of the invention, the main medium employed in a process according to the invention contains one or more complex constituent selected from the group consisting of cornsteep, soybean meal and yeast extract.

Various types of yeast may be used in a process according to the invention. These yeasts include, but are not limited to Saccharomyces cerevisiae (including Saccharomyces cerevisiae strain Y79), and Kluyveromyces lactis.

The skilled artisan will recognize that any heterologous protein can be produced using a process according to the invention. These heterologous proteins include, but are not limited to, a hirudin, a miniproinsulin, a leptin, a hirudin derivative, a miniproinsulin derivative, and a leptin derivative. These proteins further include, but are not limited to, a fusion protein comprising a protein selected from the group consisting of a hirudin, a miniproinsulin, a leptin, a hirudin derivative, a miniproinsulin derivative, and a leptin derivative.

FIG. 1 shows the nucleotide sequence for the plasmidpADHIIHirkLTY (SEQ ID NO:1), which contains the hirudin gene fragment from plasmid p.alpha.fHir inserted into the p.alpha.ADHII plasmid.

The invention relates to a novel process for using the yeast ADH II promoter system for the production of heterologous proteins in high yields.

As used herein, a "hirudin" means a peptide-like thrombin inhibitor which is derived from the known isohirudins of the species Hirudo medicinalis and which exhibits essential structural features of these isohirudins, in particular the characteristic linking of the three disulfide bridges. J. Dodt et al., Biol. Chem. Hoppe-Seyler 366 (1985) 379-385. Cf. EP A1 158 564, EPA1 168 342, DE 34 45 517, EP A2 193 175, EP A1 200 655, EP A1 158 986, EP A1 209 061, DE 33 42 199, EP A1 171 024).

As used herein, "essential structural features" of an isohirudin is used to mean features that are necessary for the biological function of isohirudin and which are common to all isohirudins. In general, these features are conserved stretches of amino acids with (1) similar or essentially identical three-dimensional structure and (2) similar or essentially identical function. As used herein, "derivatives of isohirudins" is used to mean any protein, derived from hirudin, which exhibits the biological activity of hirudin. These derivatives may be made using genetic engineering techniques that are well known to the skilled artisan, such as the introduction of mutations, deletions, insertions into a nucleotide sequence for an isohirudin so as to produce a modified protein with the biological properties of hirudin. These derivatives may be made using other genetic engineering techniques that are well known to the skilled artisan, such as the production of fusion proteins, chemical or enzymatic generation of hirudin fragments, or chemical modification of hirudin, so as to produce a modified protein with the biological properties of hirudin.

In particular, among these, a hirudin as described in EP A1 171 024, EP A1 158 986 and EP A1 209 061, all of which are incorporated by reference, is an acceptable hirudin in accordance with the invention. Such hirudins can have a range of specific activities. Advantageously, the specific activity of a hirudin produced according to the invention can be at least about 10,000 ATU/mg and advantageously at least 10,000 ATU/mg (anti-thrombin units/mg).

If the expression system described in the article by Price et al. is used for preparing the hirudin gene product described in European Patent Specification 0 324 712 B1, the specific, biomass-based product yield (grams heterologous protein/grams recombinant yeast) decreases as a result of adding ethanol, continuously or discontinuously, since an increased cell growth was not accompanied by increased product titer. See Example 1 below.

The direct addition of ethanol was also investigated on a laboratory scale. First, a culture was started with glucose as the energy source. As the glucose became depleted, ethanol was produced ("Crabtree effect"). At this point in time, additional ethanol was added to the medium. While the additional supplementation of a batch culture with ethanol during growth on ethanol increased the biomass, it did not lead to any increase in product concentration as compared with the culture without supplementation.

Similarly, laboratory cultures which were grown using ethanol as the sole carbon source did not, within the limits of measurement accuracy, result in specific yields (based on the optical density or on the biomass) which were higher than those of a comparison culture using glucose. See Example 2. From this it appears that ethanol on its own is not an inducer of ADH II.

In addition, an attempt to generate derepressed growth, and to avoid ethanol formation due to the Crabtree effect, did not increase product yield. In this regard, regulated, model-assisted glucose feeding ("following up," i.e. increasing, the feeding rate in accordance with the increasing cell density), was followed by inducing expression more powerfully by means of a pulse addition of glucose with a subsequent high level of ethanol formation as a consequence of the Crabtree effect. This method did not increase product yield.

Surprisingly, a fermentation method which is simple and self-regulating and which can be carried out on an industrial scale has now been found for using the yeast ADH II promoter system for the production of heterologous proteins in high yields. This process comprises continuously metering ("adding") glucose, as the carbon source, into a fermentation culture (main culture) in which no glucose was initially present. During the course of the culture, various phases are passed through in such a manner that optimal induction and product formation are achieved. In the light of the state of the art, for example Price et al., supra, such an induction of heterologous gene expression in yeast using the ADH II promoter system is quite remarkable since Price et al. indicate that the ADH II promoter is only derepressed after there has been a marked depletion of glucose.

Using a process according to the invention, optimal product yield coupled with high productivity is achieved when a small quantity of glucose is fed in continuously at a constant rate directly after the main culture has been inoculated with a preliminary culture containing transformed yeast. As used herein, "main medium" is used to mean the culture medium used for large-scale culturing of bacteria. Typically, the main medium culture volume will be above about 1000 liters, although the skilled artisan will recognize that other suitable volumes may be employed. "Preliminary medium" is used to mean the cuture medium used for the preliminary, or initial, culturing of the yeast. Typically, the main medium used in the invention will have the same composition, with the exception that the preliminary culture medium contains 1% glucose, but glucose is added continuously to the main medium throughout the fermentation. Typically, about 1.1 grams of glucose per liter of culture volume is added continuously to the main medium. The increase in volume of the culture due to the feeding-in can lead to a lowering, by up to 25%, of the actual glucose feeding-in rate by the time the culture has ended. At first, glucose is present in excess for the low cell population. This excess glucose represses the ADH II promoter and ethanol is formed as a consequence of the Crabtree effect. From a particular time onward, the cell density is so high that glucose no longer suffices as sole carbon source and mixed growth on glucose and ethanol takes place. After the ethanol which was primarily formed has been consumed and further growth is limited by the depletion or total absence of glucose, the ADH II promoter is switched on and product formation begins. In this way, the product yield can be approximately tripled as compared with the batch process.

Thus, in one embodiment the invention relates to a process for the production ("biotechnological fermentation") of heterologous proteins in yeast in which a main culture is inoculated with a preliminary culture of a yeast strain which can express the heterologous protein; immediately after the inoculation a small quantity of glucose, e.g. 0.7-1.4, preferably 0.9-1.3, and very particularly preferably 1.1, g of glucose/l of culture volume/hour is supplied continuously at a constant rate, the fermentation is terminated after 2 days and, finally, the heterologous protein is isolated from the fermentation culture. Aerobic culture conditions are ensured during the whole of the fermentation by not allowing the oxygen partial pressure to fall below 20% saturation. Other suitable glucose concentrations according to the invention include about 0.7 to about 1.4 grams of glucose/liter of culture volume/hour, about 0.9 to 1.3 grams of glucose/liter of culture volume/hour and 1.1 grams of glucose/liter of culture volume/hour.

The skilled artisan will recognize that there are many culture media suitable for yeast fermentation. Such media include, but are not limited to, media described in Ausubel, et al., eds. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY pp. 13.1.1-13.1.7 (John Wiley & Sons, 1995). For the processes according to the invention, the level of carbon source in the culture medium must be controlled. Typical carbon sources are glucose and ethanol. As indicated above, various medium glucose concentrations are suitable for carrying out the process of the invention. Thus in one embodiment, the medium will comprise one or more complex constituents, but will not contain glucose or other carbon sources, such as sucrose. Appropriate carbon sources will be added when appropriate. In addition, the culture medium used for fermentation will generally be free of ethanol when fermentation is started.

The skilled artisan will recognize that the process of the invention can be carried out until the desired level of heterologous protein has been produced. The process of the invention may be carried out for approximately two days. The skilled artisan will recognize that the process may be carried out for more or less time, depending on the specific experimental conditions employed to produce heterologous protein. In one embodiment, the process of the invention is carried out until the level of heterologous protein production is constant (i.e. the amount of heterologous protein does not further increase with longer times).

In another embodiment, the invention relates to the process described above, in which the media of the preliminary culture and the main culture are equivalent apart from an addition of 1% glucose to the preliminary culture medium, which addition has preferably been almost completely consumed at the time of inoculation of the main culture, and contain one or more complex constituents, for example cornsteep, soybean meal or yeast extract. As used herein, "almost completely consumed" denotes a glucose concentration of less than 100 mg/liter or less than about 100 mg/liter. As used herein, "complex constituents" is used to mean constituents such as cornsteep, soybean meal or yeast extract. Other such constituents are well known to those of skill in the art. The skilled artisan will recognize that such constituents contain a variety of components, some of which are not precisely defined.

The yeast strains used in said processes can be Saccharomyces cerevisiae, preferably Y79 (see EP 0 324 712) or Kluyveromyces lactis strains. The skilled artisan will recognize that other yeast strains, such as other strains of Saccharomyces or Kluyveromuces, may be used to carry out a process according to the invention.

The heterologous proteins which can be prepared in accordance with the process can be, for example, a hirudin, a miniproinsulin (as described, for example, in EP 0 347 781), a leptin (e.g. obese gene products as described in Zhang et al., Nature 372:425-32, 1994) or a derivative of the previously mentioned proteins or fusion proteins which contain one of the previously mentioned proteins and/or derivatives thereof. In this context, the term derivatives denotes, inter alia, functional, i.e. biologically active, fragments of the starting proteins or mutants possessing novel, in particular advantageous, biological properties. For example a derivative would possess the same biological activity as the native protein, but desirable properties such as prolonged half life (for example in blood), and altered pharmacological action profile. Such a profile may be advantageously delayed, prolonged or shortened, depending on the protein and treatment regimen used. The skilled artisan will recognize that a biologically active peptide fragment may possess one or more of the functional activities of the complete peptide.

The skilled artisan will also recognize that any heterologous protein can be expressed using a process according the invention by using the appropriate nucleotide sequence in a vector according the invention.

Techniques for producing fusion proteins are well known in the art. See, for example, EP 0489 780, hereby incorporated by reference.

The following examples are provided to illustrate the invention, but do not limit the scope of the claimed invention.

Preparation of the Expression Vector for the Recombinant Production of Hirudin in Yeast

This example describes the expression system which has been used for developing the process of the claimed invention.

The vector p.alpha. ADH 2 (see FIG. 2A in Price et al., supra, which is hereby incorporated by reference) contains, adjacent to the Spe I restriction enzyme cleavage site in the direction of the 3' end of the cDNA, a recognition site for the enzyme Nco I which is unique for the plasmid. After the DNA of the vector p.alpha. ADH 2 was reacted with Kpn I and Nco I, two DNA fragments were then obtained which are separated from each other by gel electrophoresis. The larger of the two fragments was isolated and reacted, in a T4 ligase reaction, with a Kpn I/Nco I hirudin fragment which was isolated from the plasmid P.alpha.f Hir 17 (See EP 0 324 712 B1, which is hereby incorporated by reference) by partial digestion with the enzyme Kpn I and complete digestion with the enzyme Nco I. The complete nucleotide sequence of the resulting plasmid pADH2HirkLTY (SEQ ID NO:1), which contains the hirudin gene fragment from p.alpha.f Hir inserted into p.alpha.ADHII is shown in FIG. 1.

Commercially obtainable competent E.coli K12 cells of the strain HB101 were transformed with the ligation mixture and the transformation culture was plated out on Na agar plates which contain 25 mg/l ampicillin. The plates were incubated at 37.degree. C. overnight and, on the following morning, individual colonies were picked from the plates and used to start overnight cultures. Plasmid DNA was in each case isolated from the cells of the cultures and examined by means of restriction analysis. Correct plasmid DNA, which contains the hirudin gene incorporated in the desired manner, was used to transform the Saccharomyces cerevisiae strain Y79 as described in EP 0 324 712 B1, which is hereby incorporated by reference.

Comparison of Laboratory Cultures for the Recombinant Preparation of Hirudin in Yeast Using Different Carbon Sources

Different carbon sources were investigated in a laboratory fermenter under otherwise identical conditions. The composition of the medium for the preliminary and main stages is as follows:

The main culture was inoculated with an inoculum equal in volume to 2% of the main medium and incubated for 48 hours. The cell concentration in the inoculum was from about 3.times.10.sup.7 to about 1.times.10.sup.8 cells/ml.

The following results were obtained when ethanol and glucose were used as carbon sources: Hirudin concentration Optical density A.sub.578 nm Carbon source %!           %! ______________________________________ 2% glucose 100           100 1.54% ethanol 83            71 ______________________________________

The values given in this table are relative. Identical culture volumes were used for each carbon source tested. The hirudin concentration and optical density of the 2% glucose medium at 48 hours were set at 100%. Thus, the table shows that using ethanol resulted in a lower hirudin concentration (83% of the 2% glucose level) and a lower optical density (71% of the 2% glucose level).

Comparison of the Recombinant Preparation of Hirudin in Yeast by Fermenting With or Without Glucose Feeding

a) Fermentation With Glucose Feeding (Fed-Batch Process)

1600 liters of culture medium, which comprises one or more complex constituents, for example cornsteep, soybean meal, yeast extract, etc., without glucose or other carbon sources, such as sucrose, being added, were sterilized at 121.degree. C. for 20 minutes in a fermenter having a total volume of 3600 l.

After it was cooled down, the medium was inoculated with approx. 200 l of a preliminary culture which had been grown for approximately 12 hours (optical density A.sub.540nm =3.5.+-.0.5) and which contained only very low residual quantities of glucose. In other words, the glucose was almost completely consumed and was hence present at less than 100 mg/l. The medium of the preliminary culture was equivalent to that of the main culture except that 1% glucose had been added to the preliminary medium before the yeast was grown in this medium for approximately 12 hours.

Immediately after the main stage was inoculated, 2 kg of glucose were metered in (i.e., "added") per hour, in the form of a 20% aqueous solution of glucose at a uniform rate until the end of the fermentation. The glucose was added via a device for ensuring continuous sterilization. The skilled artisan will recognize that such devices are well known in the art. Examples of such devices include a flow through heat exchanger and a continuous flow through sterilizer.

Aerobic conditions must be ensured during the fermentation; an oxygen partial pressure of pO.sub.2 greater than or equal to 20% has to be maintained. Accordingly, an oxygen partial pressure of greater than or equal to 20% was maintained during the fermentation in this example.

After 48.+-.2 hours, the fermentation was complete and was terminated by adding 0.225.+-.0.025% benzalkonium chloride, e.g. 0.45.+-.0.05% .RTM.Dodigen 226 (a 50% solution of a mixture of alkyldimethylbenzylammonium chlorides in water) to the culture medium. Completion of fermentation was determined by measuring the concentration of hirudin in the medium, using HPLC. After approximately 48 hours, the level of hirudin remained relatively constant.

The final volume (medium+condensate+preliminary culture+metered-in glucose solution-water loss due to gassing) was 2300.+-.50 l.

Following this, the medium can be worked up for isolation of hirudin from the culture broth, using procedures that are well known to the skilled artisan. See, for example, U.S. Pat. No. 5,180,668 hereby incorporated by reference.

b) Batch Process

The batch process is equivalent to the fed-batch process described in Example 3a) with the following exceptions: complex constituents.

c) Comparison of the Results Hirudin concentration Hirudin quantity Process     %!           %! ______________________________________ Batch process 100           100 Fed-batch process 228           291 ______________________________________

The numbers in this table are relative levels. The final hirudin concentration and quantity using the batch process were set at 100%. The fed-batch process yielded relatively higher hirudin concentration and quantity.

Priority application 19544233.4 (Federal Republic of Germany), filed Nov. 28, 1995, including the specification, drawings, claims and abstract, is hereby incorporated by reference. SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 1 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8491 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAATTCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTT60 CCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCC120 AGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTT180 CTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGA240 CCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCA300 CGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGC360 CATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCG420 AGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGC480 GTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATC540 GCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTA600 TGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCT660 TGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGA720 AGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTG780 CGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATC840 TCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATG900 ATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAG960 AATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACC1020 TGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAG1080 TCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGT1140 GGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGG1200 TCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGT1260 TCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCC1320 ATGAACAGAAATTCCCCCTTACACGGAGGCATCAAGTGACCAAACAGGAAAAAACCGCCC1380 TTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGC1440 TGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTT1500 ACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCC1560 CGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCG1620 CGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCG1680 GAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAT1740 GCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGC1800 TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA1860 CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG1920 AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA1980 TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA2040 CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC2100 TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC2160 GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT2220 GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG2280 TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG2340 GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA2400 CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG2460 AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT2520 TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT2580 TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG2640 ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT2700 CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC2760 TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGAT2820 AACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCC2880 ACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAG2940 AAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG3000 AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGT3060 GGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG3120 AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT3180 TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC3240 TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC3300 ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAA3360 TACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG3420 AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC3480 CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG3540 GCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTT3600 CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT3660 TGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC3720 ACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC3780 GAGGCCCTTTCGTCTTCAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAG3840 GGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCT3900 TGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGC3960 TTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATT4020 ATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGT4080 CTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGT4140 AATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAA4200 CTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAA4260 AGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCAT4320 TCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAAC4380 TCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCT4440 GGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTT4500 GATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTC4560 GTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGC4620 ACTTGCCTGCAGCTTCTCAATGATATTCGAATACGCTTTGAGGAGATACAGCCTAATATC4680 CGACAAACTGTTTTACAGATTTACGATCGTACTTGTTACCCATCATTGAATTTTGAACAT4740 CCGAACCTGGGAGTTTTCCCTGAAACAGATAGTATATTTGAACCTGTATAATAATATATA4800 GTCTAGCGCTTTACGGAAGACAATGTATGTATTTCGGTTCCTGGAGAAACTATTGCATCT4860 ATTGCATAGGTAATCTTGCACGTCGCATCCCCGGTTCATTTTCTGCGTTTCCATCTTGCA4920 CTTCAATAGCATATCTTTGTTAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGC4980 AACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAA5040 TGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAA5100 AATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAAC5160 AGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCT5220 ACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTC5280 TCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAG5340 GTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACT5400 TCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCC5460 CCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGT5520 TGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATAC5580 TACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTT5640 ACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCG5700 AGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCA5760 CAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATA5820 TTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGC5880 GCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGG5940 AATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCG6000 CACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATA6060 CATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTT6120 ATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTAT6180 CGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGA6240 TTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCATAGTACCGA6300 GAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCTG6360 GCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACATTAGTCAACTCCGTTAGG6420 CCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATGTGAGATTTTGGGCCATTT6480 TTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAGGGGGGGGGGGGGGGGGGGGGG6540 TTACTTAACCAATCTACATAGATACTATATTTAACATTCAACATAATAATAAATATTTTG6600 GGATAAATAAGTGAAACCATTTTAGAGCCCCTAGGGCTTACAAAAAGAATCATAAAAGAT6660 CCATATTTATAGTTTTAAGATTAAGAATAATAGTTACAATAGGTAGCAAACCATACATTC6720 AACAATAAATATAAAATTTAAATATTTAAATAAATAGAAGGGGTACGAGGGGGCCATGGA6780 CGAGCTCTATTACTGAAGGTATTCCTCAGGGATCTCTTCGAAGTCGCCGTCGTTATGAGA6840 CTGCGGTTTCGGGGTACCTTCGCCAGTAACGCACTGGTTCTTTTCACCGTCGGATCCAAG6900 GATGCATTTGTTACCCTGGCCGCAAACGTTAGATCCTTCGCACAGGCACAGGTTCTGACC6960 AGATTCAGTGCAGTCAGTATACGTAAGTCTTTTATCCAAAGGTACCCCTTCTTCTTTAGC7020 AGCAATGCTGGCAATAGTAGTATTTATAAACAATAACCCGTTATTTGTGCTGTTGGAAAA7080 TGGCAAAACAGCAACATCGAAATCCCCTTCTAAATCTGAGTAACCGATGACAGCTTCAGC7140 CGGAATTTGTGCCGTTTCATCTTCTGTTGTAGTGTTGACTGGAGCAGCTAATGCGGAGGA7200 TGCTGCGAATAAAACTGCAGTAAAAATTGAAGGAAATCTCATCGTGAGGTCGAGCTTGGT7260 GTATTACGATATAGTTAATAGTTGATAGTTGATTGTATGCTTTTTGTAGCTTGATATTCT7320 ATTTACCAAGAAGAAACAAGAAGTGATAAAAACAACAAGAGAGCAGTAGTAAGAGTATTT7380 CAGTGTGAAAAAAGTCGCTACTGGCACTCTATTTATATGTGATAGGCATGCTATAGCTTT7440 ACCAAAAAGTGAACCCCATTTCTATGCTCTCCTCTGCCTTTTTTTTTTTTTTTTTTTTCA7500 TTCTCTCAATCTGAAATTCTCTTATTTCTCCAACTTATAAGTTGGAGATGCCCGGTGTTC7560 CGGCAGAGGAGATCAGTCTCGTGAAGTGGATGGTTTCCCGCCTGCGGGCAAAACGTCATA7620 ACATTTTTATGAGCGAAAGCCGTTAATGAAGACAAAATCCCTTAATTAAAACATTAGAAT7680 GGTGATTAGAAAGGCAGGATTAATCAGTTACACAGGCTGTAACCGGAGAGACGGATCATA7740 AGGCAATTTTTAGATAAGACTGGTTAGAGTTCTTGGCATCAGAAAATTTGAGAAACGATT7800 TTTCCGTTTGTTTGCCCCTACGTTTTGCCCCTTTGATCAAACTATCAGTTAAGATATTAA7860 TTTTTTTGAGAAAACGATTCTTTGATTAGTCTCTTCAAACAAACAATGAGCTCTGAAGAC7920 GAATTGGGAAGTATCGGTACTGTGTTTCCCGGAAGTCCCATAGATAAGAGCATTGGGAGT7980 ATTCTCCACAATTTGATGAAGAAGTGGAGACTTTGCTGGAAGATAGCTTCACGTGGAACA8040 TTCCTGACTGGAACGAGTTAACAAACCCGAAATACAATTCGCCCAGGTTTAGAATTGGTG8100 ATTTCGAATGGGACATTCTATTATTCCCTCAGGGAAACCATAATAAAGGTGTTGCGGTAT8160 ATCTGGAACCTCATCCGGAAGAAAAATTAGATGAGACTACGGGAGAGATGGTGCCAGTTG8220 ATCCGGACTGGTATTGTTGTGCTCAGTTTGCCATTGGTATATCTAGACCTGGTAATGGTG8280 ACACCATCAATTTAATTAACAAATCGCATCACCGATTCAACGCTCTAGATACAGACTGGG8340 GATTTGCAAATTTGATAGATTTGAACAACTTGAAACATCCCTCAAAAGGAAGACCGCTTT8400 CGTTCTTAAACGAAGGGACCTTGAACATAACAGCGTATGTGCGCATATTGAAGGATCTCT8460 ACTTGTCAAATACCTTTAAATCTTATCAATA8491 __________________________________________________________________________

## Claims

1. A process for producing a heterologous protein by fermentation of transformed yeast, said process comprising: wherein said preliminary medium comprises yeast which contain an expression vector comprising a gene encoding said heterologous protein and an ADH II promoter, wherein the expression of said gene is only under the control of the yeast ADH II promoter; grams of glucose/liter of culture volume/hour to said main medium, wherein said glucose is added continuously at a constant rate; wherein ethanol is formed by said yeast as a result of the Crabtree effect; and wherein the production of said protein is initiated after (i) the ethanol that was formed in said main medium has been metabolized by said yeast, and (ii) after growth of said yeast is limited by depletion of glucose by said yeast; and wherein said depletion of glucose occurs when the cell density of said yeast is sufficiently high that said continuous addition of glucose is not sufficient to sustain growth of said yeast.

2. The process according to claim 1, wherein said fermentation is carried out for approximately two days.

3. The process according to claim 1, wherein glucose is added at a rate of from 0.9 to 1.3 grams/liter of culture volume/hour.

4. The process according to claim 1, wherein glucose is added at a rate of from about 0.9 to about 1.3 grams/liter of culture volume/hour.

5. The process according to claim 1, wherein glucose is added at a rate of 1.1 grams/liter of culture volume/hour.

6. The process according to claim 1, wherein glucose is added at a rate of about 1.1 grams/liter of culture volume/hour.

7. The process according to claim 1, wherein the compositions of the preliminary culture medium and the main culture medium are equivalent with the exception that the preliminary medium contains 1% glucose.

8. The process according to claim 1, wherein aerobic culture conditions are maintained by maintaining an oxygen partial pressure of at least 20% saturation.

9. The process according to claim 1, wherein said main medium contains one or more complex constituents selected from the group consisting of cornsteep, soybean meal and yeast extract.

10. The process according to claim 1, wherein said yeast is Saccharomyces cerevisiae.

11. The process according to claim 1, wherein said yeast is Kluyveromyces lactis strain.

12. The process according to claim 1, wherein said heterologous protein is selected from the group consisting of a hirudin, a miniproinsulin, a leptin, a hirudin derivative, a miniproinsulin derivative, and a leptin derivative.

13. The process according to claim 1, wherein said heterologous protein is a fusion protein comprising a protein selected from the group consisting of a hirudin, a miniproinsulin, a leptin, a hirudin derivative, a miniproinsulin derivative, and a leptin derivative.

14. The process according to claim 1, wherein exogenous ethanol is not added to said main medium or said preliminary medium.

15. The process according to claim 7, wherein said glucose in said preliminary culture medium has been almost completely consumed at the time of the inoculation.

16. The process according to claim 15, wherein said yeast is Saccharomyces cerevisiae strain Y79.