ES2208001A1 - Procedure is for production of polypeptide in yeast and involves first stage of fermentation of a yeast cell containing a sequence of deoxyribonucleic acid codifying polypeptide of interest - Google Patents

Abstract

The procedure is for the production of a polypeptide in yeast and involves a first stage of fermentation of a yeast cell containing a sequence of deoxyribonucleic acid codifying a polypeptide of interest, which is transcribed under control of a promoter of pH under conditions of pH permitting cellular growth. In a second stage, the pH value is increased to augment the transcription of the polypeptide. In a third stage, fermentation occurs during a determined period of time and under the same conditions of pH as the previous stage. In a fourth stage, the polypeptide produced is isolated.

Description

Procedure for the production of a yeast polypeptide.

Field of the Invention

The present invention relates to a process for the production of a polypeptide in a host cell, and the use of a promoter adjustable by p H.

Background of the invention

Recombinant DNA technology has provided the means to produce relatively quantities high levels of essentially pure proteins and polypeptides, so Efficient and economical

Using this technology, the DNA sequences that encode proteins or polypeptides could be incorporated into cells microbial host in such a way as to allow microbes express the genetic material and therefore produce the molecules Desired proteins

In addition to the ability to produce proteins and desired polypeptides, recombinant technology allows the control of gene expression when such control is desirable or advantageous.

On an industrial scale, it is often desirable control the expression of the recombinant DNA material to allow maximum growth, reproduction and / or survival of guests in fermentation systems or simply to synchronize protein production with specific needs or purification plans

A way to control gene expression heterologists in microorganisms is by regulation of the onset of transcription through the use of a good promoter / operator system characterized.

WO-92/08797 describes a promoter adjustable by p H isolated from E. coli (p. 2. lines 26-28) and states that this promoter could be used in a bacterium (p. 7, lines 7-15 ).

U.S. Pat. No. 5,830,692 describes an expression system adjustable by p H suitable for use in gram-positive or gram-negative bacteria (see page 5).

Espeso et al. (J. Biol. Chem. 271: 46, 28825-28830 (1996)) describe a promoter adjustable by p H from Aspergillus nidulans .

Hill KF et al. (Arch Microbiol 153:.. 4 355-359 (1990)) describes a Bacillus subtilis promoter menCD responds to the p H extracellular.

The use of yeast promoter systems based on temperature regulation, and systems that use different inducer / repressor molecules specific for regulation is known. The latter include systems such as GAL1-GAL10 , which is repressed while yeasts grow in a medium that includes glucose and is activated / induced by galactose; or systems based on the CUP1 promoter, activated / induced by copper.

Description of the invention

The present invention provides an improved method for the recombinant production of a polypeptide of interest in a yeast cell based on the identification of several promoters that are adjustable by p H in a yeast cell. The promoters identified comprise the sequences shown as SEQ. ID. nº 1, 2 and 3.

Based on the information of promoter sequences discovered here, in combination with the detailed description of appropriate strategies to obtain other appropriate promoter sequences adjustable by p H (see below), it is included in the field of general knowledge of specialized persons, identifying such appropriate promoter sequences adjustable by p H.

Such promoter sequence, adjustable by p H in yeast, provides the possibility of producing a recombinant polypeptide of interest in yeast, using said characteristic as an essential element in the production process.

A first aspect of the present invention is refers to a process for producing a polypeptide of interest, which comprises the following stages:

(to)

fermenting a yeast cell, which comprises a DNA sequence that encodes the polypeptide of interest, and that is transcribed under the control of a promoter adjustable by p H, in an appropriate medium, for an appropriate period of time, under conditions of p H that allow the growth of the yeast cell;

(b)

changing the p H value of the fermentation medium of step (a) to a p H value in which the promoter adjustable by p H is transcribing the DNA sequence encoding the polypeptide of interest at a level that is at least 2 times higher than the conditions of p H of (a);

(c)

ferment to the value of p H of step (b) for an appropriate period of time; Y

(d)

isolate the polypeptide of interest produced.

An advantageous feature of the described yeast production process is that a regulation of promoter activity based on a change of p H of the fermentation medium is relatively easy and cheap compared to the use of promoter systems based on temperature regulation or in adjustable systems based on inducing / activating molecules. In the latter, specific inducer / activator molecules could be quite expensive. Moreover, they may be undesirable from the point of view of, for example, environmental considerations.

This is particularly true in the case of large-scale industrial production of polypeptides, where, by example, a temperature change from a fermentation tank to large scale (such as a 1000 liter tank) could be quite expensive and slow.

Definitions

A series of definitions of specific terms related to aspects main of the invention.

The term "under conditions of p H that allow the growth of the yeast cell" of step (a) corresponding to the method of obtaining the polypeptide here denotes a growth of the yeast cell that is sufficiently effective for a relevant industrial production. Preferably, this is at least a doubling rate of yeast cells of 1 doubling of yeast cells every 90 minutes.

The term "the promoter adjustable by p H is transcribing the DNA sequence encoding the polypeptide of interest at a level that is at least 2 times higher than the conditions of p H of (a)" of step (b) of the procedure for obtaining the polypeptide is a relative term. The level of transcription under the conditions of p H of stage (a) and stage (b) is determined, and the increased relative transcription rate of stage (b) is determined. Preferably, the level of transcription at each stage is measured at the level of the mRNA by determining the amount of mRNA, within the yeast cell, for the polypeptide of interest under the conditions of each stage (a) and (b). Preferably, the determinations should be carried out in both cases at the given time when the amount of mRNA for the polypeptide of interest, within the cell, is relatively stable over time. The specific procedure for doing this is well known to a specialized person, and includes the Northern blot test, quantitative PCR procedures, etc.

In relation to stage (b) it should be understood that the change of p H of stage (b) could cause cell growth arrest, or a significantly reduced growth rate with respect to stage (a). This could actually be an advantage. For example, if the p H value of stage (a) is maintained up to near the maximum allowable cell mass in a fermentation tank, then the change of p H during stage (b) could start the production of the polypeptide of interest without the yeast cells having to use environmental resources to grow.

The term "appropriate time period" in relation to the fermentation time period should be understood here according to the general knowledge of specialized people. The specialized person knows what a "period of time is. appropriate "according to its specific fermentation protocol. In the step (a) this could preferably vary from 5 hours to 10 days, more preferably from 1 day to 5 days, and in the stage (c) normally it will be a shorter period of time, preferably from 5 minutes to 1 day, more preferably from 10 minutes up to 3 hours

The term "appropriate medium" denotes any of the numerous appropriate means for the growth (fermentation) of a yeast cell, which are known to specialized persons. Reference is made to (Sambrook et al. (1989) "Molecular cloning: A laboratory manual", Cold Spring Harbor Lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds.) "Current Protocols in Molecular Biology" , John Wiley and Sons, 1995; Harwood, CR and Cutting, SM (eds.); Becker and Guarente, in Abelson, JN and Simon, MI (eds.), "Guide to Yeast Genetics and Molecular Biology", Methods in Enzymology 194: 182-187, Academic Press, Inc., New York; Ito et al. Journal of Bacteriology 153: 163 (1983); and Hinner et al. Proceedings of the National Academy of Sciences USA 75: 1920 (1978)) to further descriptions of examples of such appropriate means.

The term "isolate the polypeptide of interest produced "from step (b) denotes that the polypeptide of interest it is isolated from the fermentation medium according to the procedures insulation standards known to specialists. The polypeptide of interest produced can be secreted into the medium of crop (for example, as a fusion product, which, for example, can be separated) or can be produced as a polypeptide not secreted The polypeptide of interest can be recovered / isolated. by well known procedures, including the separation of middle cells by centrifugation or filtration, the precipitation of protein components of the medium by a salt such as ammonium sulfate, followed by procedures chromatographs such as ion exchange chromatography, affinity chromatography, or the like.

After isolation, the polypeptide of interest is preferably at least about 20% pure, more preferably about 50% pure, even more preferably about 70% pure, more preferably approximately 90% pure, and even more preferably approximately 95% pure, as determined by SDS-PAGE.

The embodiments of the present invention will be described later, by way of illustration but not as limitation.

Stages of the process for the production of polypeptides

Apart from the individual stages (a), (b), (c) and (d) described above, the procedure to produce a polypeptide as described herein could comprise other stages

An extra step could be to readjust the value of p H after stage (c) to the original level of stage (a) before the effective isolation of the polypeptide of interest (step (d)).

Appropriate promoters, which are adjustable by p H in a cell

In the SEQ. ID. No. 1, 2 and 3 show DNA sequences of preferred promoters that are adjustable by p H in yeast. The promoter is obtained from the Saccharomyces cerevisiae yeast cell, and the sequence could be cloned from such a cell based on the sequence information provided in SEQ. ID. No. 1, 2 and / or 3, and using known standard cloning protocols. In an exemplary embodiment of the invention (see below), the p H regulation characteristics of the SEQ promoter are described further. one.

On the date of registration of this application, the sequence shown in SEQ. ID. No. 1 was in the database publicly available "SGD Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) " identified as "CHR4 Chromosome IV Sequence", having coordinates between base pairs 538,561 and 539,161, and in reference to the annotations was stated "This region of the chromosome IV that covers coordinates 538,561 to 539,161 does NOT have annotations ".

On the date of registration of this application, the sequence shown in SEQ. ID. No. 2 was in the publicly available database "SGD Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/)" identified as "CHR13 Chromosome XIII Sequence", having coordinates between base pairs 25,901- 26.501, and in reference to the annotations it was stated "YML122C (hypothetical ORF). YML122C does not have significant homologies with any yeast protein or worms, and therefore does not appear in any shared or shared clusters .

On the date of registration of this application, the sequence shown in SEQ. ID. No. 3 was at the base of publicly available data "SGD Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) " identified as "CHR2 Chromosome II Sequence", having coordinates between base pairs 25.901-26.501, and in reference to the annotations it was stated "This region of chromosome 2 that covers coordinates 798.578 to 799.178 has no annotations ".

A simple way to identify others sequences other than the appropriate promoter sequences shown in SEQ. ID. No. 1, 2 and 3, is to use a DNA probe based in the sequence shown as SEQ. ID. nº 1, 2 and 3. This probe of DNA could be used, then, to verify libraries of DNA from a yeast cell in order to Identify homologous promoters in yeast cells.

Accordingly, the embodiment of the invention relates to a process of the first aspect of the invention, in which the promoter adjustable by p H comprises a DNA sequence selected from the group consisting of,

(i) the DNA sequence shown in positions 1-501 of the SEQ. ID. nº 1, and 1-601 of the SEQ. ID. No. 2 or 3;

(ii) a DNA sequence that is at least 60% identical to one of the DNA sequences of (i)

(iii) a DNA sequence that hybridizes with a double stranded DNA probe comprising one of the sequences shown in positions 1-501 of the SEQ. ID. No. 1, and 1-601 of the SEQ. ID. No. 2 or 3; in conditions of low severity;

(iv) a DNA sequence that is a fragment of the DNA sequences specified in (i), (ii) or (iii).

The identity of the DNA sequence in the previous reference (point (ii)) is determined as the degree of identity between two sequences, indicating a derivation of the first sequence from the second. Identity could be determined appropriately by a computer program known in the art, such as the GAP provided in the package of GCG programs ("Program Manual for the Wisconsin Package", Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., Journal of Molecular Biology 48: 443-453 (1970)). Using GAP with the following parameters for DNA sequence comparison: criminalization of creation in GAP of 5.0, and penalty of extension in GAP of 0.3, a sequence of DNA partially identical to the sequence shown in SEQ. ID. nº 1, 2 or 3 (see section (ii) above) exhibits a degree of identity preferably at least 70%, more preferably at least 80%, more preferably at least 95%, more preferably at least 97% with the sequence shown in 1-501 in the SEQ. ID. No. 1, or 1-601 in the SEQ. ID. nº 2 or 3.

Hybridization conditions, mentioned more above to define a DNA sequence as defined in the step (iii) above, which hybridize a double stranded DNA probe, comprising the sequence shown in the positions 1-501 in the SEQ. ID. No. 1, or 1-601 in the SEQ. ID. nº 2 or 3, in at least low conditions severity, but preferably in conditions of medium severity or elevated, they are as described in detail below.

The appropriate experimental conditions to determine the hybridization between a nucleotide probe and a homologous DNA or RNA sequence, with low, medium and high severity, involve previously soaking the filter containing the DNA or RNA fragments to hybridize in 5xSSC (sodium chloride / sodium citrate, Sambrook et al. 1989) for 10 minutes, and prehybridize the filter in a solution of 5xSSC, 5x Denhardt solution (Sambrook et al., 1989), 0.5% SDS and 100 µg / ml of DNA of "sonicated" salmon sperm (Sambrook et al. , 1989), followed by the hybridization process, in the same solution, containing a concentration of 10 ng / ml of a probe, randomly barley (Feinberg, AP and Vogelstein, B Anal. Biochem. 132: 6-13 (1983)), labeled with 32 P-dCTP (specific activity> 1 x 10 9 cpm / µg) for 12 hours at approximately 45 ° C. The filter is then washed twice for 30 minutes in 2xSSC, 0.5% SDS at at least 55 ° C (low severity), more preferably at least 60 ° C (medium severity), even more preferably at least 65 ° C (medium severity / high), even more preferably at least 70 ° C (high severity), and even more preferably at least 75 ° C (very high severity).

The molecules to which the probe hybridizes oligonucleotide under these conditions are detected using film for x-rays

Another way to identify promoter sequences appropriate is using information derived from sequences Partial of the SEQ. ID. nº 1, 2 or 3. The information obtained from from these partial sequences could then be used in the preparation of primers for the chain reaction of the polymerase (PCR), which can be used to identify sequences appropriate promoters. For example, a reaction of PCR using such primers for PCR, using DNA from of a yeast cell as a template (for example, by way of yeast DNA library or, for example using a cell of native yeast (wild type) as a sample template). Such PCR reactions are routine work for people. specialized. The resulting PCR fragment could comprise then the complete sequence information for a promoter appropriate, or the sequence information in the PCR fragment could be used to clone the entire promoter sequence.

Still another way to identify sequences of appropriate promoters is looking for promoters who understand motives of DNA sequence derived from the information in the sequences SEQ partials. ID. nº 1, 2 or 3.

Instead of identifying appropriate promoters through a strategy based primarily on sequence information derived from the SEQ. ID. No. 1, 2 or 3, it might be preferable to identify such promoters based on strategies that use the "change of p H" as an essential functional parameter.

Examples of appropriate experimental procedures discussed below ("promoter capture" and "genomic chip" technology) have in common an essential point. The genomic DNA or cDNA libraries of a yeast cell are tested in the procedures at (1) a value of p H known to be appropriate for the growth of the specific yeast cell, and (2) at another value of p H other than (1). The specific promoter sequences are then identified, which transcribe a polypeptide at a significantly higher level under the conditions of p H in (2), compared to the conditions of p H in (1).

The "promoter capture" procedure is known in the art (Ruby, SW, Szostak, JW Murray, AW "Cloning regulated yeast genes from a pool of lacZ fusions", Methods in Enzymology 101: 253-269 (1983); Santangelo , GM, Tornow, J., Moldave, K. "Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments", Gene 46: 181-186 (1986); Dang VD, Valens M., Bolotin-Fukuhara M., Daignan-Fornier B. "A genetic screen to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p", Yeast 10: 1273-1283 (1994); Bell PJ, Davies IW, Attfield PV "Facilitating functional analysis of the Saccharomyces cerevisiae genome using an EGFP-based promoter library and flow cytometry", Yeast 15 (16): 1747-1759 (1999)).

The procedure is based on the cloning of moderately sized fragments of genomic DNA into specialized vectors that contain a revealing gene whose transcription could be easily detected. An example could be the LacZ gene, whose activity can be detected by a specific coloration around specific yeast colonies. This could be done directly or after transferring to appropriate membranes. In the present context, a library of different promoters bound to the developer gene is preferably used. Such a library could be tested under the conditions of p H (1) and (2) above, and clones expressing the developer gene (eg, LacZ ) with significantly higher levels under the conditions of p H (2) compared to conditions of p H (1) would generally include an appropriate promoter in the present context.

"Genomic chip" technology is also known in the art (DeRisi JL, Iyer VR, Brown PO "Exploring the Metabolic and Genetic Control of Gene Expression on a Genomic Scale" Science 278: 680-686 (1997); Wodicka L. , Dong H., Mittmann M., Ho MH, Lockhart DJ "Genome-wide expression monitoring in Saccharomyces cerevisiae " Nat. Biotechnol. 15: 1359-1367 (1997); Jelinsky SA and Samson LD "Global Response of Saccharomyces cerevisiae to an Alkylating Agent "PNAS 96: 1486-1491 (1999); Stewart, DJ" Making and Using DNA Microarrays: A Short Course at Cold Spring Harbor Laboratory "Genome Res. 10: 1-3 (2000)).

The procedure is based on the complete known sequence of the genome of the Saccharomyces cerevisiae yeast cell. Through the use of this sequence information, hybridization systems have been developed in which the PCR fragments or oligonucleotides, specific for each gene in the S. cerevisiae cell, have been fixed on a solid support in an organized manner (genomic matrix ). The solid supports could be silicone chips, glass plates (similar to those used in microscopy) or nylon membranes. Such hybridization systems are commercially available (eg, Affymetrix Inc. (Santa Clara, CA, USA), Invitrogen (Carlsbad, CA, USA).

In the present context it is preferable to obtain total mRNA from a yeast cell (preferably a Saccharomyces cerevisiae cell) cultured under the two conditions of p H (1) and (2) mentioned above. From the two populations of total mRNA, cDNA is prepared, which is radioactively or fluorescently labeled. Next, these populations / cDNA libraries are hybridized with the matrix system as described above. In the case of different fluorescent labeling it is preferable to mix equal amounts of the two cDNA libraries, which will result in competitive hybridization. The result of such a protocol will be the identification of mRNA produced with a significantly higher level in the condition of p H (2) compared to the condition of p H (1). This mRNA will probably be transcribed by a promoter adjustable by p H, appropriate in the present context. Consequently, the information in the sequence of an mRNA identified in this way can be used to identify the promoter that transcribes said mRNA, according to the standard protocols known to specialized persons.

In both of the above procedures, "capture of the promoter" and the "genomic chip", it is preferred that the p H of step (a) be a value of p H from p H 4 to p H 7, and the p H is increased in step (b) up to a value of p H from 6.5 to 9.5.

This is mainly due to the fact that a certain number of yeast strains, known to be very suitable for large-scale production of the polypeptide of interest, have an optimal growth rate at p H with slightly acidic values. Such yeast strains include a strain of Saccharomyces cerevisiae and, perhaps, a strain of Pichia pastoris .

Preferred p H values of step (a) and step (b), (c) of the first aspect of the invention.

As mentioned above, a number of yeast strains, known to be very suitable for large-scale production of a polypeptide of interest, have an optimal growth rate at slightly acidic p H values.

Accordingly, it is preferred to ferment the yeast cell in step (a) of the process of the invention under conditions of p H similar to these, in order to ensure sufficient growth of the yeast cells; while in step (b) it is preferred to slightly increase this condition of p H. An example of this is shown in example of procedure 2, where the SEQ promoter is used. ID. No. 1

Therefore, one embodiment of the invention relates to a process wherein the conditions of p H of step (a) is a value of p H selected from p H 4 to p H 7 and p H is increased in the stage (b) up to a value of p H from 6.5 to p H 9.5. The term "increased", mentioned above, denotes that if the value of p H of step (a) is, for example, 7.25, it cannot be, for example, 6.75 in step (b), since This is not an increase in the value of p H.

Preferably the conditions of p H of step (a) comprise a value of p H from p H 5 to p H 7, and p H is increased in step (b) from a value of p H 7.25 to 9 , 0.

Relative increase in the transcription level of the promoter adjustable by p H

A preferred embodiment of a process, as described in the present invention, is that in which the level of transcription by the promoter adjustable by p H of step (b) is at least 3 times higher than under the conditions of ( a), preferably at least 5 times higher than under the conditions of (a), more preferably at least 10 times higher than under the conditions of (a), and even more preferably at least 50 times higher than under the conditions conditions of (a). See example of procedure 2 as an example of the latter case.

Yeast cell

Any yeast cell could be used in the process of the invention as described herein.

However, it is preferably a yeast cell selected from the group consisting of: Hansenula polimorpha, Pichia pastoris, Candida utilis, Kluyveromyces lactis , and Saccharomyces cerevisiae . From the previous list; more preferably it is a yeast cell selected from the group formed by Pichia pastoris, Candida utilis and even more preferably a Saccharomyces cerevisiae cell.

As mentioned above, a yeast cell used in the process, as described in the invention, includes a DNA sequence encoding the polypeptide of interest, which is transcribed under the control of a promoter adjustable by p H .

The DNA sequence encoding the polypeptide of interest and the promoter adjustable by p H could, for example, be located on the chromosome of the yeast cell or, for example, be located in a vector (for example, a plasmid) in The inside of the cell.

Moreover, the yeast cell could include other appropriate elements such as an eligible marker gene, by example, an antibiotic resistance gene.

Large scale production

As mentioned earlier, a procedure as described here, is very appropriate for the large-scale production (see section "Summary of invention").

Accordingly, an embodiment of the invention it refers to a procedure such as the one described here, in which Fermentation in stages (a), (b) and (c) is carried out in a tank of fermentation comprising at least 100 liters of medium appropriate, preferably in a fermentation tank that It comprises at least 500 liters of an appropriate medium, and more preferably in a fermentation tank comprising at least 10,000 liters of an appropriate medium.

Be part of people's knowledge specialized scale a fermentation process, preferably by step-by-step scaling, where each step involves the fermentation in growing fermentation tanks.

Polypeptide of interest

Any polypeptide of interest could be advantageously produced according to the process of the invention such As described here. Preferably, the polypeptide of interest is a polypeptide suitable for use in the preparation of a medical product, and more preferably the polypeptide is insulin human, human growth hormone, cholecystokinin human, interferon, the human plasminogen activator (u-PA), interleukins 2 and 3, or different types of recombinant subunit vaccines.

A yeast cell comprising a promoter adjustable by p H

A final aspect of the invention relates to a yeast cell that includes a p H-adjustable promoter operably linked to a heterologous gene, which includes DNA encoding a polypeptide of interest, wherein the p H-adjustable promoter includes a sequence of DNA selected from the group consisting of:

(i) the DNA sequence shown in positions 1-501 of SEQ. ID. No. 1, and 1-601 of SEQ. ID. No. 2 or 3;

(ii) a DNA sequence that is at least 60% identical to one of the DNA sequences of (i);

(iii) a DNA sequence that hybridizes with a double stranded DNA probe that includes one of the sequences shown in positions 1-501 of I KNOW THAT. ID. No. 1, and 1-601 of SEQ. ID. No. 2 or 3; to low severity;

(iv) a DNA sequence that is a fragment of the DNA sequences specified in (i), (ii) or (iii).

All of the above preferred embodiments are also preferred embodiments of a yeast cell that Present these final characteristics.

The term "heterologous gene" refers here to a gene under the regulation of a promoter that does not control it from natural form. A heterologous gene can encode any peptide, polypeptide or protein, except that which is encoded by a gene which presents a promoter that controls its expression normally in the nature.

Examples Materials and procedures Strains

Yeast strains: The strain of Saccharomyces cerevisiae used was W3124 (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prc1 :: HIS3; prb1 :: LEU2; cir +) . The E. coli strain was: DH10B (Life Technologies).

Plasmids

pUC18: (Life Technologies), pJS205 (not commercial, is described in the document. Sch ü ller HJ, Hahn A, Tröster F, Schütz A, Schweizer E (1992) "Coordinate genetic control of yeast fatty acid synthase genes FAS1 and FAS2 by an upstream activation site common to genes involved in membrane lipid biosynthesis "EMBO J 11: 107-114).

General molecular biology procedures

Unless otherwise mentioned, DNA manipulations and transformations were carried out using standard molecular biology procedures (Sambrook et al. (1989) "Molecular cloning: A laboratory manual", Cold Spring Harbor Lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds.) "Current Protocols in Molecular Biology", John Wiley and Sons, 1995; Harwood, CR and Cutting, SM (eds.) "Molecular Biology Methods for Bacillus", John Wiley and Sons, 1990; Becker and Guarente, in Abelson, JN and Simon, MI (eds.), "Guide to Yeast Genetics and Molecular Biology", Methods in Enzymology 194: 182-187, Academic Press, Inc., New York ).

Enzymes for DNA manipulations are used according to the specifications of the suppliers.

Enzymes for DNA manipulations

Unless otherwise mentioned, all enzymes for DNA manipulations, such as, for example, restriction endonucleases, ligases, etc. were obtained from New England Biolabs, Inc.

Example 1 Construction of a plasmid pPH1 that includes the promoter adjustable by p H of the sequence with identification number 1, operably linked to a LacZ reporter gene

Plasmid pPH1 was constructed as described below. A 0.5 kbp DNA fragment, which includes the SEQ, was amplified. ID. No. 1, from genomic DNA by PCR using oligonucleotides A1 fw and A1 rev.

A1_ {fw}: 5'-CATGCTCGAGTTTATCTCGTCACTCTTGTAC-3 '

A1_ {rev}: 5'-CGATCTCGAGTCCCTGTATATATCTATGTA-3 '

This fragment was purified, digested with Xho I, and cloned into plasmid pUC18 previously digested with the same restriction enzyme. The insert was sequenced to verify unwanted mutations, and then cloned into the Xho I site of plasmid pJS205. The proper orientation of the insert was evaluated by restriction analysis and / or DNA sequencing.

Example 2 Proving that the promoter of the sequence with identification number 1 is adjustable by p H in yeasts

Plasmid pPH1 of Example 1 was transformed into a strain of Saccharomyces cerevisiae , and recovered in CM medium lacking uracil.

The transformed strain was grown, at 28 ° C in CM medium lacking uracil, to an OD 600 of 1.0-2.0. An aliquot of the culture was added to a standard YPD medium at p H 6.5 (condition of p H (1)) to achieve an OD600 of 0.2, and growth was resumed to an OD 600 of 0.5 -0.6.

An aliquot of the culture was taken, and the value of p H was increased to p H 7.75 (condition of p H (2)) and the fermentation was continued for 1 hour, and the amount of LacZ gene expressed was measured.

The results are shown, then in the Table I:

TABLE I

       \ nobreak \ vskip.5 \ baselineskip \ centering \ begin {tabular} {ll} \ hline \ multicolumn {2} {l} {
data correspond to the mean ± standard deviation
of} \\\ multicolumn {2} {l} {three independent clones, and they express themselves
as units of} \\\ multicolumn {2} {l} {activity of
 β-galactosidase according to Ausubel, F.M et
to the. } \\\ multicolumn {2} {l} {(eds.) Current Protocols in
Molecular Biology, John} \\\ multicolumn {2} {l} {Wiley and Sons,
1995.} \\\ hline Condition (p H) \ + Activity
 β - galactosidase \\\ hline p H 6.5 \ +
4.0 ± 0.9 \ p p 7.75 \ + 353 ± 84
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

These results clearly demonstrate that the promoter of the SEQ. ID. No. 1 is adjustable by p H in yeast cells. Moreover, it can be seen that the level of transcription is more than 50 times higher under the condition of p H (2) ( p H = 7.75) compared to that of the condition of p H (1) ( p H = 6 ,5).

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<220>

         \ vskip0.333000 \ baselineskip
      

<221> promoter

         \ vskip0.333000 \ baselineskip
      

<222> (1) .. (601)

         \ vskip0.333000 \ baselineskip
      

<223> PHO89 SEQUENCE in chain address above, from -700 to -100

         \ vskip0.333000 \ baselineskip
      

<400> 3

3

Claims (9)

1. Procedure for producing a polypeptide of interest that includes the following stages:

(to)

fermenting a yeast cell, which includes a DNA sequence that encodes the polypeptide of interest and is transcribed under the control of a promoter adjustable by p H, in an appropriate medium, for an appropriate period of time, and under conditions of p H that allow the growth of the yeast cell;

(b)

changing the p H value of the fermentation medium of step (a) to a p H value in which the promoter adjustable by p H transcribes the DNA sequence encoding the polypeptide of interest at a level that is at least 2 times higher than in conditions of p H of (a);

(c)

ferment to the value of p H of step (b) for an appropriate period of time; Y

(d)

isolate the polypeptide of interest produced.

2. A method according to claim 1, wherein the promoter adjustable by p H comprises a DNA sequence selected from the group consisting of:

(i) the DNA sequence shown in positions 1-501 of SEQ. ID. No. 1, and 1-601 of SEQ. ID. No. 2 or 3;

(ii)

a sequence of DNA that is at least 60% identical to one of the sequences of DNA of (i);

(iii)

a sequence of DNA that hybridizes with a double stranded DNA probe that includes one of the sequences shown in the positions 1-501 of SEQ. ID. No. 1, and 1-601 of I KNOW THAT. ID. No. 2 or 3; at low severity;

(iv) a DNA sequence that is a fragment of the DNA sequences specified in (i), (ii) or (iii).

3. The method according to claims 1 and 2, wherein the condition of p H of step (a) is a value of p H from p H 4 to p H 7, and p H is increased in step ( b) up to a value of p H from 6.5 to 9.5. 4. The method according to claim 3, wherein the condition of p H of step (a) is a value of p H from p H 5 to p H 7, and p H is increased in step (b) to a p H value from p H 7.25 to p H 9.0. 5. A method according to any one of claims 1 to 4, wherein the level of transcription by the promoter adjustable by p H of step (b) is at least 3 times higher than under the condition of p H of (a ), preferably at least 5 times higher than under the condition of p H of (a), and more preferably at least 10 times higher than under the condition of p H of (a). 6. A method according to any one of claims 1 to 5, wherein the yeast cell is selected from the group consisting of Hansenula polymorph, Pichia pastoris, Candida utilis, Kluyveromyces lactis , and a Saccharomyces cerevisiae cell, and preferably from the group formed by Pichia pastoris, Candida utilis and more preferably a Saccharomyces cerevisiae cell. 7. Procedure, according to any of the claims 1 to 6, wherein the fermentation in the stages (a), (b) and (c) is performed in a fermentation tank comprising at least 100 liters of appropriate medium, preferably in a fermentation tank comprising at least 500 liters of an appropriate medium, and more preferably in a fermentation tank comprising at least 10,000 liters of appropriate medium. 8. Procedure, according to any of the claims 1 to 7, wherein the polypeptide of interest is a appropriate polypeptide to be used to prepare a product doctor. 9. Method according to claim 8, in which the polypeptide is human insulin, growth hormone human cholecystokinin human interferon activator human plasminogen (u-PA), interleukins 2 and 3, or different types of recombinant subunit vaccines.