Yeast of the genus Kluyveromyces modified for the expression of preprothaumatin or its various allelic and modified forms or their maturation forms.

DESCRIPTION OF THE G1STBROGADB5 £ * .V. Based at 2600 KA DELPT, The Netherlands for the Invention:

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ICLUYl ^ EHOKYCSS clone-forming system

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A novel isoform-forming system is described, capable of reproducing (generating) a gene. material derived from a recombinant DI4A material, which contains a KLUYVEROMYCES genus yeast as a host.

Suitable bodies)^:are, for example, vectors containing autonomously rearranged sequence (ARS) and vectors containing homologous DHA KLUYVEROMYCES which have a backbone for recombination after the chromosome of the host. New and preferred carriers are those containing the ARS series derived from KLUYVEROEYCES (KARS carriers)., • Genetically engineered new KLUYVEROEYCES strains produce, among others, lactase and jujube.

Field of the Invention This invention relates to the field of D1TA recombinant technology. Particular reference is made to the use of biotechnology combination with DITA for the production of polypeptides. More precisely, the present invention provides for novel D1IA recombinant cloning vectors and their suitable host organisms, which may be used in high yield polypeptide production, e.g. enzymes such as β-galactosidase (lactase) and chymosin and their precursors.

* At the beginning of the invention

In the recent past, microorganisms have been shown to produce xenapeptides and proteins encoded by artificially-inserted foreign genes using a transform system and unregulated controls.

Some of the basic techniques of this process have been described, for example Am. double 4 237 224.

The key components of recombinant DHA technology are formed by: the gene encoding the desired property and provided with a sufficiently controllable sequence required for the imprinted organ / organ;

-3f £? ·^p / J - / o *¹ 3 3 β β £ £ · · · · fp ^f ^ • rr ^ Mv * '⁷· '·'. &lt;"Jl / '; '-'&gt; '- · *'; a vector, usually a plasmid, within which the gene is capable of '^: ; to ensure that a fixed, imprinted and reproducible guarantee of said nid is provided, at a level of 1 °,

- a suitable micro-organism-host within which the vector is. The carrier of the gene can be transformed and has cellular systems to mimic the information in the gene. ,

Among the products thus formed are enzymes, hormones, antigens and other useful peptides and proteins.

Some of these products serve as pharmaceuticals, e.g. the growth hormone and / or deroferrone, hereinafter as an adjunct to the food industry, e.g. β-galactosidase (lactase), juice and amyloglucosidase and others, also as biological catalysts for the conversion of certain compounds.

Any contamination of medicines or foods by pests or substances should be excluded. Host organisms should not be 'harmful' to humans who handle microbes during experiments or production methods you have scaled up. * Therefore, a prerequisite is that the host is unaware. .

· The first years of work on recombinant DI7A were characterized by strict rules and restrictions, to prevent or minimize the effects of undesirable side effects, especially on the uncontrolled spread of micro pathogens.

If, or in view of the alleged dangers, it appears to have been exaggerated too much, there is nevertheless a constant need for hosts with no association with any harmful effects.

Until now, commercial efforts involving the manipulation of antisense genes of plasmids for the production of various substances have been concentrated on E. coli as an organism. The main reason is that E. COM is historically the best studied micro-organism. First, the discoveries and inventions of the D1TA recombination technology have been carried out with X. GOL1 as a host. / ••• However, E. COL1 is no longer suitable for the commercial production of substances for use in the pharmaceutical industry. It may still result in an inappropriate ESvCSsSSo name;

-4 pyrogenic agents. These eliminations can often create problems. Therefore, E. col1 has a very limited utility as a production organism:. fermentation industry. Also, the proteolytic activity of E. col1 may severely limit the yield of certain useful products.

These and other aspects have led to increased interest in alternative host / vector systems. Of particular interest is the use of eukaryotic systems for the use of eukaryotic products. There is a continuing search for new hosts suspected of producing chemicals, in particular products used in food and pharmaceuticals, and which are further suited to the industry by large-scale fermentation.

The names of suspected harmful micro-organisms are listed in the so-called GRAS (generally recognized as safe). However, only a few newborns of the genetic technique are known to date, for the formation of clones and gene imprinting -GRAS organisms are used.

Among the eukaryotic organisms suitable for commercial use yeast are probably the most convenient. * Brewers, especially BAKER brewers, are cheap, inexpensive, easy to grow in large quantities and have a highly depleted genetic system.

Serum brewer (the yeast) is often used for SACCHAROMYCES

CEREY1S1AE alone, or BAKER brewer, is one of the most common and well known species. It is conceivable that the term brewer is used in this specification, that is, not all genera, and is not limited to the species SACCHAROMYCES 'CEREV1S1AE.

It has recently been discovered that the cells of SACCHAROKYCES CEREV1S1A3 are susceptible to plasmid transformation (A. Hlichen et al., PROC. KATI. ACAD. SCI. USA 75 (1.978), 1929). and imprinting on this brewer, bacterial resistance genes for ampicillin, chloramphenicol and kanamycin but also for ovalbumin, leukocyte interferon (CP) HOLLEMBERG, CURREBT TOPICS IN MICROBIOLOGY ARD 119-144).

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-5_g G. To date, only one species of yeast has been investigated as a host for "D";

'Z&gt;' ξ, ~^j '' yy yy '' / 'ΎΊ • Impressive brewers. The gene, Electrin. 'SACCHAROETYCES. CEREV1S1AE. has been successful: formed a clone and imprint on SCHIZOSACCHAROHYCES ROMBE (D. * BEACH AND P. NURSE, NATURE 290 (1981) 140-142). .

Brewers who have described that they (have a successful transformation, are based on the 2 µm (2 µm) natural plasmid which each of them has the most strains of S. CEREV1S1AG 27, NAT JD (acts e.g. (1973) 104-109), and on autonomous sequencing sequences (ARs) of yeast-derived chromatosomal DNA (acting e.g., K. STRUHL et al., PROC. NATL. ACAD. SCI. USA 76 (1973)). 1979), 1035-1039), respectively. CEBEV1S1AE capable of being used for transformation have also been described by C.P. HOIiLENBBRG, CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY 96 (1932) 119-144.

* The transformation of non-fully characterized or industrial yeast species is seriously hampered by a lack of knowledge of the transformation conditions and appropriate vectors. * In addition, are nototropic notes often (important) or are they unwanted by direct selection? by auxotropic complement formation.

Summary of the Invention It is an object of the present invention to provide a system of dual yeast vectors, such as to encode a polypeptide coding sequence.

Further antique, the subject of the invention is to be sure. to provide genomically produced yeast strains of the genus NJUYVYSROMYCES, capable of producing polypeptides, for mass production. .

Further, the object of the invention is to provide new, genetically engineered yeast strains of the genus ELUYVERQeIYCES, which can produce chymosin and precursor forms. this, a mass production crop.

It is an object of the invention to provide formulated yeast strains of the genus NLUY. 'EROMYCBS to induce lactase in mass production.

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&gt; -j + * 4 &amp; * *. '&gt;_PThe object of the invention - is to provide methods, par., treatment. policy, ".⁴ ·,%. *? · · *. "X". 'P' peptides and particularly enzymes with organism-derived KLUYVEROEYCBS strains obtained from techniques; DHA compound. 7

Another object of the present invention is either the delivery of the whole of the modified cells: KLUTVEROKYCES for use in the preparation of prelipeptides derived from certain enzymatic activities.

-. And so on. other items are to be described in more detail following the description.

Description of the Invention

--- i - II I I ----.- 11 1IUI — 1 ... IH — WHI. II I. , yeasts of the genus KLUYVEROKYCES; ·}, and in particular Mr LACS1S and Mr PRAG111S are important from a biotechnological point of view and are of commercial interest. * For example, K. LACT1S and K. ERAG1L1S, are used commercially to produce the enzyme lactase (β-galactosidase).

KLUYVEROHYCSS organizations are listed in the GRAS directory.

* In contrast to most bacterial species studied in transformation experiments, yeast cells have a cell wall that is not permeable to plasmids. Thus, a preparatory step during the transformation of the brewer's yeast is the removal of the cell wall of derived protoplasts, which may recruit plasmids. The cell wall lysing enzymes that can be used advantageously are selected from the β-1,3-glucanase group. A suitable example of an incomplete or helicase, unclean enzyme formulation derived from the offal of the snail HELIX ROCAT1A. Another suitable antisense representative enzyme is zymolyase.

/ 7 /; . It is well known that the cell wall may be unborn at a later stage under appropriate conditions. However, only part of the protoplasts are regenerated for KLUYVEROliYCES LACT1S, or this procedure appears to be even 20 times lower than for SACCHAROHYCES¹ CEREV1S1AE under similar circumstances.

Although the transformation of brewers using protoplasts has been described by several authors, it appears that particularly free-living strains and species of KLUYY3RO may be regenerated. '

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-7 ”0 HOLLENBERG (CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, 96 / (1982);

119-144) describes the regenerative pattern of S. prototype CEREV151AEs which may also occur if the standard osmotic stabilizer sorbitol is highly volatile, potassium chloride. Surprisingly, it has been found that by applying this method to KLUYVEROKYCES prototypes, the proportion of regenerated zymocytes is increased by three to five times.

Recently, it has described a method under 1T0 et al. (j. BACTER1OL. 153 (1985) 163-168), in which all cells are used instead of protoplasts, bypassing the anisogenesis stage. * This method has been shown to be applicable to SL CEREV1S1AE with certain types of plasmids.

It has also been found that this method is surprisingly effective in volume. That are used

- KLUYVEROHYCES 'own uterus when the plasmids viruses have a KARS sequence (as you will describe here). ',. Understanding the specifics or availability of a suitable carrier is crucial. On the other hand, it is uncertain if you are an expert_: the host / vector combination works successfully as a transformation system. For example, it is known from E. ERHART and CP .HOLLENBERG, CURRENT. . GENETICS 3 (1981) 83-39, where the plasmid pEP81 can be transformed into S. CEREV1S1AE YT6-2-1L (thiol) but not on KLUYVEROMYCES LACT1S.

D. BEACH and P. NURSE, described: in NATURE 290 (13S1) 140-142, that plasmid pJDB219 shows a large number of copies in S. cerev1S1AE, but in other SCKLZOSACCHAROMYCES mutants. with DEA.

To date, no known carriers of KLUYVEROMYCES are known. The result of extensive research and experimentation is the uneasiness of new players who can transform the KLUT.TBROKYCES host organism and can then reproduce autonomously.

Young, vectors, which are particularly suitable for KLUYiEROMYCES LACT1S and K. FRAGILIS, preferably in two classes, depending on the components of the LILA, have the function of,

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-81. 'T' in vectors containing autonomously expressed sequences (ARS) ^ and

· ''. · 3 ··: ^ t · Li * · .j. · · / ' ¹ '' .V · *: · ... ··· .. · ► ·,. ·· · '

A &lt;7 L „V. · Ί · - ·.) · - ·· - · -. ./y '' ··, ·· ·: ·: '· · .. ·

2 g are vectors that do not have self-replicating sequences, but contain homologous DHAKLUYVEROMYCES which are positioned as receptors for recombination after the host chromosome.

. Suitable and preferred ARS carriers are the originators. of the species KLUYITSROSYCES, also referred to as KARS vectors.

The KARS type transmitters used are preferably used because of their high frequency of transformation. Carriers of the second class usually transform at a low frequency but are held tightly within the host cells.

Its preferred carriers. of the first category are, for example, KARS carriers originating from K. LACT1S of which the most preferred are carriers. pKARS2 and pKARS12. PKARS 12 and pKARS2 are hybrid plasmids. fractional. DNA of K. LACT1S containing the KARS12 and KARS2 sequences, respectively, inserted into the YRp7 plasmid of C. CEREV1S1AS, respectively.

A preferred carrier of the second class is, for example, pL4 -, a hybrid plasmid consisting of the ARS1 carrying plasmid.

YRp7 and one fragment of DHA XI) 1 kL of K. LACT1S carrying the LAC4 gene.

Before Transforming Inside. KLUYVEROMYCES, THE FOLLOWING GENES, IN EXAMPLES, MAY BE ADVANTAGED TO BE ELIGIBLE AS MARKED ON VICTORIES:

1. ; The tryptophan gene (TRP1) derived from S. CEREY1S1AE, the lactase gene (LAC4) derived from K. lact1S / gene, which encodes resistance to the E4 antibody and the related antibody G4. 00L1.

Suitable restriction sites are present on the vectors which allow for further branching of the gene.

* The integrity of the transformed plasmids can be improved Significantly if a central region (CEt1) of the X. LACT1S or Tg2-_ chromosome is present. .

S. CEREV1S1AE, inserted into the vector.

2.

A.

E. coli, also, is a suitable host, eubiquitous. I? », 5₍ 1 Cloning and storage. Are you in this case?

P. . 1 - An ampicillin antisense gene (Amyl j also constitutes

-9'T &lt;em&gt; plasmids multiply and are preferably maintained in-cells. E. COL1, particularly those of strains DG75 and JA221. The transformants (mutants) are selectively grown on zymodi-L containing:

kanamycin / (20 / µg / h) for E. coil DG75 (PTY75-1AC4, and ampicillin (100 µg / l for E. coil DG75 (P14) and E. 0011 JA221 (pKARS12)).

The so-called mutant strains are deposited under Regulation 28, Ref. 28a EUROPEAN PATENT CONVENTION at the Central Office n. SCHBESELCULTURES, 003TERSTRAAT 1, 3742 SK BAARN, THE NETHERLANDS, under CBS 351.82 (- 1MD 82.18), CBS 352.82 (- Lffl&gt; 82.19) and CBS 355.82 (LLlD 82.20), dated 1982. Plasmids can be isolated from cells, e.g. by the method of L. KATZ et al., J. BACTER10L. 114 (1973) 577..

The yeast-host prototypes are transformed by the above vectors over conventional suppression medium containing TR1S (cal) and calcium chloride and TEO with a molecular weight of from 4000 to 6,000;

Prokaryotic mutations can easily be undermined by the well known means of primary selection. Even though the desired identity is not indistinguishable from the phenotype of the mutation, the vector usually contains a multitude of genes encoding primary selectable identities such as the antisense, antisense, or anti-antibodies. In the latter case a corresponding shuttle should be available. silk of the host. '* While there is a large number of prokaryotic prokaryotic organisms, q the number of industrial prokaryotes is limited? Mutation of a gene by one strain generator often adversely affects the important growth and production characteristics of that strain. '.

According to the method of transformation according to the invention, it is convenient to perform the following as appropriate.

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- The method of the invention comprises; KLUYVER cells OMYCES within a typical brewer growth medium and harvesting cells between 1 and 25. "Excellent results are obtained between 4 and 10.

Ta_; KI1UYVBRO1.TYCBS cells are also washed. are preheated after certain types of chaotropic ions, e.g. Li *, Ω *, Kb *. LiCl and L ^O 'are conventionally used at final concentrations of about 20 mM - 0.2, preferably about G.1 M $. ,

KLUYVEROiriCBS cells are incubated with these monovalent ions for 5-120 minutes, usually about 60 minutes, followed by DMA incubation. The transformation may be promoted by subsequent addition of polyethylene glycol. .

In general, an equal volume of 70% polyethylene glycol 7000 is used. The transformation of KLUYVEROLIYCES can be further promoted by exposure of the cells to heat effect. For example, by treatment for about 5 minutes at 42 ° C, or improvement is about 20-fold.

The usefulness of this route according to the invention will be shown in detail :: some examples with the KLUYVEROMYCIS LACT1S SD11 strain, K. PRAG1L1S

Yea 24 and K. PRAG1L1S 012, as their corresponding stressor organisms.

In contrast to prokaryotic organisms or useful antistatic markers for yeast antibiotics, it is easy to avoid. Only a small number of antibiotics are active against yeast. Further, antistatic agents are predominantly bacterial and are not at all certain that they can be imprinted on yeast cells and used. as an eclectic major. ':

..jZh kanamycin and the aminoglycoside G418, related to gentamicin, have been shown to be deleterious to freely living yeast strain cells.

Further, it is known from the HOLLEHBERG, EXTRACER0K0S0KA1 DMA, ldi^Mr UCLA SYKP. '(1979) 15: 325-338, ACAD. PRESS. MEW YORK, that the transposable antisense element Tn601 (found on the bacterial plasmid j&gt; CR1) contains a gene that transfers resistance to kanamycin in the SACCHASO1 / SACCHASV1 mutations of CAC. J1KEMES AMD J. DA (133O) 869-871, '': showed later resistance to kanamycin, may also be carcinogenic: S. CERE71S1AE, against antibiotic G418, potent inhibitor

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1; "-11-; /; *, -7, '4-7" A. AAAAAA: :: AAAAAA -'77^{: =} • PTY75-LAC4 plasmid, hybrid-plasmid constituting 'ectoplasm' ^x * &lt;* 7 1 u- * ·· '' '·' '7 ⁿ · ··· ''&gt; z7'Z. '^ 7? 77' f '3 * d. ® i n₄ ...,. * .. ·&gt; .- π · τ, - 7 ·:; ': &lt;

2-jpCRI, 2d plasmid from S. CEREVISIAE and Sal fragment 1 cm.

- l-plasmid plasmid, carrying the LAC4 gene- K. lactis, contains. - the same antisense gene itself., Has it already been found that the gene is also self-coding (replicated) in K.? LACTIS, allowing the strain to be inactivated - the G418 recruited from the growth medium and so obtained for the main selection of KLUYVEROUYCES LACTIS mutants.

If plasmid TT75-I; As4 does not contain any autonuclear replication; Sequence of KLUYVEROIilYCES, surprisingly, found plasmids containing the 2-fold plasmid from S. cerevisiae, such as PTY75-LAC4, can be reproduced autonomously in K species.

The selection of the G418-resistant yeast strains, transformed by PTY75-LAC4, was performed on regeneration plates containing glucose, sorbitol and 0.2 MG / ml of G418. KC1 is not suitable here because, for example for reasons of high salt concentration, KLUT? EROLIYCBS LACTIS cells are insensitive to G418 even at concentrations up to 0.8 KG / LH.

Resistant colonies appear within 5-6 days after transformation to PTY75-LAC4. Actual mutations can be distinguished from six colonies which have become resistant to spontaneous mutation by controlling the presence of DMA PTY75-LAG4,. by hybridizing the colony with DMA-labeled pCR1. or do you miss K. LACTIS Lac4, which is used as a host strain, by controlling its ability to grow on M1H1MAL (D1FC0 basic yeast nitrogen source), with a unique carbon source.

On average, 5% of the resistant colonies were found to contain BTY75-LAC4 DMA or Lao *. Approximately 4 mutations (mutants) per microgram (µg) of plasmid DMA were obtained by the selection method.

* Direct selection on K. lactis SD69 1a4 "for the presence of the LAC4 gene, using lactose-containing plates as the sole carbon source and O.ffl KC1 as a Lao osmotic stabilizer, provided 20 min. 5 day of incubation at 30 ° C. * 3b comparative backgammon jv; '. 7 no DMAs, no Lac colonies found within up to

First Lac colonization of direct selection, it was shown to be metal / ®

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The significant KirrjE on G418 plates was described above.

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When plasmids pL4 or KARS-type plasmids are used, there is / is also a choice to enable tryptophan mutagenesis. By comparison: with plasmid PTH5-LAC4, the use of plasmid PL4 has caused a significant increase in transformation efficiency:

30, mutant strains were found without DNA. However, the KARS plasmids with mutations without DNA have been shown to be much superior.

The PTK75-LAC4 plasmids and KARS-containing plasmids were found to mutate independently in mutant cells. This has been shown to aid DNA analysis. Fallen metallic microlysis products were diluted accordingly to allow SOUTHERN. BIOT, by hybridization with⁵²P-labeled pCR1, i.e., the bacterial component of plasmid PTY75-LAC4 or with labeled pBR322, the bacterial portion of pKARS plasmids.

Comparative electrophoresis -PROCEEDS mikrolyseos non-mutated '- V KLUYVEROKYCES LACKT1S LaC4 and what is purified plasmid preparations, indicated that alone In the mutagenized strains exist strips hybridization non ilektrofiritikin mobility corresponded in convoluted and open ring form of the used dia'ton ​​transformation plasmid.

The presence of the plasmid in the mutant cells was further confirmed by transformation of E. coli with DNA preparation from yeast mutations and isolation of the same plasmids from the E. coli mutations.

The method of the present invention may be applied to strains of KLUYVIKOHYCES 1ACT1S and to strains of KLUYVER0MYC2S &lt;ERAG1L1S. Both species are safe organisms and are listed in the GRAS catalog.

'Especially useful for beginners are the mutated KLUY' / EROLiYCES IACT1.S SD11'1β4 4 / P strains.¹ and SD69 1a04, derived from the free-living type CBS 2360 and regulated by Regulation 28, RSSP 23a of the EUROPEAN PATENT CONVENTION NATIONAL CENTRAL BUREAU VOOR SCKlI.aiELCUIM OOSTERSTRAAT N ° 37 OSTERSTRAAT_;, SO93, and CBS 8093, respectively, of 19 May 1982.

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Typically, the transformant plasmids remain within the host cell as individual assemblages capable of self-generation. However, is it emphasized that genes? after insertion into the plasmids (with or without sequences);^:/^L.· · '· /// ·. ; · '. DHA, non-derivative;) 7 can then also be incorporated into the cell / chromosome of the cell.

The so-called trans-integrin transformation appears to have occurred in the K. mutant LACT1S SD11 -transformant mutations after transformation for plasmid pL4. In this case there is no free EIT plasmid within the mutations. * The incorporation of the LAC4 gene can be demonstrated by analysis of DMA by SOTOTHER1T BLOT of whole DMA cell, which is digested by restriction enzymes, where the plasmid I, pL4, acts as a significant activator.

) To preserve the plasmids within the yeast mutants, the following selection media may be used, for example: Yeast Nitrogen Basic Nutrient (D1PC0) + 2% lactase, instead of glucose1 / K.

SD69 lac4 (PTY75-LAC4) and for K. LACT1S SD69 1β4 (pl &lt;4) and basic yeast nitrogen medium (LipoC) 2% glucose for X. LACT1S SD11 ^ ePl lac4 (pKARSl2).

“They are made of hybrid plasmids consisting of KAES12 'LAC4 and KARS12-2 mAbs — LAC4 strains. "When the novel microorganisms of the invention are used to produce a large scale, it is desired or removed" / Z · '/ · of all DMA bacterial strains from vector plasmids.

The genes remain on autosomal recessive plasmids from the forearm. '

'.K; . within the cell or integrate - within the DEA chromosomal cell. boiling?

- * The invention may be used to form clones and imprint prokaryotes and. eukaryotic genes within XLUYVEROMYGES as a host; Preferably using a plasmid vector of one of the above-described types Suitable prokaryotic genes useful in accordance with the invention are, for example, lactase, α-amylase, amylase and amyloglycase. Suitable eukaryotic genes according to the invention are

P; lactase, chymosin, invertase and interferon. For import of the coders. products at appropriate locations (suspect i

; exist on the cargo as described above.

<img file="GR78852B_D0011.tif" />

14. According to the present invention, prokaryotic and eukaryotic genes can be used / as many homologs as heterologous / * H. to use it advantageously for us to scale production. chemicals, in particular polypeptides. A preferred embodiment of the invention is the preparation of chymosin, a milk clotting enzyme.

- * The choice of vector and regulators for cloning and gene expression in KLUYVER0MYCES species may of course be different depending on the case of the gene used.

Also, the selection of a strain KLUYYEROMY0ES as a host and the conditions of work can be achieved. to differ by analogy, inter alia, between the gene and the vector selected. If conditions and method and selection are met, they can be determined by ordinary 'experimentation.' Variables such as the above are included in the invention.

The invention is further illustrated by way of a detailed description of the formation of clones and imprinting:

a. a homologue of the β-galactosidase (lactase) gene in K. LACT1S b. a prokaryotic heterologous gene, Kai, toc, LACT1S and

K. ERAG1L1S, '

c. a eukaryotic heterologous gene in K. LACT1S, or TRP1,

L. a eukaryotic heterologous gene, U3U2 by K. ERAGIL1S. . a eukaryotic heterologous gene encoding pro-prokymosin and these natural forms in K. lact1S and

g. a eukaryotic heterologous gene encoding the pro-lethal and both natural and modified forms. This is Mr LACT1S.

The following examples illustrate some. forms of the present invention.

Pzb-

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Example 1i: * BTY75-BAC4 Recombinant Plasmid j: ..: c. -., - ·, ·,. .. · / - ', ·&gt;. . · Srn · '' ·, - .., ...- • sq.m ·,? 0.5 µg of plasmid HK 16 described by R. DICKSON (GENE, 10 (1980) 347-556) and 0.5 pG of plasmid PTY 75 Described by CP HOLLENBERG.uk. (GENE 1 (1976) 33-47), were digested by the restriction enzyme Sal 1. The two digestion products were mixed and after inactivation of the restriction enzyme the solution was quenched with T4-ligase (ligase), a solution of the recombinant DNA obtained.

The coupled mixture was used for transformation, within E. coli L1 strain DG75 (i | g &lt;1 S1 fec-6α-14 galK2 xγ1-5 nGl-1 &lt;gpp2020 hg-1 fSE44-f-lag Z 39). to RC DICKSON et al., CELL 15 (1978) 123-130, thereby obtaining resistant colonies to kanamycin (Ka). And colonies were further selected on plates enriched with ITLNlilAL medium containing lactose as the sole carbon source for formation of lag colonies. Plasmid PTY75-LAC4 was isolated from one of the Kag Lag mutants by the L. kattz et al.

J. BAGTER10L. 114 / (1973) 577-591. .

Example 2

,. * Recombinant plasmids pKARS '. .

G of plasmid YRp7 (STRUHL et al., PROG. NATL. ACAD. SCI. 76 (1979) 103539) were downregulated by Sa1-1 restriction enzyme. 14 pG DNA from the free living. strain of K. LACT1S CBS 2360, were digested with the Xljol enzyme. The plasmid and DNA fragments of K. lact1S were immobilized in a molar ratio. 1: 3. After inactivation of the peripheral enzymes, the solution was concentrated,

DNA of 25 µg / ml and post-T4 - / Igase was lost under standard conditions (30ETR1NGSR). .

Transformation, the E. coli DG75 from the usual mix

J- R. · condition, provided 4.5 x 10 mixture³ AIP (mutant strains), of which any

.. · G.

X 10² content. insert fragments of K. lacs1S, as can be demonstrated from sensitivity to tetracycline. The proportion of tetracycline-sensitive cells can be increased to 85% by post-cycloserine treatment, see BOLIVAR AND K. 3ACHJAN, METHODS IN ETYMOLOGY S3 (1979) 245-267.

According to the method of KATZ et al., (Present example 1); impregnated rafts which are referred to as pKARS 1-14. All had t

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yak-16Ttr * ^ with a frequency of .10-10 **. mw KIA. / Plasmid VKAHSI 2 showed no

-yah- · - ·· '·' · ·^{; ;} · 7 ···. U - '' Higher '' transformation frequency, 3 U. 10; / pG / DNA., but polychmid 'pKARS. appeared more user-friendly during further processing. ; . „· Ϊ́, Received · Unbound_;plasmids may also be transformed within

E. COLI JA221 (Δ TRPE5, LEU 36, lap y, /? Hs &amp; 'V?, K§cL R^- ).

Example 3. &lt;'; .

* PL4 recombinant

A mixture of YHp 7 and K. 1ACT1S DNA fragments was prepared as described in Example 2. The E. 0011 DG75 strain was transformed into the linked mixture and subsequently plated on plates with M9 medium alone. lecine was added. A colonel. They appeared after 8 days at 30 ° C. Plasmid P14 was isolated from one of these LaC '' colonies by the KATZ method et al. (act example 1).

Example 4

KLUYVEROMYCES LACT1S SD69 lac4 transformed to G418^R Lac4 * through the plasmid PTY75-LAC4

Cells of the KLUYVEROMYCES LACT1S SD69 lac4 strain were suspended in growth medium (pH 6.8) containing 1% yeast extract,

2 peptone and 2% glucose. * Growth continued until the exponential phase was reached. of cell growth (3-5X10¹ cells / iii). The yeast cells were harvested by centrifugation, washed with water, and suspended in a solution (pH 8.0) containing 1.2 M sorbitol, 25 ml / mTT® and 0.2 M freshly prepared mercaptoethanol.

After·^;less than 10 minutes. 30 minutes^The0 The cells were centrifuged, washed twice with a 1.2 M sorbitol solution, and suspended in a solution (20 mL) (pH 5.8) containing 1.2 M sorbitol, 10, EDTA, 0.1H sodium citrate and 10 Mg of propellant; They were centrifuged for 20 minutes, washed three times with 1.2 M sorbitol and resuspended to concentration; about 5 · 10 cells / SD, in 0.1 ml of a solution containing 10 mM calcium, calcium and 1 x 2 M sorbitol. , pG PTY75-LAC4, were added and the mixture was quenched for 15 min, then 0.5 mL of a solution (pH 7.5) containing 10 mM CaCl2 and 20% (W / V) polyethylene glycol 4000 was then added;

<img file="GR78852B_D0014.tif" />

The pellets were centrifuged and resuspended again. . : 'Z' · Abs. 'A' to a concentration of about 5.1 ° C^Huh . protoplast i / l, in solution (pH 6.8) containing 7 mM CaClg, 1.2 M sorbitol, 0.5 Mg / ta yeast extract, 1

-peptone and 2 MG / kg glucose.

After 60 min at 60 ° C, the prototypes were centrifuged, washed with KCl O.6K solution and suspended in 0.6 K KCl solution.

To enable the selection of G418-resistant mutants, 1.10 g protoplasts were coated on 3% agar surface, 2% agarose containing 2% glucose, 1.2 g / l sorbitol and 0.2 luG / kg 18 g / kg. To achieve or simultaneously select Lac mutants, 5 · 1i protoplasts were coated on a 3% agar surface, 2% agar substrate (EHIHMAIi), (B1PC0, YEAST - NITROGEN BASE Iq. 2% by weight). and 0.6K KC1 instead of 1.2L of sorbitol.

The colonies appeared within 4-5 days. * Sorbitol plates without G418 or protoplast formation were usually 0.2-0.5%, whereas for plates with C.SH; K31 reduced with glucose as carbon source and / or its ratio increased to 0.5 - 1.5%; 418 was used in the selection, a mutant strain of 10 was obtained.⁽ regenerated protoplasts. * Simultaneous selection on lactose plates provided 10 mutant cells over 10 * regenerated protoplasts or mutant DBA plasmid.

* In the presence of BT75-1AC4 in yeast cells, it could be improved by the SOUTKERH hybridization method, with²P-labeled pCR1.

The BHA preparations were prepared according to the STRUHL et al. (Proc. HAIL. ACAB. SCI. 76 (1979) 1035-1039).

/ Example 5 .ICLUrTEROHYCES. -LACTIS SB11 1αα4 TRP1, mutated TPP * for 1,. plasmid -pKARSl2.

E. LACTIS SD11 strain cells were transformed to Example 4 for 10 G BHA pKARS12. The mutants were selected in triplicate; 2% hippikab agar containing 2% glucose and Ooil KC1. 'Receive &amp; nbsp; &lt;3 * 4X10 TRP' '' mutations with pKAESl2 BHA.

/ * $ + f + C fa;

«\

- I.

-18Example ^ d ¹

KLUYVEROUYCSBS LACT1S SD69 1x4 ~ transformed-plasmid children Plasmid. EL4 ::¹'.

The strain K. LACT1S. SD69, 1x4, was formed from plasmid E4 'by the method of description and di. PTY75-LAC4 in Example 4.

Mutations: selected, on plates with 2% lactic acid (D1PC0) basic agar plates. The frequency of mutations was 20 mutations. of MS plasmid DNA. Examples-7-15

KLUYVEROMYCES: LACT1S SD69 1α4 transformed Itr ”with KARS-type plasmids.

Depending on the method described in Example 5, they were searched. KARS type plasmid transformation plates._;The results of the experiments are summarized in the table below.

^: : US

<td>Delivery '</td><td>Stem</td><td colspan="2">Genotype</td><td>Plasmid</td><td>Mutations with no DHA</td><td>Hego-c bleed (Kb</td>

<td> 4.</td><td>SD69</td><td>1oq4</td><td></td><td>PTY75-LAC4</td><td> . 20</td><td></td>

<td> 7. :</td><td>SD11</td><td>1a4</td><td>itpi</td><td>'pKARS1</td><td>1.5 X10³</td><td> 2.24</td>

<td> 8.</td><td>Yi</td><td>II</td><td> «1</td><td>^: pKARS2 ^:</td><td>5X1O⁵</td><td> 1.24</td>

<td> 9... </td><td>II</td><td>II</td><td>^11;</td><td>pKARS7 </td><td> 10³</td><td> 2.5</td>

<td> 10.</td><td>. THE</td><td>It</td><td>II</td><td>P-KARS8</td><td>5X10³</td><td> 1.85</td>

<td> 11.</td><td>II</td><td>II</td><td>II.</td><td>pKARSIO</td><td>2.4X10⁴</td><td> 5.15</td>

<td> 5.</td><td>II</td><td>II</td><td>II</td><td>pKARS12</td><td>5.4X1O⁴</td><td> 5.0</td>

<td> 12.</td><td> «</td><td>II.</td><td>II</td><td>pKARS13</td><td>- 1.5X10⁴</td><td> 2.0</td>

<td> 13. .</td><td>II</td><td>II</td><td>Pi</td><td>pKARS14</td><td>1.8X1Q⁴</td><td> 2.15</td>

The molecular weights of the pKARS plasmids were determined by digestion with the ECo R1 and Hindu intronocytes. 111, 0.8% agarose gel used and significant molecular weight markers .

Example.14

KLUYVER0I5YCES LA0T1S SD11 1β4 Transformed transcripts were transduced with plasmids containing the KARS-2 sequence, using a cell-transfection pathway.

Plasmid pREK2-7 was used as a wild-type transformant. ^: 3'ⁿ',, *, ~ I .15 *

SD 11. This plasmid is one of the well-known plasmid 1 whose clone is formed by the 1.2 Kb fragment: / ^; having = = - 14-19. The K. lactis SD11 strain did not grow overnight 30 ° C in 1% yeast medium, 2 / l peptone and 2 / l glucose (pH 5.3) 4 Cells were harvested at room temperature.₁4-8 minutes

·. ·. same, centrifuge. you have 1000 cj. for 5 minutes. The cells were then washed with TE buffer (10 mM TR1S-HCl, 1 mM EFTA, pH 8.0) and the pellet was resuspended in TE buffer at a concentration of 2 X 10 cells / fflL. This suspension was diluted with a volume of 0.2 L LiCl and incubated at 30 ° C for 60 min. Then, plasmid pEK2-7 DNA (10 µL) was added with 0.1 ml of lysed cells and 30 cells. ° C for 30 minutes. A volume of 70% polyethylene glycol 7000 was added and the mixture was stirred for another 60 minutes at 30 ° C. The mixture was heated at 42 ° C for 5 minutes, and the cells were washed with sterile, deionized water. The cells were plated on MINIMAL agar plates containing 2% glucose and 0.67 / g yeast-nitrogen base.

The mutations were observed after 36-48 h at 30 ° C;

Example 15. KLUY7ER014YCES ERAG1L1S plasmid-transformed KARS-2 series

Two types of plasmids were used to transform K. PRAG1L1S.

The first plasmid, pGB 180, was constructed by cloning with 3.5 IOb Bgl 11 fragments of plasmid pEK2-7 (Fig. 2) containing KARS-2 autonomously rearranged from K. lactis and with the S. trp1 gene. CEREV1S1AE within the Ba) H1 site of plasmid pJBB 207 (JD BEGGS, ALFREI3D BEZZON SYMPOSIUM J6 (1981) 383).

Approximately 36 mutations in K. PRAG1L1S leu, obtained after treatment with UV in K. PRAG1L1S, were transformed with pGB by the LE * treatment method, - as described in Example 14. One mutant, K. leu1, 'transformed to Leu' with a frequency of about 10 mutations per | JG plasmid D1TA The second plasmid pGB 181 was constructed. by cloning the 3.5 lib fragment of plasmid pRK2-7, as described above. Within the BamHI site of the well-known plasmid pA0Y0184, which contains the Tn6O1 transporter, it provides resistance to the conamycin and the derivative of "fiber" * - - | ¹ WNP ·&gt; \ &lt;· ~

The strain K. rKASlllS C12 was transformed with a plasmid cast XIII; lyjygl. Treatment with IL * is described in Example 14. Tam, Ash, Tissue-cells were transfected with 50 µg G418 / 1H: The mutations ... c, were obtained. after suppression for 48 hours, a highly resistant mutant strain was obtained after 6 days. Of the mutations in K. PRAG1L1S, extract. ENA cells and were transformed into appropriate E cells. COLI DG75. BPA extracted from E. cells. Kanamycin resistant col1 showed the presence of plasmid pGB 181.; .

Experiments have shown that K. fragilis strains can be transformed into plasmids containing the KARS series and that plasmids are automatically reproduced within K. fragilis. . ;

Example 16 * 0 KLUYVEROKYCES LACT1S SD11 la (j4 Itri, which encodes the pro-prochymosin and its various mature forms, after transformation by plasmids containing the KARS-2 codon sequence, the codons these and various motifs directing the synthesis of the structural genes in question.

This example includes a number of stages, most of which are:

1. Addition of the Sal1 ligands to the front of the transcribed structural genes encoding pro-prochymosin, prochymosin, pseudohymosin.

and juicy. .------- '.

'2. Introduction, of a portion of DHA into plasmids obtained above and containing the autonomously-replicating KARS-2 sequence, was obtained from K. iiACTlS and the TRP.1 gene from S. cerey1S1AE.

3. Introduction of various sub-motifs into the above-obtained plasmids, which direct the synthesis of the various mature forms of the pre-pro-prokosmin. stacked&gt; »single genes

-Methionyl-octahydroquinoline, described as pUR 1531

- Ketionyl chymosine, hieroglyphic as pUR 1522

- Phthionyl-prochymoamine, described as pUR 1525

- N -propylene-sulfuric keto-sulfur, described as. p'JR 1524.

<img file="GR78852B_D0015.tif" />

-21- * The construction and syntax of these plasmids has been described in detail in European Induction. No. 82201272.0, published April 20, 1983 under the serial number 0077109. The genes were isolated and the plasmids were constructed according to the specification in question.

D. Introduction of Sal 1 ligands into plasmids pUR 1531, pUR 1522, pUR 1523 and pUR 1524 (ΐ)

Plasmids pUR 1531 ,. pUR 1522, pUR 1523 and pUR 1524, contain an ECo R1 restriction site, precisely at the front of the ATG start codon. Because one additional site restriction ECo R1 exists within the chymosin gene, the insertion or binding of the Sa1 1 binding molecule precedes the first site of Eqo R1 to facilitate insertion of the various subunits. .

Approximately 50 µg LIAA were knocked down after 50 ECo R1 endonuclease units in the presence of Ί25 µg / ml ethidibbromide. within 10 ml of TR1S-HC1. 50 ml / ml, 6 ml of β-mercaptoethanol, 10 ml of magnesium chloride and 100 µl / ml of bovine albumin, pH 7.5 at 37 ° C, for 60 minutes. The plasmid was converted mainly to linear and open-loop molecules in the tau condition. The DNA was extracted with one step of phenol and one step of chloroform and precipitated with one step of propanol-2.

The RNA was passed into TE-buffer and completely digested with Salon endonuclease I. A portion of the DIIA of approximately 1800 Bqp was isolated by electroelution agarose gel. . · / '; '

The fractions were extracted with phenol and chloroform and precipitated by

2-propanol. Ta. precipitates were dissolved in buffer-TE.

The sequences were then sequenced (filled in between these pools) by ONE polymerase as follows:

• In 15 µl (solution) containing: SNA fraction 1800 Ip (about 1-2 µL), 1 µL of 21 µl dATP, dGTP, d.CTP and dTTP solution was added. 6.5 HI KICK 4x Buffers Containing 0.-2C TR1S-HC1 (PH 7.2), 40 ml M Magnesium Sulfate, 4 ml L-Dithiobutyrate .200 LIG / l, bovine serum albumin and 2.5 µl of water. DNA polymerase '(KLENOW fraction) was added and the mixture was lost at 20 ° C and inactivated ENA polymerase by heating at 70 ° C for 5 min.

<img file="GR78852B_D0016.tif" />

WJ. -c * - *

-22 ···,;. '·',. '·,! · ':&gt; ·. ···, · · ..j,'. M,. . ·, C., ·. . r-; ·. ., ..Y __ * · ν 7 · ', * * f e&gt; S,. · - «·« (»Λ' ·¹* .'-- x ·, -: ·:., L * - .7 ·¹' -7' '7 ·:3-'&lt;·/» _r¹. ' , "An phosphorylated Sa1 1 ligand (prepared as described;^:ii. MAKPATIs et al., MOLECULARCLOYING, CSH). (Did you mix this one again?).

T4 DNA ligase (10¾ units). Then quench - 22 ° C for 4 hours the mixture^: lost at 4 ° C for 16 more hours. Then the mixture was lost to the Sai l and Hiia intra-nuclei. Ill and in a BHA fraction of about 1500 gp were obtained by electroelution agar gel.

The fractions (A, · B, C, D ') were purified by phenol and extraction with chloroform, and by precipitation with 2-propanol. The fragments were then ligated to a 3.3 Kb fragment of Ill-Sa1 1 (approximately 0.5 pG), - ex. From plasmid pPAL55-209 containing a thermosensitive replication unit and an antisense gene (ampicillin). and the product was purified by agarose gel and electroelution. '.

The connected ones. molecules were transformed within. E. coli HB 101 and the ampicillin resistant, tetracycline sensitive, tetracycline-resistant clones, etc., were also extracted -BIA of the plasmid. Pepsi: ·: /; /, \ '-? DBA of the plasmid by endonucleases Sal 1, ECo R1 and Hind. Ill, confirmed the reception of plasmids pGB131, pGBl22, pGBl23, pGB124 (Fig. 1).

Preparation of KARS2 and TRP1 Ion I Plasmids pG3131, pGB122, 'pG3123 and pGB124, respectively. .

It is legally reproduced, strings derived from and reproduced within KLUY-,

VEROHYCES, were obtained as described in Examples 2 and 7-15 '. * The self-replicating sequence in plasmid VKAK8-2 is present in a fraction of 1Y 24 Kb and this fraction v / v; ., .... clone the well known plasmid YRp7 '^L. and a new plasmid pREK2-7 was obtained (Fig. 2). Digestion of pREK2-7 by a Clon 1 isonuclease resulted in the acquisition of 3 · 5 and 5 · 5 Kb fragments, respectively;

The 3.5 Kb fragment containing the TRP1 gene was derived from S. CEREV1S1AE kVX or KARS ^ 2 sequence from K. lact1S (Fig. 2) and was then isolated by agarose gel electrophoresis and Cl-1 conjugates. pGB131, pGB122, pG3123 and pG3l24, respectively. The resulting methylmercury azimuth was formed within E. coli '3FA3OO (inert) and the characterization of IgG &amp; Mn' kagit '! Ironi? flush /; $ C &amp;^;* ··; Species: pG3151, pGB152, pGB153 and pGBl54 respectively (fig. 2).

-the 'extracted. of Ttr mutations, identify it'

, - '. ; . '' ·.-23C. Introduction ... of various promoter sequences within the plasmids, which guide the synthesis of the various mature forms of pro-prokymosin /

The 3a1 1 fallen plasmids containing the KARS-2 sequence and the TRP1 gene as well as the structural gene of pre-prochymosin or its various mature forms are well suited for their receptor binding to Sa1 cells. of the synthesis of the longest (longest-lived) structural gene of the K. lact1S mutation. ~

In most cases the displacement sequences must be provided with connector 3a1 1. Any displacement sequence may be of this kind, i.e. 3a1 1, and the following examples are illustrated below:

1. isocytochromic motility έκ 1 cm from S. CEREV1S1AE

2. the lactase promoter by K. LACT1S.

C1-.

: Adding Sa1 ligands 1 to the isocyte promoter q! from there

S. CEESV1S1AE and in: plasmid in situ (Fig. 3)

Plasmid pYeCYCl consisting of the isocytochromal G1 gene, inserted as a clone into plasmid U pBR322; used as a primer (G. PAYE et al., USA 78: PROG. SCI. NATL: 1981). .2258).

From the information of the nucleotide sequence it is known that within the isocytoma gene q! at the +8 nucleotide, there is a position -ECo R1

The plasmid pYuClU was cleaved with ECo R1 hydonuclease, ligated with T4DNA ligase and transformed. within E. coli HB1O1, to obtain plasmid pC1 5, containing, in 1930 Ip fragment, carrying the promoter and 8 nucleotides of the same gene; of; isocyte Ql · ''

The pC15 plasmid was cleaved by the Eco hclononuclease, and then was cleaved with a Ba1 nucleus 31 min shortly to remove a few single nucleotides. ·.

The Ba1 bottom 31 fallen edges were converted to an outer membrane embolism

(KLEHOT fraction) and in an ECo R1 phosphorylated ligand /

DHA this. After reduction with ECo R1 eukonucleoside, binding and / or Btm2:

<img file="GR78852B_D0017.tif" />

in E. 00L1, a pC15-R12 mutant was detected in which L2 from the cytochrome C1 gene had been removed.

_T.24 'An Sa1 1-linker was introduced by cleavage of plasmid p0.15 -Ri2 / u.iiij-endonuclease ECo R1, complementation of consecutive ends by DBA polymerase, binding of a phosphorylated linker of Sal'1 each 'Cloning the resulting 1070 bp fragment' into the subunit of Sal 1 Falling Plasmids p © 1517 pGBT52, pGBl53 and pGB154, respectively, obtained herein from the cytochrome c subunit PGB162 pGBl63 and pCBΐ64, respectively, were detected by colony hybridization with ³²P-labeled 1070, 3rd fraction as probe. . '

BBA plasmid was prepared from positive clones and correct isocytochemical orientation &lt;31 was ascertained from herein. of a fraction of 850 gpp, digested with Srna 1 endonuclease.

&lt;32 · Adding Sa1 1 ligands to the lactase promoter from KLUYVEROLTYCES LACT1S and inserting into plasmids' starting material; (RC B1CKS01I Al © JS KARKLH, CEIL 15 (1978) 123). - * The sequencing of large fragments of the lactase structural gene and its motif, identified 7tapouoiav of a Cla site. about 450 rpm ///////// 0) within the lactase structural gene.

The plasmid pK1s was digested by endonuclease Cla 1 and the fragment containing the promoter and approximately 450 rpm of the structural gene were re-cloned into plasmid pBR322, each with Cl. and Aqo 1 (partially). In a plasmid, pGB 182, or at a Cla 1 restriction site of approximately 450 kb of the lactase structural gene, was cloned by endonuclease Cla 1 and reduced to the required size by clot 31; BAL 31 became blunted by BBA polymerase deprivation and this purified fragment was bound to an ECi R1 ligand.

Peptide after ECoRl endonuclease and cloning of the cysteine ​​residues.

fraction resulted from the reception of plasmid pGB 183 u / e ligand- * * 'A ~^: lactase promoter inserted and structural parental '-' ** jSTeK '”removed

-25-g -, &lt;1 h '-.- 77

7-7

In this fraction added was added 8α1 Equi., Described in an exemplified example (16.C2), '0 on the Sa1 I' bound lactase-affinity promoter. · To / from Sort 1 cleaved plasmids pGB 151, p © 152, p © 153, and pGB154, respectively. be taken correspondingly. new plasmids p © 171, p © 172, pGB 473 and p © 174. //.

The plasmids obtained as described in the present example 16 were inserted into the KLUYVEROHYCES LACT1S SD11 1a4 Itri by the method of Li * as described in example 14, selected for the Ttr mutations.

The presence of pre-pro-prozymosin or the mature forms; these KLUYVEROEYCES extracts, were demonstrated by EL1SA immunoassay and by staining part of the extracts on the Nitrocellulose AB filter media P. COTOAIT (PROC. BAIL.ACAD. SCI. USA 78 (1981) 4520-4524).

The cell extracts were prepared as follows: K. lact1S mutants were grown at 30 ° C for 16-24 h in YBB medium containing 2x dextrose. Cells were harvested at 0B61C between 2.2 and 6.0 by centrifugation at 6,000 rpm, for 10 minutes with a SORVALL G-S3 centrifuge.

. The pellet was suspended in sterile-distilled water for up to 0D600 h.

600 and it was chilled with ice. '0.5 ml of the suspension of these cells' was diluted with 0.5 ml of ice-cold water and the solution was mixed. after 2 G of beads 3ALL0T1N1 '(diameter 0.25 -0.35 ME' BRAUH-HELSUBGEB .'GMBH, GPR). '

The cells were then shaken by shaking for 4 minutes on immobilized VORTEX at high speed. More than 95% of the cells were fractured, as observed by a microscope under a phase-contrast field microscope. The fragments of the cells were removed by centrifugation for 1 min in a centrifuge. EPPEMDORE apparatus. Portions of the extracts were frozen in liquid nitrogen and maintained at room temperature. -80 ° C.

Sections 1-5 of the extracts of the cells were cultured as nitrocellulose filter membranes. The filters have dried up.

192 1 ml of glycine, 25 g of THIS, 20% ethanol (pH 8.3) were added at 22 ° C.

<img file="GR78852B_D0018.tif" />

r * ·&gt;.

-r «3-26- SS / SS -77 -: - 7 7. 7.7T &lt;

'Afterwards the filters were lost with buffer ^' ''

NaCl, 10 mM TR1S-HC1 (H * 7.6), 2% bovine albumin / strain). 30 minutes. ,; '7; / * ·; filters were washed&gt; three times for 5 minutes with RiA buffer (0.125 M NaCl, 10 mM).

TR1S-HCl, pH 7.6, 0.1 n H 1 H PKSP, .1% TRITON^:X 100,. 0.5% sodium desoxicholate,. '0.1% dodecyl ether sodium and' 0.3% gelatin). Overnight filters were incubated overnight at 4 ° C in 10.1 g, R1A containing 10 µg of chymosin staining. * 0 anthracite .. removed by. wash-in rate R1A (three times) and lost after 1 min

125 L of Protein A in 1 L, buffer R1A, at 60 ° C at 22 ° C.

· A was removed by washing with buffer. R1A (5-fold).

The filters were dried and self-radiographed overnight. There was no clear evidence of the presence of pre-prochymosin or these mature forms of K. lact1S mutations.

The presence of chymosin in K. lact1S cell extracts (mutants) was determined. by high performance liquid chromatography (EPIC) as described by ACH H00YD0EK and C. 0L1SLAN, NETHERL. KLLK DAIRY 36 (1982) 153.

5Q of an enzyme extract or extract were added to 1 ml of a 10% milk fat solution (D1PC0) in 10 ml of M CotOlg. The solution was lost for 15 minutes

31 ° C. The reaction was interrupted by the addition of 2.MU 12% trichloroacetic acid (TCA).

Nearly α1 proteins were precipitated under TCA except_C.glycomacropeptide (GMP) which had been cleaved by the effect of chymosin on it. casein. ··. .; ,

Deny yourself, protein. are pelleted by centrifugation and 1 pg. besides. The clear supernatant was neutralized by the addition of 0.4 Ml of 1N NaOH.

The solution was centrifuged. and again or the quantity produced. SIP was detected for 7 / HELC by monitoring the damping at 214 nm. .

Extracts of K. lact1S mutants containing prohymosin were first suppressed in 7 pH 2 eps. 2 hours and then neutralized by performing the chymosin activity assay. * Chymosin was only found after treatment at pH 2. '

Example 17

KLUYYEROMYCSBS SD11 1 'q4 ρtrI, which encodes prothymatine of certain forms, after transformation with the plasmid pURI, the structural gene encoding pro-proctrautamine.

i '' 7-

<img file="GR78852B_D0019.tif" />

LACT13. ir.'fn.'vXAoHAn - a -. 'v_:vr. · n- ·· ^ ι.ΐ! ττ &lt;· AeiV

-27- ·. ;

= Example 'this. comprises a number of stages, most of which are:

Isolation of clones containing the glyceraldehyde-3-phosphate dehydrogenase coefficient of S. CEREV1S1AE (GAPDH)

DNA content of S. cerevisiae was prepared in the yeast EwCOLl-yeast plasmid pP1 '(ii.R. CHEVALL1ER et al., GEHE 11 (1980) 11-19) by a method similar to that described by M. CARLSON Al. ® D. 30TSTE1H, SELL 28 (1982) 145-154.

The purified yeast DHA was partially digested with Sau 3A restriction endonuclease and the obtained DEA fractions (average 5 kb long), conjugated with T4 DEA ligase at the downstream phosphorylated Ca 1+ site. of E. col1 cells with the associated material, containing&gt; 30,000 ampicillin resistant clones. They were infected by the hybridization process of the colony (RE

THAYER, AHAL. B10CHEK. 93 (1970) 60-63), with a chemically bonded and^&gt;2P-labeled oligomer, with 5'TACCAGGAGACCAACTT3 'sequence;

According to the data published by JP HOLLAED and LI.J. HOLLAED (J. BIOL. CHECI., 255 (1980) 2596-2605) this oligomer was complementary to the DEA sequence of coding amino acids 306-310 (or unstable base of the last amino acid, was omitted at half-time). Through the hybridization conditions of RB WALLACE et al., EUCLE1C ACID RES., 9 0981) 879-S94, • 6 positive mutations were detected or detected. Six of these contained plasmid pP1 1-33. The last plasmid contained the GAPDH gene including its promoter / regulatory region and its transcription-polyadenylation domain. In the posterior length, about 9 Kb, gpPI 1-33 has been characterized by restriction enzymes analysis (Fig. 4) and partial nucleotide sequence analysis (Figs. 5 and 6).

2. . Isolation of the GAPDH Susceptibility / Regulatory Region and Insertion of It into Plasmaproteumatine * Based on Enzyme Restriction Analysis and Their Nuclide53 Sequence. Isolated / | 5 ^ C ~ regulating - inserting / regulating the DEA of the GAPDH gene, as k, v, Z Z '··· -, X r- Z z ~. z I i £ M. r nok / ne, y, y, ie Dae 1. Who's t-pumping

XN&gt; '-28ΐ .......-; ··. ·, --- 5 / ---' .'9 '^r · Synthetic-oligomer (SiMi / SOUTHERE, J. EOL. BIOL. 98 (1975) 503-517) ··· · ///, gg * 7 7 .ΐ / ;. · ".^:·/ ,// · 7-.7.//./_

A 4 * 3'Kb hybridization restriction fragment was isolated, over-scaled, electroelectric, 0.7% agarose gel and subsequently cleaved with Dd-ei. Of the fractions obtained, &amp; lt; 1 only the largest of these had a recognition site for Pvu, 11-, a cleavage site located within the GADPH co-region (Fig. 1). Larger fraction 1 was isolated and lost by LNA polymerase-KLEK0W and four d. IFR (AR DAVIS et al., GEHE 10 (ΐ93a) 205-218) to generate a D1IA blunt-ended molecule. Keta extract of the mixture by means of a flask / chloroform (50/50 V / V), passing the aqueous layer through a SEPilADEX G50 column, and ethanol precipitation of the material A in the vacuum column, 5'3GAATTCC3 * β Epo 51 labeled with P, by incubation with T4 D5A ligase. Evidence of reaction with KLEiJQW polymerase. and the subsequent coupling to the ECo'Rl connector, and the starting positions 1 &amp; e, were reconstructed at the end of the fraction fraction. Prior to inactivation of the ligase, the reaction mixture was heated to 65 ° C for 10 minutes, then sodium chloride (final concentration 50'MOL / liter) was added and the resulting mixture was quenched with ECo R1. The reduction was terminated: by phenol / chloroform extraction, D11A was ethanol-precipitated; * it was suspended again and then coupled to the appropriate carrier molecule. As soon as the coefficient of cDNA is provided by the ECo R1 sites, it can now be easily inserted into the EGo R1 site of pUR 528 (EP-PA 54331) to generate a plasmid at any time. a structural gene encoding pro-proauthumatin. The final plasmid was obtained by cleavage of pIK 523 by ECo R1, treatment of the phosphatase-aligned plasmid (six bovine intestine) to inhibit the autoclaving and degradation of each of the four D-like molecules. 'Transforming the various epoxy bonding mixtures.' E. COL1 EB10.1 cell treatment provided several colony-resistant ampicillin. Of these colonies, they plated the DHA plasmid (HC BlHEBOUi ACIDS RES. 7 (1973) 1513-1523) and lost it with Pvu, 11 to six insertion inserts.

<img file="GR78852B_D0020.tif" />

f 5l ·

Whis^t Nomenclature does not contain: ^;BC .Replicable BCo Ri (Dtie ΐ) GADPH in the correct, orientation (i.e., transfer out of the GAD? H region takes place against, the address of, the downstream structural gene) 01 in the original code of the plasmid (e.g. pUR 528, modified to pUR 526-01, oper. Fig. 7).

To facilitate manipulation of plasmids containing the coefficient of BCo R1, or one of the two BCo R1 sites, destroyed, 2 µg of plasmid INA (e.g., pUR 528-01) were partially digested with Eco R1. and then the product was lost after 5 units of IIx cj. nuclease (obtained. of PL B10CKEK1CALS INC.) Under a total volume of 200 µl, in the presence of 0.05 Wl / liter sodium acetate (PL 5; i), 0.05 Eq / liter sodium chloride or 0.001 HOL / liter zinc chloride, at 30 min. room to remove the welded edges. Clonuclease was inactivated by addition of SDS to a final concentration of 0.1% (D. KOWALSKI et al., B1CCHED51STRY 15: (1976) 4457-4463 and BPA was precipitated by the addition of 2-volumes of ethanol (in this case, the addition or addition of 0.1 volume 3. HCl / liter acetate). The aligned DNA molecules were re-injected with T4 DNA ligase and used for cell transformation. CaClg-treated COL1 · plasmid RTA isolated from them. Ampicillin resistant colonies, increased by dissociation after BCo.Rl and one. with respect to the presence of a single ECo R1 locus L-thiomatin parent (Fig. i). “

Plasmids containing the GAGPH fragment were only one site unknown; of ECo. R1 near the ATG start codec located below. of the structural gene are referred to as plasmids of type -02 (e.g., pUR 528-01.

. converted to pUR 528-02, act, min. 7) ·

3. Reconstruction. of the GADBH origin of the downstream / downstream region within the plasmids encoding pro-pro-proAutamine by introducing a synthetic fragment; (i) As shown by the nucleotide sequence shown in Fig. 1, i.^-. 5, the GAEPH ECo cellular subunit, R1 (With ΐ), contains the nucleotide &lt;7;

-650 to -39 of the 'original' GABPR stimulation / regulation region. In the present section, this promoter is the 39 nucleotides of the ATG inducer of the GAHPII coding gene.

-V?

«-30- λ '·&gt;: ·' 7-c

The last 38-nucleotide long fragment contains the sequence PeCACACA .. found in several yeast genes. Said sequence of PuSACACA located approximately 20 ip above the decoding start site (I3.J.

DOBSON et al., NUCLEIC ACID RES. 22 Monitor⁸²(2625-2637) provides the above nucleotide sequence of the ATG encoded that is or is excellent for initiating protein formation (K. KOZAK, NUCLEIC ACIDS RBS. 9 (l9Sl) 5233-5252). Further, the nucleotides then allow for the formation of a small bronchoid structure (loop) that should be involved in the regulation of GADPH gene expression.

In the light of the foregoing assumptions, or insertion of the 38 nucleotides between the Vahe 1-promoter fragment and the ATG encoded below the structural gene found, it was considered necessary to enhance the activity;

* 2 is illustrated in Figure 9, the DNA fragment removed, obtained by. of the chemical composition of the two partially overlapping oligomers * The SaQ 1 site encoded by the collision portion of the two oligonucleotides was introduced for two reasons:

(i) to allow the nucleotide sequence to be immediately above the ATG coding, including, to construct the poly-A terminated yeast imprinting vectors, (ii) to obtain an enzyme-cleavable cleavage site; can be easily and reproducibly removed by T4 DNA polymerase depletion in the presence of the four dETPs. Equivalent amounts of the two purified oligomers reduced phosphorylation at -5 ', collapsed (JJ ROSS1 et al., J. BIOL. CHEK. 257 (1982) 922 (5-9229) and converted to DNA double strand (helix) by cleavage with DNA polymerase KLEN0W and after four &lt;3ETPs under the conditions described for double strand DNA synthesis (AR DAV1S et al. GESE 22 (1980) 205-218); Analysis of the acrylamide gel electrophoresis reaction products and subsequent autoradiography, showing oligonucleotide primers, were transformed into a helix. DNA was isolated by passage of the reaction mixture / h

SEPHADEX G5O and Ethanol Precipitation Material

<img file="GR78852B_D0021.tif" />

The DMA was then subjected to phosphorylation by suppression, polynucleotide drive, and digestion with 3 d / 1 to remove the split second nucleotide reaction, the two-stranded mixture. .

In Figure 8, the clone formation of the obtained synthetic DMAs was performed by simultaneously binding this fragment to a GAJ3DH B1 -111-I-E1 subunit-regulated fragment within the molecule. or ECo R1 site or existing thereto. ATG-starting conicers were removed by digestion with 1IUMG nuclease ((E.). The 'Bjlll-Bae' promoter / regulatory fragment was obtained by digesting plasmid pUR 528-02 and DcLel₃111. . Separation of the obtained limiting fractions, by electrophoresis in $ 2. of agarose gel and subsequent isolation of the gel fragment to provide the 793 nucleotide clear / regulatory fragment.

In plasmid pUR 528-02 or, the nucleotide sequence encoding the ATG encoder, is 5'-GAATT0 (T) ATG-3 '(DP-PA 54330 and EP-PA 54331), which is different from probiotic. given by H. KOZAK (KUCLEIC ACIDS RES. 9 (1981) 5235-5252). If the purpose was or did not constitute the original GAPDH region of subunit / regulation / initiation of protein formation, with as high precision as possible, or the position of ECo R1 was abolished in association with the terminal D synthase. IT removal of the ECo R1 site was achieved by digestion with the MJ3JG nuclease of EGo R1-cleaved DMA / pUR.528-02.

The plasmid was then digested with .bg.Hl and lost after phosphatase. After separation of the two DMA fractions by electrophoresis mes. 0.7? » of the agarose gel, the largest fraction was isolated and used as the carrier to which the Bcpll -Ddel excitation fraction and the -Ddsl.-treated DMA synthetic fraction were attached.

The plasmids in which they are inserted: the fraction&gt; 1e1 promoter / regulatory, but also ~; The synthetic DMA fragment containing the Sac I recognition site is indicated by concentration -03 (e.g., pMMz2? ~ 02mM; &lt;p&gt; 528-03). ///

<img file="GR78852B_D0022.tif" />

-32Z - \ »4 · Enter what, what to write? KARS2 from KiLACTlS and the TRP1 gene.

/ S. CEREVISIAS, within plasmids; pro-proauthumatin coders /

The KARS2 copy and the TRP1 gene were cleaved from pER 2-7 · .// :: by digestion with Bg111, followed by 0.7% agarose isolation; g. '' 'Z Fraction 3.5 Kb. This purified fragment was inserted with the phosphorylated thiol Bcp 11 cleavage site pUR 528-03 by suppression with T4 DMA. ligase.

Transformation of the ligation mixture into E. coli, provided the plasmid pURK 528-03 (Fig. 10D). The mutations generated by the insertion of the plasmid pURK 528-03 into Ki LACT1S SD41 cells, processed by the Li method, were shown to synthesize a thiamine-like protein identified as ED 18. 1982) 1-12, op. 11.