DE3347772C2 - Process for producing a polypeptide - Google Patents

Abstract

The invention relates to a method for the production of a polypeptide using an expression vector with a DNA fragment which contains a gene coding for a phosphate binding protein and a replicon from the group of plasmids and bacteriophages, wherein the DNA fragment is ligated to the replicon and wherein the The following procedural steps are observed: ligating a DNA fragment leading to a gene coding for a polypeptide to the expression vector on its section located below the DNA sequence of a promoter for the gene coding for a phosphate binding protein transforming the cells of a bacterium from the Enterobacteriaceae family with the expression vector , to which the gene coding for a polypeptide is ligated, into transformation products - reading out the transformation products from stem cells of a bacterium from the Enterobacteriaceae family - incubating the transformation products, with the transformation products expressing sion of the gene coding for the polypeptide to be caused to form the polypeptide and - isolating the polypeptide from the incubated transformation products.

Description

The invention relates to a method for producing a polypeptide using an expression vector with a DNA fragment which encodes a gene for a phosphate binding protein and contains a replicon from the group of plasmids and bacteriophages.

The term “gene expression” here means translation from a gene coding for a polypeptide sequence understood in the polypeptide. In gene expression, the coding in a DNA chain for the

nucleic acid transcribed into a so-called messenger RNA, and then the translation of the so transcribed messenger RNA into the aforementioned polypeptide instead.

1970 the technique of transforming Escherichia coli (M. Mandel and A. Higa, Journal of Molecular Biology, 53, 159-162 [1970]) and found a restriction enzyme (H. O. Smith and K.W. Wilcox. Journal of Molecular Biology, 51,379-391 [1970]). These Findings led to rapid progress in genetic engineering and cell technology. For example The basic technique of genre combination was developed on the basis of this knowledge, as described in US Pat. No. 4,237,224. In addition, new vectors and methods have been developed for the production of beneficial substances with the help of genetic engineering. The aforementioned new vectors are for example in DT-OS 27 12 615. in FR-PS 24 41 659, US-PS 42 73 875, in the PCT application WO 79/01169, GB-PS 20 52 516 and EPC-PS 32 238 are described. The aforementioned procedures for the production of beneficial substances are described, for example, in US Pat. No. 4,375,514.

From all of the above publications, however, it only emerges that the desired beneficial substances, z. B. polypeptides, can be produced with the aid of gene expression. The techniques used in the Cited publications of the prior art are described, however, have the disadvantage that the Yield of the desired useful substances is not high enough. Because of this, insists on this Field of desire for a method of producing useful polypeptides in high yield.

The basic techniques for genre recombining are outlined in the Proceedings of the National Academy of Science, USA, 70, 3240-3244 (1973) and 71, 1743-1747 (1974), but these publications no general method for isolating a gene that is characterized by a high level of gene expression has not been proposed and is isolated from a large number of naturally occurring genes, from which difficulties arise in solving the stated problem.

Extensive intensive studies and studies to solve this have been carried out on the present invention Problem, with the result that a gene coding for a phosphate binding protein was found which was an expression vector with extremely strong gene expression - compared to the previous ones known vectors - results. Such an expression vector, which encodes a gene for a phosphate binding protein using the cloning technique with great success. The invention is therefore based on the object of a method for producing a polypeptide using to provide an expression vector. The expression vector used is the subject German patent application 33 20 339.3-41.

The object is achieved with the measures of claim 1. Claims 2 and 3 name embodiments the invention.

The expression vector used is prepared by a method comprising the following steps:

1) Obtaining a chromosomal DNA (DNA) with

_Λ : / -> ι j: r. ··. : "Ni u ~ * u: ~ j

v »iii \ * i vj ^ itivuui% .i uiig lui viii ι iiuapiiaiuiiiuuiigapiu *

tein from bacteria of the Enterobacteriaceae family: 2) splitting of the chromosomal DNA into DNA fragments with the aid of a restriction enzyme;
3) Ligate the DNA fragments to a replicon

the group of plasmids and bacteriophages;
4) Transformation of the cells of a bacterium from the Enterobacteriaceae family with the help of the replica

Kons, to which the DNA fragment is ligated, in Transformation products, including transformation products which are a recombination vector with the DNA fragment containing the gene coding for a phosphate binding protein carries with him;

5) Reading out transformation products which encode the recombination vector with the one gene for a DNA fragment containing phosphate binding protein, from the transformation products, and

6) Isolation of the recombination vector with which the gene coding for a phosphate binding protein leading DNA fragment from the read out or selected transformation products.

The method according to the invention is explained in more detail with reference to the following drawings. It shows

F i g. 1 the restriction maps de: ¹ plasmid pSN401. pSN507, pSN508 and pSN53S and

F i g. 2 the flow chart for the manufacturing process of the plasmid pSN2507, with the restriction maps of the plasmids pKN402A, pSHIOl and pSN2507.

In Fig. 1 and 2 is with the symbolic designation »PhoS« is a gene coding for a phosphate binding protein of the Enterobacteriaceae, about which a detailed explanation is given elsewhere. The symbolic names »phoT«, »pstA« and "phoU" each stand for genes that are attached to the chromosomal DNA of E. coli together with phoS are; also, a detailed explanation of this follows later. To identify the recognition sequences at the following symbols are used for restriction enzymes:

EcoRI, Hpal, Pstl, BgIII, MIuI
Hindlll, Aval, Hindi and Smal
(The numbers attached to these symbols are used as shown in the drawing to identify the respective recognition sites for a restriction enzyme).

Further abbreviations:

Tn3 (Ap ^R ) (indicated with V ///// M) ·. Transposon, which contains a gene coding for ampicillin resistance;

Replication gene
Replication origin
1000 nucleotide pairs
erased part

The term “replicon” here denotes a vector that is capable of replication and a gene for a phenotypic one Has a feature and a restriction site that is suitable for attaching a donor DNA fragment is.

A replicon is understood to be a carrier of genetic information, the replica unit of a Nucleotide chain is known, so that at the beginning of a replication process the replication is sequential takes place gradually until the end of the nucleotide sequence. Let us take as examples of such a replicon call themselves plasmids, bacteriophages, viruses and the like. Replicons include DNS replicons and RNA replicons, but practical he

considerations in the present invention RNA replicons not used with preference. DNA replicons can be plasmids and replicable ones Name DNA fragments derived from DNA viruses, as well as procaryotic and eucaryotic cells. Of these, the plasmids and bacteriophages are preferred for use in the present invention Conventionally, widely distributed DNA replicons have been used to clone various genes

ίο used, which carry a gene with them, the resistance to antibiotics. The widely used method of cloning a gene includes Dissolving a chromosomal DNA with a restriction enzyme to produce a DNA fragment, which carries the desired gene, the ligation of the DNA fragment to the DNA replicon, the transformation a unicellular organism with the DNA replicon, which comprises the DNA fragment, into transformation products, the isolation of the transformation products from the unicellular parent organisms below Use the antibiotic resistance conferred by the DNA replicon and incubate the isolated Transformation substances, the DNA replicon being cloned with the DNA fragment.

The gene coding for a phosphate binding protein is responsible for phosphate transport and phosphate metabolism and is present in development in procaryotic cells and unicellular eucaryotic cells. Among other things, the gene coding for a protein for phosphate binding in Escherichia coli has been investigated relatively thoroughly, and in particular the gene in Escherichia coli is called the phoS gene.The phoS gene is not only present in Escherichia coli, but also in bacteria from the Enterobacteriaceae family . Accordingly, the phoS gene of any bacterium belonging to the Enterobacteriaceae family can be used in the present invention. The phoS gene in Escherichia coli is together with phoT, pstA and phoU at 83 minutes on the standardized gene map for E. coli (Backman, BJ and KB Lx) W, Microbiol. Rev, 44, 1-56 [1980]). The genes phoT, pstA and phoU are genes whose function is related to phosphate transport and phosphate metabolism. As already mentioned, the genes phoS, phoT, pstA and phoU are attached in clusters on a chromosome of E. coli, and if now the DNA fragment carrying the phoS gene is extracted from the chromosome DNA of E. coli with the help of a restriction enzyme is obtained, the DNA fragment also contains the phoT, pstA and phoU genes. The gene coding for a phosphate binding protein in / ZgImS phage has also been investigated and can be used in the present invention.
In addition, genes whose function is related to phosphate transport and phosphate metabolism can be used in yeast and a bacterium other than one of the Entet obacteriaceae family. In this case one has to find a replicon and a host suitable for genes whose function is related to phosphate transport and phosphate metabolism in yeast and a bacterium other than one of the Enterobacteriaceae family.
The expression vector used in the present invention is produced by a method in which

1) a chromosomal DNA is produced, which is a gene coding for a protein for phosphate binding

contains manure from a bacterium of the Enterobacteriaceae family;

2) the chromosomal DNA cleaved with a restriction enzyme to generate DNA fragments will;

3) the DNA fragments to a replicon chosen from the group of plasmids and bacteriophages be ligated;

4) cells of a bacterium from the Enterobacteriaceae family transformed with the replicon to which the DNA fragments are ligated to form transformation products, including those that contain a recombination vector, including the DNA fragments, which carry a gene coding for a phosphate binding protein;

5) Transformation products that encode the recombination vector, including a gene coding for DNA fragments carrying a phosphate binding protein, from the total transformation products can be selected, and

6) the recombination vector, including the gene coding for a phosphate binding protein leading DNA fragments, is isolated from the selected transformation products.

In step 1 of the process, bacteria from the Enterobacteriaceae family are prepared using a conventional isolation technology such as lysis, centrifugation or the like to produce a chromosomal DNA, which contains a gene coding for a phosphate binding protein (hereinafter simply referred to as "phoS gene")

In the second step, the chromosomal DNA produced in the first step is treated with a restriction enzyme cleaved, whereby DNA fragments are obtained which contain a DNA fragment (DNA fragment) carrying the phoS gene include.

In the third step of the process, as already mentioned above, every plasmid and every bacteriophage use as a replicon. The connection of the DNA fragments to the replicon can be used carry out a ligase according to the conventional method (see, for example, US Pat. No. 4,237,224). At least one of the DNA fragments carrying a phoS gene and a replicon has a gene for a phenotypic Characteristic.

In the aforementioned fourth step, cells of a bacterium from the Enterobacteriaceae family with the the replicon carrying the DNA fragment, which was previously prepared in step 3, is transformed into Als Bacterium from the Enterobacteriaceae family can be Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Name Salmonella, Serratia and Shigella. In addition to the bacteria mentioned, mutant cells can also be found use all of these bacteria. Of course, the corresponding bacterium must be taken into account the ability of the replicon to replicate in the cells of a bacterium. For example the species Escherichia colL, Serratia or Salmonella can be used as a host in a suitable form, if as a replicon, a well-known relaxation type plasmid such as pBR322, pBR325, pACYA177 or pKN410 is used.

In the aforementioned fifth step, the transformation products are grounded initially from the stem cells of a bacterium from the Enterobacteriaceas family The help of the phenotypic trait isolated, for example by taking advantage of drug resistance, which is mediated by the replicon that carries the corresponding trait genes. Then be the transformation products that contain the recombination vector - including the DNA fragment, which carries the phoS gene - selected from the transformation products isolated as above, where the amount of phosphate binding protein in the transformation products is used as the criterion

ίο will. The amount of phosphate binding protein in each of the transformation products is determined as follows. First, the phosphate binding protein is isolated from the transformation product and using a conventional technique, e.g. B. lysis, centrifugation or salting out, purified. The amount of the purified phosphate binding protein is then determined using a conventional technique such as SDS-agarose gel electrophoresis. The amount of phosphate binding protein in each of the transformation products is compared with the amount of phosphate binding protein produced by a stem cell of a bacterium from the Enterobacteriaceae family which was not transformed with the replicon carrying the DNA fragment. The transformation products with a greater amount of phosphate binding protein than in the stem cell of the bacterium of the Enterobacteriaceae family are then selected.
If a mutant cell is used as the cell of a bacterium of the Enterobacteriaceae family in step 4 described above, which produces alkaline phosphatase not only under low-phosphate conditions but also under high-phosphate conditions, the selection of the transformation products which the replicon with the phoS gene leading DNA fragment, carry out the fifth step described above very easily without determining the amount of phosphate binding protein in each of the transformation products. The reason for this is as follows. If such a mutant cell is transformed with the replicon carrying the DNA fragment, as produced in the third step described above, the mutant cell generates alkaline phosphatase under low-phosphate conditions, but under phosphate-rich conditions due to the function of the DNA fragment that ligates to the replicon is, the phoS gene does not contain alkaline phosphatase. Thus, if the transformation is carried out under phosphate-rich conditions, transformation products negative in terms of alkaline phosphatase are selected out

In the sixth step explained above, the recombination vector with the one carrying the phoS gene DNA fragment from the transformation products read out in the fifth step by means of a conventional Technique such as lysis, centrifugation or the like isolated

On the other hand, it should be noted that an expression vector that carries a gene, its function in context with the phosphate transport and the phosphate metabolism, in yeast or another bacterium as one of the Enterobacteriaceae family by essentially the same procedure as that described above can be produced. Furthermore, it should be noted that instead of the steps 1 and 2 of the DNA fragments produced by the method described above, a DNA fragment can be used, that from phages such as the / ZgImS phages

was won.

The recombination vector produced by the method explained above can be determined with regard to partially erase the DNA fragment portion and / or the replicon portion with the help of a restriction enzyme, while the phoS gene is retained, whereupon the resulting, erased recombination vector is tied again with the help of a ligase so that a reconstructed recombination vector with lower Size forms. The reconstructed recombination vector can be manipulated more easily because the number of restriction sites in the reconstructed recombination vector versus the same number in the original Recombination vector was reduced. If, in addition, a gene coding for a certain Polypeptide is attached to the reconstructed recombination vector, the ratio of the length the polypeptide gene coding to the total length of the recombination vector is greater than when the polypeptide gene coding is attached to the original recombination vector which was not reconstructed. It is it is well conceivable that with an increasing ratio between the length of the polypeptide gene coding and the Total length of the recombination vector, the amount of protein obtained per cell and the amount of protein per cell increases resulting amount of by-products becomes smaller. Thus, if the above reconstructed recombination vector is used to obtain a particular desired polypeptide, so can not only achieve the high productivity for the polypeptide, but also a higher purity of the recovered polypeptide.

A DNA fragment carrying the phoS gene can be extracted from the recombination vector which is after prepared using the above procedure and attached to a different type of replicon than the original recombination vector was tied to produce a different type of reconstructed recombination vector. Such a different type of reconstructed recombination vector can advantageously be used, if an effortless selection of the transformation products by changing a phenotypic Feature of the replicon and / or an increase in the amplification number of a recombination vector is intended are. The use of such a different type of reconstructed recombination vector is desirable, if the transformation products produced with the aid of the original recombination vector are those from which the desired ones result It is difficult to read out transformation products and / or for which the replication capability of the original replication vector is low in the transformation products

As already mentioned, the reconstruction of the recombination vector is carried out using one of the usual techniques executed (see. For example, US-PS 43 40 674, 43 49 629 and 43 56 270).

The expression vector obtained by the above process can be generated by building up the restriction image mark as follows. The expression vector is used to obtain DNA fragments with different Restriction enzymes partially or completely dissolved. The fragments obtained become one Subjected agarose gel electrophoresis, taking size and restriction patterns of the expression vector are determined. The location of the phoS genes on the above Expression vector is erased according to the invention by building a large number of different ones Expression vectors by means of partial or complete resolution with an appropriate restriction enzyme and with subsequent self-binding and through Examination of the deleted expression vectors determined by complement formation tests in which the phoS gene negative mutants can be used as the host.

The expression vector carrying the phoS gene has the following advantages when the gene coding for a desired polypeptide on the expression vector on it below the DNA sequence for the promoter a phoS gene is linked and the recovery of the desired polypeptide by genetic engineering is carried out:

1) The yield of the aforementioned desired polypeptide is lxl0 ⁵ -10 ⁶ molecules / line, that is several to 100 times higher than when using the vector usually used.

2) The gene expression of the expression vector used here can be adjusted without restriction control the phosphate concentration in the culture medium. This also allows production control of the desired polypeptide by adjusting the phosphate concentration in the culture medium.

3) The desired polypeptide can enter the periplasm together with the proportion of phosphate binding protein of the host. In this way, the desired polypeptide can be removed without difficulty Isolate the host.

For this reason, the expression vector can be used industrially on a large scale. Below an example of the method according to the invention for producing a desired polypeptide is now shown Using the expression vector explained in more detail above. The gene coding for the desired polypeptide is obtained from the genes of eucaryotic cells, procaryotic cells or viruses, and that way The gene obtained is linked to the expression vector. Subsequently, the host will use the previously obtained Expression vector can then be converted to form transformation products the transformation products obtained in this way are incubated to a large extent in order to produce the desired polypeptides with to gain extremely high yield.

In the practical implementation of the method according to the invention, organic synthesis is the first step a DNA fragment containing a gene coding for a polypeptide is obtained or by conventional techniques such as lysis, centrifugation, dissolution with a restriction enzyme or the like isolated from organisms It is also possible to produce a DNA fragment with a gene coding for a desired polypeptide from a Obtain RNA fragment from a messenger RNA and RNA virus by converting an RNA fragment with the desired gene into the complementary DNA fragment with the help of well-known Reverse transcriptase is produced. The DNA fragment carrying a gene coding for a polypeptide is connected to an expression vector leading to the phoS gene at its below the DNA sequence of a promoter bound for the phoS gene lying section. At least one of the gene coding for a polypeptide leading DNA fragments, one of the DNA fragments leading the phoS gene of the expression vector or the replicon of the expression vector has a gene for a phenotypic trait, for example the

Resistance to drugs, enzyme activity or the like. The section at which this is a gene coding for a polypeptide leading DNA fragment is ligated to the expression vector, can between the proximal and the distal part of the DNA fragment carrying the phoS gene, so that a fusion protein can be generated. This section can also be found below the das DNA fragment leading to the phoS gene, so that a hybrid protein can be generated. The one described above The attachment process is carried out with the aid of a ligase using conventional techniques (See, for example, US Pat. No. 4,237,224).

In the aforementioned transformation step, cells of a bacterium from the Enterobacteriaceae family are produced with the expression vector with the gene coding for a polypeptide produced by conventional methods is ligated to it, transformed (see, for example, US Pat. No. 4,237,224). As examples of a bacterium from the Enterobacteriaceae family include Erwinia, Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Name Serratia and Shigella.

In the above-described readout, the transformation product is obtained from the stem cells of the bacterium singled out from the family Enterobacteriaceae on the basis of a phenotypic trait, such as e.g. B. Resistance to drugs mediated by the expression vector.

In the aforementioned incubation, the transformation products incubated under suitable nutrient conditions for this, thereby producing the transformation products to express the genes and produce the desired polypeptide.

The desired polypeptide is then isolated from the transformation products and purified of the polypeptides using a conventional technique such as lysis, salting out, column chromatography or similar.

If the gene coding for a desired polypeptide is attached to the below the DNA sequence containing the coding represents the section lying for the signal peptide of a phoS gene during ligation (first process step) of the expression vector is ligated, the desired polypeptide which is present in the transformation products was obtained in the aforementioned fourth step, deposited in the periplasm of the transformation products. The reason for this is as follows. The said signal peptide is in the phoS gene on the side of the Encodes the N-terminal amino acid sequence of a phosphate binding protein and its expression occurs together with that of a phosphate binding protein. The signal peptide is designed to interact with the inner membrane of a cell and is required for secretion by it. In other words, the signal peptide plays a role in the Deposition of a phosphate binding protein into the periplasm of a cell through the inner membrane Role.

Thus, if the transformation product containing the expression vector to which the gene coding for a desired polypeptide at the one below the DNA sequence coding for the signal peptide of the phoS gene is incubated, the desired polypeptide, which is in the transformation product was generated, into the periplasm of the transformation product due to the function of the signal peptide divorced. The polypeptide deposited in the periplasm can be isolated without lysing the transformation product, and thus is in comparison to FIG Polypeptide production in which there is no deposition into the periplasm, the individual representation of the secreted Polypeptide very simple.

The expression vector with a gene coding for a polypeptide bound to it, which when ligated can be made in essentially the same way as previously in connection with the reconstruction of the expression vector described above. For example, the expression vector having a gene coding for a polypeptide bound thereto at a portion which is used to form of a hybrid protein, converting it into an expression vector, the gene coding for a polypeptide is connected to a section. which leads to the formation of a fusion protein.

It is important to ensure that the phoS gene is attached to a Coding for a desired polypeptide in viral genes can be ligated so that the desired encoded in the virus gene Polypeptide can be produced with good performance.

As explained and described above, the expression vector can be used to generate physiological active substances, enzymes, antigens, toxins, amino acids, metabolic by-products and use the like. Examples of physiologically active peptides include hormones such as somatostatin, Insulin, corticotropin, growth hormone and the like, kalmodulin, the cell growth factors, interferons and the like. The enzymes can be urokinase mutanase («-1,3-glucan 3-glucanohydrolase), Amylase, glucose isomerase, hyaluronidase, reverse transcriptase, Name enzymes for fixing nitrogen and the like. Among the antigens are the antigen for the flu virus HA, the surface antigen for hepatitis B and the like. As examples of Toxins can be the toxin for Bordetella pertussis. Name snake venom and the like, and as examples for amino acids, L-glutamic acid, L-lysine, L-methionine and the like are mentioned here. Antibiotics, Mycotoxins and the like are examples of metabolic by-products, and examples of other proteins as the substances listed above, opioid peptides, ovalbumin and the like may be mentioned. From it It is found that the invention in the production of food, medicine, feed, fermented Is of great use to products, energy sources and the like

The invention will now be illustrated in more detail using the following examples.

The culture media used in the examples. Buffer solutions, methods of DNA extraction and methods for DNA purification are each summarized below.

A: Composition of the T medium

Bacto-Tryptone 10 g

NaCl 5 g

both dissolved in 100 ml of water.

B: Rapid alkali extraction of the plasmid DNA

0.1 ml of a lysozyme solution (2 mg lysozyme / mL 50 mM glucose, 100 mM CDTA (cyclohexanediamine tetraacetate), 25 mM Tris-HCl (pH 8.0) were added to the bacterial cell granulate, which was obtained by centrifuging 1.5 ml culture prepared after overnight incubation

became. The resulting suspension was incubated at 0 ° C. for 30 minutes. Subsequently, the suspension 200 μΙ of an alkaline SDS solution (0.2 N NaOH, I w / w ° / o SDS (sodium dodecyl sulfate) was added and touched. After five minutes of incubation at 0 ° C, 150 μΐ 3 M sodium acetate (pH 4.8) was added to the suspension and carefully mixed with it. The resulting The suspension was allowed to stand at 0 ° C. for 60 minutes and then the precipitates were centrifuged for five minutes long at 5000 rpm. From the upper layer of the supernatant, 0.4 ml aliquots were extracted with the pipette deducted. The aliquot was mixed with 1 ml of cold anhydrous ethanol to precipitate the DNA The resulting mixture was centrifuged for 2 minutes and the supernatant which formed was removed; the resulting granules were 100 μl of a sodium acetate solution (0.1 M sodium acetate, 0.05 M Tris-HCl [pH 8.0]) added. The resulting solution was with double the amount of cold anhydrous ethanol to reprecipitate the DNA. The precipitated one DNS was dissolved in an appropriate buffer. The details of the above operations are described in Nucleic Acid Research, Vol. 7, No. 6, pp. 1513-1523 (1979). The after the above Crude plasmid DNA obtained in the workflow can be used for physical mapping analyzes and to Use transformation purposes.

C: Removal of ethidium bromide from DNA

The DNA fraction was after the CsCl density gradient centrifugation with the same volume of an isopropanol solution mixed with the aqueous

5 M NaCl - 10 mM Tris-HCl-1 mM Na ₃ EDTA (pH 8.3) was saturated. This process was repeated until the aqueous phase containing the DNA decolorized. Two parts by volume of water and then

6 parts by volume of anhydrous ethanol were added to the resulting aqueous phase. The mixture was allowed to stand at -20 ° C for one hour to several days for the DNA to precipitate. The precipitated DNA was collected by centrifugation and dried to produce a DNA sample. The DNA sample was dissolved in 100 μl of a Tris-EDTA solution (10 mM Tris-HCl, 1 mM Na ₃ EDTA [pH 7.5]) and the Use fed. Details of this procedure can be found in Advanced Bacterial Genetics, p. 126, Cold Spring Harbor Laboratory (1980).

D: Composition of the buffer for the EcoRI restriction enzyme (five-fold concentrated) (5.0 M CsCI, 10 mM MgSO ₄ , 10 mM Tris-HCl [pH 8.0], 0.1 mM Na ₂ EDTA [floating density = 1.60] ) a. 3 ml of a 3.0 M CsCl solution (3.0 M CsCl, 10 mM MgSO ₄ , 10 mM Tris-HCl [pH 8.0], 0.1 mM Na ₂ EDTA) were placed over the solution in the glass [Floating density = 1.40]). Then 1 ml of a phage suspension was introduced over the CsCl solution layer, and the whole thing was centrifuged at 30,000 rpm in a rotor of the SW 50.1 type at 20.degree. C. for one hour. Subsequently, less than 0.5 ml of the phage fraction was removed from the side walls of the glass with the aid of a 1 ml syringe and a 15.8 mm needle (caliber 25). Details of this procedure are described in Advanced Bacterial Genetics, pp. 80-81, Cold Spring Harbor Laboratory (1980).

G: CsCl equilibrium density gradient centrifugation

The phage fraction prepared according to step F above was washed with a 4.0 M CsCl solution (4.0 M CsCl, 10 mM MgSO ₄ , 10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA) added and centrifuged at 30,000 rpm in a rotor of the SW 50.1 type for 20 hours.

A 1 ml syringe and a 15.8 mm (25 gauge) needle were removed from the side walls of the centrifuge tube whole 0.5 ml phage fraction removed. Details of this procedure are in Advanced Bacterial Genetics, p. 81, Cold Spring Harbor Laboratory

(1980).

H: Composition of the LB medium *)

Bacto-peptone 10g Yeast extract 5g NaCl 5g

These three ingredients were dissolved in 1000 ml of water and the pH was adjusted by adding NaOH set to 7.2.

I: Composition of the T-agar medium *)

Bacto-Tnpton 10g NaCl 5g Culture medium (agar) 15g

NaCl

Tris-HCl

MgSO ₄

50OmM

250mM (pH 7.4) 50mM

E: composition of the ligase buffer

Tris-HCl
MgCl ₂

Dithiothreitol
ATP

66 mM (pH 7.6)

6.6 m M 1OmM

0.5 mM These three ingredients were dissolved in water, and then water was added to make a total of a solution volume of 1000 ml has been reached.

*) After the medium had been sterilized in the autoclave, 1/1000 volume of an aqueous tetracycline solution of 10 was added mg / ml or an aqueous ampicillin solution of 40 mg / ml. The media containing the antibiotics were used to select the antibiotic-resistant transformation products and should be the maintenance of the antibiotic-resistant recombination vector in the bacterial cells.

J: Obtaining the DNA for plasmid pBR322 and cleavage of the DNA for plasmid pBR322

with the restriction enzyme EcoRI

F: CsCl block density gradient centrifugation

At the bottom of a cellulose nitrate centrifuge tube with a diameter of 12.7 mm and a length of 50.8 mm brought 1 ml of a 5.0 M-CsCl solution 11 of the T medium was germinated with 10 ml Escherichia coli Κ-12 strain C600 inoculated (B. J. Bachmann, Bacterial. Rev, 36, 525-557 [1972]) (carrier of the plasmid pBR322) and cultured with shaking at 37 ° C until the cloudiness of the cultural outlines is called fiflilnm

0.6 A. Chloramphenicol was then added to the broth up to a final concentration of 250 μg / ml the culture was incubated for 15 hours at 37 ° C. and centrifuged to obtain cells cells obtained were once with 100 ml Tris-ED-TA solution (the 100 mM Tris-HCl buffer [pH 7.5] and

1 mM EDTA contained) washed and rapidly extracted with alkaline; this resulted in a crude plasmid DNA. In the so prepared crude plasmid DNA, the above Tris-EDTA solution was added in such an amount that the total volume 15 ml. 16 g of crystalline CsCl and

2 ml of ethidium bromide in a concentration of 5 mg / ml were added to the solution, whereupon the specific gravity d ^{25 of} the mixture was adjusted to 139 by adding water or crystalline CsCl. The mixture produced in this way was then ^{centrifuged for 40 hours at 20 °} C. at 33,000 rpm. The position of the plasmid DNA tape was determined with the aid of long-wave UV light and the plasmid DNA was obtained by fractionation. The ethidium bromide was extracted from the plasmid DNA fraction by the method described in point C. 0.05 ml of the restriction enzyme buffer for EcoRI was added to the 0.2 ml pBR322 DNA solution (100 μg / ml) prepared in this way and then the restriction enzyme EcoRI was added in such an amount that the activity of the restriction enzyme EcoRI became one unit per µg of the plasmid DNA. The enzyme reaction proceeded at 37 ° C for 60 minutes. The mixture was then heated to 65 ° C. for 5 minutes in order to inactivate the restriction enzyme. The mixture was then dialyzed against a Tris-EDTA solution (containing 10 mM Tris-HCl [pH 8.0] and 1 mM EDTA). After the completion of the dialysis, 4 units of Escherichia coli alkaline phosphatase was added to the mixture, followed over 30 minutes was incubated at 65 ° C. An equal amount of phenol solution (prepared by saturating the above Tris-EDTA solution with phenol) was added to the mixture, followed by shaking. The shaking mixture was then centrifuged at low speed and separated into the aqueous phase and the phenol phase. The aqueous phase was collected. This centrifugation with collecting the aqueous phase was repeated twice. An equal volume of solution I (24: 1 by volume, mixture of chloroform and isoamyl alcohol) was then added to the aqueous phase collected. It was then mixed thoroughly and the aqueous phase was collected. This process was repeated four times, whereupon the collected aqueous phase was dialyzed against a ligase buffer for the subsequent binding reaction, a cleaved DNA solution for pBR322 being obtained.

Second step:

Production of the DNA fragments with a DNA containing the phoS gene of Escherichia coli Κ-12 strain

1 1 of the T medium was mixed with 10 ml of germs from Escherichia coli K-12 strain KLF48 / KL159 (CGSC No. 4302) Journal of Bacteriology, June 1975, Vol. 122, No. 3, pp 1153.1154) and cultured with shaking at 37 ⁰ C. The culture broth in the phase of logarithmically increasing growth (turbidity: 0.6 A at 600 nm) was centrifuged to obtain cells. The cells thus collected were suspended in 10 ml of a lysozyme solution (containing 2 mg / ml lysozyme, 100 mM EDTA and 0.15 M NaCl), and the suspension was allowed to stand at 37 ° C. for 15 minutes.

The suspension was quickly frozen on a dry ice / acetone bath at -20 ⁰ C. 50 ml of a Tris-SDS buffer (which contained 0.1 M Tris-HCl [pH 9.0J 1 w / v% SDS [sodium dodecyl sulfate] and 0.1 M NaCl) were added to the suspension frozen in this way.

ίο added, and the mixture was melted at 60 ⁰ C. This freezing over a dry ice / acetone bath and melting at 60 ⁰ C was repeated five times the cells were completely lysed. An equal amount of a phenol solution (prepared by saturating the aforementioned Tris-SDS buffer with redistilled phenol) was added to the solution thus obtained, in which the cells were lysed, and then stirred. This solution was slowly centrifuged into an aqueous phase and separated into the phenolic phase. The aqueous phase was collected. The aqueous phase collected in this way was admixed with twice the volume of anhydrous ethanol. This mixture was then centrifuged at 12,000 rpm for 20 minutes, whereupon the granules were obtained. These granules were dissolved in 20 ml of a mixture of NaCl and citric acid (containing 0.15 M NaCl and 0.015 M sodium citrate at pH 7.0) to give a solution containing the DNA. An equal amount of solution I (cf. first step) was added to 10 ml of the DNA-containing solution prepared in this way, and the mixture was then stirred. The aqueous phase was obtained from this. The aqueous phase obtained in this way was mixed with twice the volume of ethanol and the precipitated DNA was dissolved in 5 ml of Tris-EDTA solution (which contained 10 mM Tris-HCl [pH 7.5] and 1 mM Na ₂ EDTA). The above-described precipitation with anhydrous ethanol was repeated and 5 ml of the DNA solution were made up. 0.05 ml of the restriction enzyme buffer was added to 0.2 ml of the DNA solution prepared in this way (200 μg / ml), and then the restriction enzyme EcoRI was added in such an amount that the activity of the restriction enzyme EcoRI was one unit per μg of the DNS reached. The mixture was then incubated at 37 ° C. for 60 minutes, followed by a 5-minute heat treatment at 65 ° C. to inactivate the restriction enzyme. The mixture was then dialyzed against the ligase buffer. an E. coli DNA solution was obtained.

L: ligating the pBR322 DNA and the
DNA fragments from E. coli with a phoS gene

The pBR322 DNA solution that was prepared in the first step was adjusted to 30 μg DNA concentration / ml diluted with an installation buffer. The E. coli DNA solution that was prepared in the second step was on 100 μg DNA concentration / ml diluted with the same installation buffer. 50 μΐ of the pBR322 DNA solution

T4 ligase was added to this mixture in an amount corresponding to one T4 ligase unit per μg of DNA to be ligated. The enzyme reaction for ligation of DNSen was conducted for 8 hours at 8 ⁰ C. After the end of the ligation, the solution obtained was dialyzed against a Tris-EDTA solution (which contained 10 mM Tris-HCl [pH 7.5] and 1 mM EDTA).

M: selection of the plasmids carrying the phoS gene A: E. coli strain C75

The strain C75 from E. coli (Garen, A. and N. Otsuji, J. MoL Bio! .. 8,841-852 [1964]) was cultivated in an LB medium with shaking until the culture broth became turbid at 600 nm Reached a value of 0.6 A. Then 2.5 ml of the broth were slowly centrifuged to obtain cells. The cells obtained were suspended in 24 ml of an aqueous MgCfe solution of 0.1 M and this suspension was then slowly centrifuged to obtain cells. The cells obtained were in 1.2 ml of an aqueous CaCb Solution of 0.1 M was suspended and this cell suspension was allowed to stand for 30 minutes at 0 ° C., after which it was then slowly centrifuged to obtain cells. The cells obtained were resuspended in an aqueous CaCl ₂ solution of 0.1 M in an amount of 0.1 ml. 10 μl of the solution of bound DNA prepared in the third step were added to this cell suspension. This mixture was left on ice-cold water for 30 minutes stand, and then heated for 5 minutes in a water bath at 40 ⁰ C 1.0 ml of pre-warmed to 37 ° C LB medium were added to the heated mixture, and then 90 minutes at 37 ⁰ C was incubated. Then were applied 0.1 ml of the culture broth thus prepared, and 0.1 of ten times with LB medium ml diluted culture broth in each case on T agar plates containing tetracycline or ampicillin, followed by overnight incubation at 37 ° C and 30 ⁰ C. to form colonies of transformation products resistant to tetracycline and ampicillin, respectively. The resulting colonies of the transformation products were sprayed with an alkaline phosphatase staining solution (which contained 2 mg tx-naphthyl phosphate / ml and 20 mg Fast Blue B / ml Salt [tetrazot-treated o-dianisidine]) which was dissolved in 0.5 M Tris-HCl (pH 8.0) was dissolved. The white colonies of transformation products which had not turned brown when the dye solution was sprayed on were removed as the cells which contained the plasmids in which the E. coli phoS gene was ligated into the pBR322 DNA.

B: E. coli strain C2

To select the white colonies of transformation products that contained plasmids in which a E. coli phoS gene was ligated into the pBR322 DNA - with the difference that the transformation of the C2 strain of E. coli (Morris, H., MJ Schlesinger, M. Bracha and E. Yagil, J. Bacteriol., 119, 583-592 [1974]) was used as an indicator in the selection of the plasmids - essentially became the the same process sequence as the one just described is repeated.

N: Construction of the restriction maps for the

plasmids carrying a phoS gene

(pSN401 and pSN402)

The transformation products selected in the previous fourth step were cultivated in LB medium. Subsequently, plasmid DNAs were extracted from the obtained cell culture and purified as follows. To extract the plasmid DNAs from the previously set cell cultures, the procedure for rapid alkaline extraction used. Then the crude plasmid DNA solutions were by equ weight density gradient centrifugation in the same Purified as in the first step The ethidium bromide was leached out of the thus purified plasmid DNA solutions and the DNA was precipitated (see first step).

The plasmid DNAs obtained in this way were digested with various restriction enzymes, and then the length of the DNA fragments was determined by electrophoresis on an agarose gel

ίο The restriction maps of the plasmids were then It was found that two types of phoS gene-carrying plasmids had been obtained.

In the following, one of the plasmids is referred to as "pSN401" and the other as "pSN402". The Restrik The illustration of plasmid pSN401 is shown in FIG. 1 shown The plasmid pSN402, the EcoRIi.-EcoRIr fragment of pSN402 was ligated with the opposite orientation to the plasmid pSN401

Production of the DNA fragments with the phoS gene for the bacteriophage yJglmS DNA

150 ml of the T medium were germinated with 1.5 ml Escherichia coli KY7388 strain (T. Miki, Annu. Rep.

Inst Virus Res, 21, 1-26 [1978]), that is to say one Lysogen of the bacteriophage / iglmS, and cultivated with shaking at 37 ° C. In the middle of the phase of logarithmic growth, the culture mitomycin C bis added to a final concentration of 0.5 μg / ml Lysis was cultivated for about two more hours. The culture was cooled on ice and added dropwise one added (in several drops) chloroform. Then air was drawn into the culture broth with the pipette given to the formation of bubbles, causing the cells in the Culture broth were completely lysed. The attached Phage lysate was then slowly centrifuged. The supernatant was collected, while the precipitated material, which consisted of cell debris, was discarded. The obtained supernatant was rotated at 25,000 rpm Centrifuged for 90 minutes to recover the / ZgImS phage particles deposited at the bottom of the centrifuge tube. The phage particles obtained were suspended in 4.0 ml / Z diluent (which contained 10 mM Tris-HCl [pH 7.5], 10 mM MgSO ₄ and 0.1 mM Na ₂ EDTA) dated. The prepared phage particle suspension was subjected to a CsCl block density gradient centrifugation, followed by CsCl equilibrium density gradient centrifugation, whereupon a fraction was obtained which contained only / iglmS phage particles.

The iglmS DNA was extracted from these phage particles with formamide in the manner explained below. 0.05 ml of a Tris-EDTA solution (the 2 M Tris-HCl [pH 8.5] and 0.2 M Na ₂ EDTA contained) and then 0.5 ml of formamide. The set mixture was left at room temperature for about 3 hours stand. Then one set successively 0.5 ml of distilled water and 3 ml of anhydrous ethanol Mixture and mixed this to precipitate the / igimS-Fhagen-DNA. The precipitated / igimS phage DNA was collected by centrifugation, with 70 Washed v / v% ethanol and dried in vacuo, whereupon a dry / ZgImS DNA was obtained. The dried / ZgImS DNA was in 5 ml of a Tris-EDTA Solution (which contained 10 mM Tris-HCl [pH 7.5] and ImM Na ₂ EDTA) and then cleaved by means of a restriction enzyme EcoRI, and dialyzed in the same way as in the second step in Example 1. In this way

a solution was prepared with DNA fragments carrying the phoS gene

Ligation of the pBR322 DNA and the / iglmS phage DNA fragments with the phoS gene

This was done essentially the same as in the third step in Example 1, except that a solution was ligated with DNS received, but with the difference that the iiglmS phage DNA solution as described above The first step was set up instead of the E.C0I1 DNA solution was used.

Selection of the plasmids carrying the phoS gene

A: E. coli strain C75

Substantially the same procedures as in Step 4A of Example 1 were repeated to obtain whiteness Colonies of transformation products with the plasmids containing the phoS gene from the iglmS phages to be selected, but with the difference that the solution used in the second step above has ligated DNA was used instead of the solution with ligated DNA that was obtained after the third step of Example 1 was scheduled.

B: E. coli strain C2

The procedure for the white colonies was essentially the same as in step 4B in Example 1 read with transformation products with plasmids in which the phoS gene obtained from / ZgImS phages was ligated into the pBR322 DNA, with the difference, however, that the solution with ligated DNA, as stated in the previous second step, was used instead of that in the third Work step in Example 1 prepared ligated solution.

Construction of the restriction maps of the plasmids carrying the phoS gene (pSN401 and pSN402)

Substantially the same operations as in the fifth step in Example 1 were repeated to obtain the Create restriction maps of the plasmids, but with the difference that those in the preceding Plasmids obtained in steps 3A and 3B were used, and not those obtained after step 4A and 4B plasmids prepared in Example 1. It was found that these are the ones won here Plasmids were pSN401 and pSN402.

Construction of the plasmid pSN507 carrying the phoS gene from pSN401

The pSN401 DNA, as it was obtained in the fifth step in Example 1, was partially disrupted with the restriction enzyme HindIII and then ligated again with the aid of T4 ligase, essentially in the same way as explained in the third step in Example 1. The E. coli strains C75 and C90 (Garen, A. and N. Otsuji, J. Mol. Biol., 8, 841-852 [1964]) were essentially used as recipients in transformed in the same way as described in the fourth step in Example 1, whereupon strains were obtained which carried the DhoS ⁺ plasmids. The plasmid DNAs were then extracted from the cultures of the various independent transformation products and purified in essentially the same manner as in the fifth step in Example 1, whereupon a restriction map of the plasmids was constructed. The purified re-ligated plasmid is referred to below as “pSN507”; its physical structure is shown in FIG. 1 can be found.

Construction of the plasmid carrying the phoS gene
pSN508 from pSN507

The pSN507 DNA obtained in the previous first step was tested with the restriction enzyme MIuI unlocked and then the plasmid and the Transformation of each of the Ecoli strains C75 and C90 with the relegated plasmid, essentially in the same way as in the third step in Example 2, resulting in strains which have the umligiert plasmid (hereinafter referred to as "pSN508"). The pSN508 DNA was obtained from the cell cultures these strains are extracted and purified in essentially the same manner as in step 5 in Example 1.

A restriction map of plasmid pSN508 was constructed. The result is FIG. 1 can be found.

Construction of the plasmid carrying the phoS gene
pSN518 from pSN508

The pSN508 DNA obtained in the previous second step was with the restriction enzyme pstl digested and then found re-ligating the plasmid and transforming each of the Ecoli strains C75 and C90 with the relegated plasmid held in essentially the same manner as in the above second step, strains with the umligierten Plasmid (hereinafter referred to as "pSN518"). The cell culture was then used of the strains obtained in this way, the pSN518 DNA was extracted and essentially the same as in the fifth Cleaned step in example 1. When constructing the restriction map of the plasmid, pSN518 was obtained that from FIG. 1 result to be taken.

Construction of the plasmids carrying the phoS gene
pSN407 and pSN408 from pSN402

The pSN402 DNA obtained after Step 5 of Example 1 became essentially the same Relegated as in the first step in Example 3. The plasmid obtained is hereinafter referred to as "pSN407". A restriction map of the plasmid pSN407 was then constructed. The restriction map of pSN407 is essentially the same as that from pSN507, with the difference that the orientation of the EcoRli-EcoRh fragment from pSN407 is in pSN507 is exactly the opposite.

The pSN407 obtained in this way was re-ligated in the same way as in the second step of Example 3. The won Plasmid is hereinafter referred to as "pSN408". The restriction map of pSN408 was created; it essentially corresponds to that of pSN508. with the difference that the orientation of the Eco-Rli-EcoRh fragment with pSN408 is exactly the opposite of that with dSN508.

Construction of the plasmids carrying the phoS gene
pSN1507 and pSN1508 from pSN507

The plasmids pBR325 (Bolivar, F. et al. Gene, 4, ■ 21 [1978]) and pSN507 each cleaved with the restriction enzyme EcoRI. Then the split Plasmid pBR325 into the cleaved plasmid pSN507 in essentially the same way as in the third step in Example 1 was then inserted - essentially as in the fourth step in example 1 - the selection of the bound DNA obtained below Use of the E. coli strain ANCC75 (a transduction product of the type Pl: E. coli strain C75X E. coli strain CSH57 (Miller, J. H, Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. "pp. 13-23 [1972]) as recipients carried out

Essentially in the same way as in the fifth step in Example 1, the extraction and purification were now carried out of the plasmids obtained in this way. The restriction maps were then created the plasmids, and found that two kinds of plasmids containing the phoS gene were obtained. One of the plasmids is hereinafter referred to as "pSN1507" and the other is called "pSN1508". It was also found that the EcoRIi-EcoRh fragment of pSN507 ligated into the EcoRI installation site at pBR325 had become, thereby forming pSN1507.

Construction of the plasmid carrying the phoS gene
pSN2507 and creation of a restriction map of the plasmid pSN2507

The plasmid pKN402A (B. E. Uhlin et al. "Gene, 6, 91-106 [1979]), whose DNA is shown in the restriction map in Fi g. 2 was shown in part with the Restriction enzyme HincII digested and re-ligated with the aid of T4 ligase. Strains with umligiert plasmids (hereinafter referred to as "pSHIOl") were essentially the same as in the fourth step obtained in Example 1 and in the fifth step in Example 1, whereupon a restriction map of pSHIOl was established. Figure 2 shows this restriction map. Now pSHIO1 and pSN507 were each digested with EcoRI. The recovered fragments from pSHIO1 and pSN507 were integrated with the aid of T4 ligase. The ligated plasmid obtained was now subjected to a selection in order to remove the white colonies of the transformation products, which contained the plasmids carrying the phoS gene and by ligating the pSN507 fragment into pSHIOl were formed. The resulting transformation product was set in a culture and turned out to be the plasmid DNAs extracted from the cultured transformation products and essentially in the same way cleaned as in the fifth step in example 1. Now it became essentially the same as in the fifth step in example 1 a restriction map of the plasmid pSN25C7, one of the plasiriidc obtained in this way, is constructed, and determine the location of the phoS gene in pSN2507. F i g. 2 shows the restriction map of pSN25O7. It was found that a plasmid in which the orientation of the EcoRI i-EcoRl2 fragment was opposite to the orientation in pSN2507, also in that described above Incorporation of the pSN5O7 fragment into pSHIOl had been established.

example 1

By calculating the number of molecules of the phosphate binding protein that per host cell were produced, the gene expression power of the expression vector was determined The mechanism of Production of a phosphate binding protein using phoS gene expression is briefly discussed below The precursor of the phosphate binding protein becomes

ίο first produced by expression of the phoS gene The precursor has a signal peptide N-terminal on the mature phosphate binding protein. The signal peptide is for Interaction with the inner cell membrane and necessary for secretion through it.

Therefore the precursor of the phosphate binding protein is first transported to the inner membrane, whereby the signal peptide comes into operation. The signal peptide part of the protein is then split off while it passes through the inner membrane and thus becomes the mature phosphate binding protein in the periplasm of the cell secreted in the split state

The number of molecules of the phosphate binding protein, produced per cell was determined as follows

The E. coli strain ANCC75 was transformed with the plasmid pSN507 and incubated overnight in a Tris / glucose medium, i.e. a minimal salt solution buffered with Tris / HCl (pH 7.2) containing 0.2% (w / v) Contained glucose and made up with 0.64 mM KH2PO4 (hereinafter referred to as "high phosphate medium"). After the incubation, the host cells were harvested and suspended in two types of culture media, namely the aforementioned high phosphate medium and the same Tris / glucose medium as described above, with the difference that the concentration of KH ₂ PO _{4 was} 0.064 mM ( hereinafter referred to as "low phosphate medium"), followed by incubation for six hours with shaking at 37 ° C. The cell density of each culture was determined from the absorption power at 600 nm by estimation. It was found that the number of cells in each culture was 1.3 χ 10 ⁹ was. The cells of each culture were then collected by centrifugation at 2000 g for five minutes.

The cells obtained were washed once with 1 ml of buffer solution containing 10 mM Tris-HCl (pH 7.2) and 30 mM NaCl, and again in a centrifuge tube in 0.1 ml of 33 mM Tris-HCl (pH 7.5). suspended. 0.1 ml of the buffer solution containing 33 mM Tris-HCl (pH 7.5), 40% (w / v) sucrose and 0.1 mM EDTA were added to the aforementioned cell suspension, mixed rapidly and incubated at 37 ° C. for 10 minutes . The cells were obtained by centrifugation for three minutes at 8000 χ g and immediately resuspended in 0.1 ml of 0.5 mM aqueous MgCl solution. The cell suspension was shaken vigorously in an ice water bath for 10 minutes and then centrifuged for five minutes at 8000 χ g. The supernatant obtained (hereinafter referred to as "periplasm fraction") was given 20 μ! one

«.— ff I · * C" J "Γ»

rcDcnpuftcrolung rar d: e Pc

lyacrylamide electrophoresis added (the 1 ml of 0.5 M Tris-HCl [pH 6.8], 1 ml of 10 w / v% sodium dodecyl sulfate [SDS], 0.1 ml ^ mercaptoethanol, 1 ml glycerine, 2.5 ml 0.1 g / g% strength aqueous bromophenol blue solution and contained 4.4 ml of water) with subsequent five-minute heat treatment at 100 ° C. The granules (hereinafter referred to as the »intracellular fraction«), which is separated during the centrifugation described above

21 22

stood, was twice with 1 ml of 0.5 mM aqueous gene coding for the /? - balactosidase (together

MgCb solution washed and again in 20 μΙ the lyso slope with the plasmid pSN 1403 is on the Journal of

zymlösung (the 2 mg lysozyme in 1 ml water, 50 mM Bacteriology, 143, 971-980 [1980] referenced) with the help

Glucose, 10 mM EDTA and 25 mM Tris-HCl [pH 8.0]) from EcoRI to form a recombination vector

contained suspended, with subsequent 30 minutes In-5 was prepared.

incubation at 37 ° C. The sus- The recombination vector (the

Pension was selected with 180 μΐ 0.5 M aqueous MgCl ₂ solution (hereinafter referred to as "pSN5081")

diluted and the mixture was stirred thoroughly. and essentially in the same way as with the

120 μΙ of the aforementioned pro-steps 4 and 5 in Example 1 were isolated from this mixture,

benbufferlösung for polyacrylamide electrophoresis. Then the plasmid pSN5081

added and then a heat treatment partially extinguished, whereupon the two terminals of the

Carried out at 100 ° C for 5 minutes. resulting DNA fragment of the plasmid

The fractions prepared as above were partially pSN5081 with the aid of the nuclease BAL31 (Haneiner SDS polyacrylamide gel electrophoresis according to the dels designation; Deoxyribonucelase) unlocked Subjected to procedures as split by Laemmli, U. KL, Natuis and with the help of Smal. The derelict, 227, 680-685 (1970), where the cleaved DNA fragment of the plasmid was obtained with T4-Li yield re-ligated to phosphate binding protein in the cell gases.

was determined. The results are shown in the following- Now the E. coli strain CSH26 (here can be found on

summarized in the table. J. H. Miller, Experiments in Molecular Genetics, Cold

20 Spring Harbor Laboratory, p. 17 [1972].

Table den), which represents a Lac mutation that lactose in

- Can not process a culture medium in one

Amount / cell) _with lactose added tetrazole bed as culture medium

Niedngphosphat- Hochphosphat- _m j _t the aforementioned recombination vector transform-

^{medlum medlum} 25 miert, which contains a gene coding for /? - galactosidase

leads, whereby transformation products emerged.

Phosphatbin- 8, OxIO- ⁸ 1.0x10 "By transformation with this recombination vector

generation protein was derived from the phenotypic trait Lac ~ beim

E. coli strain CSH26 the phenotype Lac ⁺ , and thus

30 the previously won transformation pro

As can be seen from the table above, the product has been changed so that it now contains lactose production of the phosphate binding protein and its lactose-added tetrazole bed. Precursors - in other words, the expression of the accordingly could be the transformation pro-gene coding for a phosphate binding protein of the product without difficulty from the stem cells of the expression vector according to the invention - find out by setting 35 Ecoli strain CSH26, with size and The concentration of phosphate in the culture medium is indicated by the color of the lactose-enriched tetra influenced zolbett served as selection criteria and one in

Subsequently, the number of molecules became essential in the same way as in the experiments

Phosphate binding protein per cell was calculated using Molecular Genetics, Cold Spring Harbor

determined by the following equation: 40 Laboratory, p. 49 and p. 52-54 (1972).

The reincorporated plasmid is hereinafter referred to as

Number of Molecules, e _P roZel, e - A χ C ^ e ^ Lm ^ SN ^ became the gene coding

tion for ^ -Gaiactosidase leading DNA fragment in

whereby 45 inserts the DNA fragment, which at its un

below the DNA sequence of a promoter for the

A - Yield of phosphate binding protein phoS gene the phoS gene resulted in so that in the form of a (g / cell) fusion protein /? - galactosidase are formed

B = molecular weight of the phosphate binder.

dungsproteins (35,000), 50 The Ecoli strain CSH26 was then with

C - Avogadro's numeral ²³ ). the plasmid pSN5084 to form transforma

transformation products The selection of the

When cultivated in the low phosphate medium, transformation products from the stem cells were from the number of molecules of the phosphate binding protein Ecoli strain CSH 26 was carried out in the same way as per cell 13x10 *. 55 previously described. The selected transformations

products were incubated in low phosphate medium, in the form of the fusion protein / galactosidase

Example 2 was created. Then the specific gravity was

the / f-galactosidase in the transformation products

Production of /? - galactosidase using 60 according to the method determined in experiments in of the plasmid pSN508 Molecular Genetics, which carries the phoS gene, Cold Spring Harbor Laboratory. S.

352-355 (1972) describes The number of MoIe

The plasmid of / 2-galactosidase prepared as in step 2 in example 3, which is in the form of the fusion propSN508 was cleaved with the help of EcoRI, resulting in a teins, was calculated per cell. EcoRI-EcoRI DNA fragment with a phoS gene 65 containing the specific activity of the in the form of the fusion will. / 9-galactosidase with the specific ment with the phoS gene, the DNA fragment II activity of a standard - ^ - galaclosidase with a which was compared to the molecular weight of 145,000 by digestion of the plasmid pMC1403. Included

23 24

it was found that the number of molecules of / ■ galactosidase formed in the form of the fusion protein was 1 χ ' ¹ per cell.

For this purpose 2 sheets of drawings

10

10

J5

40

45

$ 0

t5

CO

65

DRAWINGS BLAn

Int. Cl. ⁴ : C 12 P 21/00

Release date: December 4th, 1986

FIG.2

EcoRI

Smal

Hpo 1 (Hindi)

Tn3 (Ap ^R )

EcoRI Hpal (Hindi) Hin di digestion and llgation

EcoRI EcoRI

Hpal (Hindi) Tn3 (ApR)

EcoRI digestion and ligation

EcoRI

Smal

Hpo I - (Hincll)

Tn3 (Ap ^R )

EcoRI 2

PBR322

EcORI ₂

iii lit

Claims (3)

Patent claims: 1. A method for producing a polypeptide using an expression vector with a DNA fragment which encodes a gene for a phosphate binding protein and a replica from the group of plasmids and bacteriophages, the DNA fragment attached to the replicon is ligated and the following procedural steps are followed: Ligating a DNA fragment carrying a gene coding for a polypeptide to the expression vector, Transforming the cells of a bacterium from the Enterobacteriaceae family with the expression vector to which the gene coding for a polypeptide is ligated into transformation products,
Reading out the transformation products from stem cells of a bacterium from the Enterobacteriaceae family; Incubating the transformation products, causing the transformation products to express the gene coding for the polypeptide to form the polypeptide, and
Isolating the polypeptide from the incubated transformation products, characterized in that the DNA fragment carrying a gene coding for a polypeptide is ligated to the expression vector at its section located below the DNA sequence of a promoter for the gene coding for a phosphate binding protein. 2. The method according to claim 1, characterized in that when ligating the portion between the proximal and the distal part of a gene coding for a phosphate binding protein leading DNA fragment. 3. The method according to claim 1, characterized in that that when ligating the section below a gene coding for a phosphate binding protein leading DNA fragment.