JPH0458954B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a complex plasmid and a calf prorennin-producing host microorganism containing the same. More specifically, the present invention contains cDNA (complementary DNA) of calf prorennin (prochymosin), which is a precursor of the milk-clotting enzyme rennin (chymosin), which is secreted from the abomasum of calves and is essential for cheese production. The present invention relates to a complex plasmid and a calf prorennin-producing host microorganism having the complex plasmid. Rennin secreted from the calf abomasal mucosa is a type of acidic protease with 323 amino acid residues, and its precursor, prorennin, is a rennin precursor protein with 365 amino acid residues.
It is an enzyme protein with a molecular weight of approximately 37,000 that autocatalytically converts to the milk-clotting enzyme rennin under acidic conditions. In recent years, the field of technology that utilizes genetic recombination technology that allows microorganisms to produce the above-mentioned useful substances using a host microorganism that has a complex plasmid in which a cDNA containing useful substance production information has been incorporated into the host microorganism's vector plasmid has rapidly expanded in recent years. Technology has been developed to produce useful substances such as growth hormone and interferon using Escherichia coli as a host microorganism. The present inventors have continued their research with the aim of cloning the entire chain length of the structural gene of prorennin, which is a precursor protein of the milk-clotting enzyme rennin, which is essential for cheese production. Proceedings of the 1980 Conference of the Japanese Society of Agricultural Chemistry, page 408 (published March 5, 1980);
th International Fermentation Symposium,
179 pages, F-21・5(L), 1980; progress reports have been published in the Japanese Society of Agricultural Chemistry, 1980 Conference Abstracts, pages 259 and 569 (published March 10, 1980), etc. . However, hybrid arrested
Translation method (hybrid-arrested)
It was not possible to create a clone that had substantially the entire chain length of the prorennin structural gene confirmed by translation assay (translation assay), that is, a clone that could definitely be expected to have the ability to produce calf prorennin. The clones in the progress report above were hybridized (+) strains, but they contained approximately
A partial fragment of prorennin cDNA with a partial chain length of about 0.2Md, which has a prorennin structural gene,
It did not contain prorennin cDNA. As a result of further research, the present inventors have
As will be described in detail later in the Examples, we succeeded in generating a calf prorennin-producing clone carrying a composite plasmid containing substantially the entire length of the prorennin structural gene identified by the hybrid arrested translation method. The present inventors succeeded in synthesizing a complex plasmid containing the cDNA of calf prorennin using a method described in detail later, and obtained a host microorganism containing this complex plasmid. A new host microorganism with this new complex plasmid was developed by Fermentation Research Institute, Agency of
Industrial Science and Technology, Japan)
Microorganisms belonging to the P2 level that do not accept deposits such as Escherichia coli C600rm (pTACR1)
It is preserved in the Fermentation Laboratory, Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, to which the present inventors belong. Calf prorennin is an enzyme protein secreted from the abomasum of calves within a few weeks of birth, and is essential for cheese production, but in recent years its production has not been able to meet demand, and microbial rennet has been used as a substitute. Therefore, the success of the present inventors in providing a host microorganism capable of producing calf prorennin using genetic recombination technology has extremely great industrial significance. Therefore, the object of the present invention is to improve the production of calf prorennin.
A complex plasmid containing cDNA, and furthermore,
The present invention provides a host microorganism containing the complex plasmid. Another object of the present invention is that calf prorennin
The present invention provides a method for producing calf prorennin, which comprises expressing cDNA in a recombinant host microbial cell. More specifically, an expression vector/plasmid capable of expressing calf prorennin cDNA in a recombinant host microorganism cell is prepared, the host microorganism cell is transformed to obtain a recombinant host microorganism, and the recombinant host microorganism cell is transformed into a recombinant host microorganism. The object of the present invention is to provide a method comprising a series of steps of culturing to produce calf prorennin and recovering it. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. According to one embodiment of forming a complex plasmid of the present invention and a host microorganism containing the same, prorennin is extracted from the abomasal mucosal layer of a calf within several weeks of birth.
After extracting mRNA (messenger RNA) and using a method using reverse transcriptase to synthesize complementary single-stranded DNA using the mRNA as a template, the template mRNA is removed by alkali treatment. The single strand thus obtained
DNA is used to create complementary double strands using techniques such as DNA polymerase or reverse transcriptase.
Synthesize cDNA and use nuclease S1 (nuclease S1)
S1) is used to selectively remove only the hairpin structure of the formed complementary double-stranded cDNA, resulting in a complete prorennin-specific double-stranded cDNA.
After cDNA synthesis, terminal deoxynucleotidyl transferase (TdT;
Using a method using terminal deoxynucleotidyl transferase, for example, poly-dG
(deoxyguanine) homopolymer is added. On the other hand, if a suitable vector plasmid such as Escherichia coil vector pBR322 is used, Sal
A method using DNA polymerase is applied to linear pBR322DNA, which can be obtained using a method that uses restriction enzymes, to repair both 3' end single-stranded portions, and then TdT is used. For example, polydC (deoxycytidyl) homopolymer is added to both repaired 3' ends using a technique. Polymers at the 3′ end that can be obtained in this way
Linear pBR322DNA with dC homopolymer added,
Polymer at the 3′ end, which can be obtained as described above.
Mixing and annealing of prorennin-specific double-stranded cDNA with dG homopolymer added
A complex plasmid containing the calf prorennin cDNA can then be formed. Next, E. coli is treated with calcium and mixed with the complex plasmid obtained as described above to form a transformed strain. Clones with some inserted DNA on the Sal site of pBR322 were selected by a method that utilizes the drug resistance of the vector plasmid, and from among these selected strains, prorennin
Colony hybridization is performed to select clones with cDNA.
dization). Prorennin by this method
Selection of clones with cDNA was performed by radiolabeling the previously obtained prorennin-specific mRNA.
This can be carried out by colony hybridization using mRNA, such as 125I-mRNA or 32P-mRNA, as a probe. The calf prorennin obtained in this way
The presence of the inserted prorennin structural gene in host microorganism candidate strains that are thought to have a complex plasmid containing cDNA is confirmed by the hybrid arrested translation method.
As shown later in the Examples, the present invention provided a clone having substantially the entire chain length of the prorennin structural gene confirmed by the hybrid arrested translation method. Furthermore, the existence of a base sequence corresponding to the N-terminal amino acid sequence of prorennin was confirmed by the Maxam-Gilbert method. One aspect of obtaining the complex plasmid of the present invention and the host microorganism containing the same has been described.
Various modifications can be made to the plasmid and the like. Other examples of host microorganisms include:
Yeast such as Saccharomyces cerevisiae,
Examples include Gram-positive bacteria such as Bacillus subtilis and Gram-negative bacteria such as Pseudomonas bacteria. For example, a method of synthesizing a complex plasmid using a restriction endonuclease ligase method is also available. or,
Other examples of vectors and plasmids include pMB9 and other known vectors and plasmids when E. coli is the host microorganism, and 2μDNA or various known vectors derived from yeast chromosomes when yeast is the host microorganism. When using a vector plasmid or Bacillus subtilis as a host microorganism, for example,
known vector plasmids such as pUB110,
When using bacteria of the genus Pseudomonas as the host microorganism, various drug-resistant plasmids can be used. Hereinafter, a more detailed example of the formation of the complex plasmid of the present invention and a host microorganism containing the same will be shown. Prorennin mRNA (Metssenjar RNA)
Isolation Prorennin mRNA is extracted and collected from the abomasal mucosal layer of calves within a few weeks of birth. Extraction was performed using the method of Uchiyama et al. (Agr.Bial.Chem., 44 , 1373-1381,
This can be done using a modified method (1980). For example, the abomasal mucosal layer of a calf is collected, powdered at low temperature, and then total RNA is extracted using the phenol method, preferably using the phenol method in the presence of bentonite.
The resulting aqueous extraction phase containing polymeric RNA can be treated with a precipitant, such as lithium chloride, and the formed precipitate containing polymeric RNA can be collected. For example, forming an aqueous solution of a polymeric RNA-containing precipitate that can be obtained as described above, e.g., poly(U-Sepharose)
Prorennin mRNA-containing components can be separated and collected by affinity chromatography using an adsorbent such as or oligo (dT) sepharose. When the aqueous precipitate solution containing high molecular RNA is allowed to flow down through the adsorbent column as exemplified above, mRNA containing prorennin mRNA is detected.
The fraction is selectively adsorbed, and ribosomal RNA and other contaminant components are washed out and removed. Prorennin by elution with aqueous formamide
An aqueous formamide solution containing mRNA is obtained. It is preferable to repeat this operation multiple times. The prorennin mRNA-containing fraction obtained as described above was subjected to molecular weight fractionation using a sucrose density gradient centrifugation method according to a method known per se to obtain a fraction having a molecular weight centered on 15s. (prorennin mRNA fraction) can be collected. Prorennin mRNA fraction can be confirmed using an in vitro protein synthesis system. This fractionation operation can also be repeated multiple times if desired. Preparation of prorennin cDNA. Using the prorennin mRNA obtained as described above as a template, a method using reverse transcriptase was applied to synthesize complementary single-stranded DNA of the mRNA, and then the template mRNA was removed by alkali treatment and the single-stranded DNA was synthesized. Full-stranded prorennin cDNA can be obtained. For example, by treating the prorennin mRNA with reverse transcriptase using oligo(dT) as a primer in the presence of four types of deoxynucleotide triphosphates (dXTP's), the prorennin
Complementary single-stranded DNA can be synthesized using mRNA as a template. Degradatively removing the template mRNA by heating the synthesized complementary single-stranded DNA to about 70°C in the presence of an alkali,
Single-stranded prorennin cDNA can be formed. DNA polymerase and reverse transcriptase were added to the single-stranded prorennin cDNA obtained as described above.
Complementary double-stranded cDNA can be synthesized by acting in the presence of dXTP's. The synthesized complementary double-stranded cDNA has a hairpin structure, so
Enzyme neclease that selectively excises the structural part
The hairpin structure portion is removed by the action of SI, resulting in a complete prorennin-specific double strand.
Can synthesize cDNA. Ligation of prorennin cDNA and vector plasmid. Both 3' ends of the complete prorennin-specific double-stranded cDNA synthesized as described above, and both 3' ends of the linear DNA derived by cleaving the vector plasmid of an appropriate host at the desired site. 'At the end,
Complementary deoxynucleotidyl homopolymers are added. For example, by acting TdT on both 3' ends of the prorennin double-stranded cDNA in the presence of dGTP (deoxyguanosine triphosphate),
A poly-dG homopolymer can be added to the 3' end. On the other hand, a suitable vector plasmid,
For example, the E.coli vector plasmid pBR322
A linear pBR322DNA is formed by reacting with the restriction enzyme Sal nuclease. By allowing DNA polymerase to act on both 3'-end single-stranded parts of this linear DNA in the presence of four types of deoxynucleotide triphosphates (dXTP's), both 3'-end single-stranded parts are After repair, a poly-dC homopolymer can be added to the end of the repair site by acting with TdT in the presence of deoxycytosine triphosphate (dCTP). A complex plasmid containing the cDNA of calf prorennin of the present invention can be prepared by combining a prorennin-specific double-stranded cDNA with a poly-dG homopolymer added to the 3' end, which can be obtained, for example, as described above; By mixing and annealing linear pBR322 DNA to which a poly-dC homopolymer has been added to the repaired 3' end obtained by A complex plasmid containing substantially the entire chain length of the prorennin structural gene can be synthesized by combining the prorennin structural gene. Transformation of host microorganisms and production of calf prorennin
Selection of clones carrying complex plasmids containing cDNA. Host microorganisms such as Escherichia coli ( E. coli c600r ^-
m ^- ) was treated with calcium, mixed with the complex plasmid containing the calf prorennin cDNA obtained as described above, and the complex plasmid was incorporated into E. coli to form a transformed strain. do. Among the transformants thus formed, clones having a complex plasmid containing prorennin cDNA are selected. For example, if a prorennin cDNA was inserted into the Sal site of pBR322 as exemplified above, this selection would be expected to result in a composite plasmid containing the cDNA of calf prorennin that is ampicillin resistant and tetracynoline sensitive. The transformed strain to be transformed and other transformed strains that are resistant to ampicillin and tetracycline can be first preliminarily tested by utilizing the difference in drug resistance to ampicillin and tetracycline. In order to select and isolate transformants that truly have the composite plasmid from the transformants that are expected to have the composite plasmid containing the cDNA of calf prorennin that has been preselected in this way, Perform colony hybridization and hybrid arrested translation methods. Colony hybridization is
For example, radiolabeled prorennin-specific mRNA that can be obtained by the method described above.
Using 125I-mRNA and 32P-mRNA as probes,
This can be carried out by a method known per se. The presence of the inserted prorennin structural gene is confirmed for the hybridization (+) strain obtained by this colony hybridization by a hybrid arrested translation method which is known per se. Hereinafter, an example of the synthesis of the complex plasmid of the present invention and the formation of a host microorganism containing the same will be described in more detail using Examples. Example (1) Prorennin Methsenjar RNA (mRNA)
Extraction and purification of mucosal layer of two-week-old calf abomasum (for two animals)
Peel 140g, 0.025M EDTA, 0.1M NaCl,
It was washed with a cooled 0.1M Tris-HCl buffer (PH9.0) containing 1% SDS, dehydrated and frozen in acetone-dry ice, and further pulverized using a homogenizer. The obtained powder was suspended in the above buffer solution 1 containing 0.7 g of bentonite, and a mixed solvent of phenol, chloroform, and isoamyl alcohol (50:
After adding 500 ml of 50:1) and stirring, the mixture was centrifuged and the upper aqueous phase was taken out. After repeating the same operation six more times, LiCl was added to the final aqueous phase to a final concentration of 2M, and after standing at 4°C overnight, the precipitate was collected by centrifugation. of NaCl
and 15mM citrate trisodium salt) in 60ml. After repeating the same operation once more, double the amount of ethanol was added to the resulting solution to make a polymer.
The procedure for precipitating RNA was repeated three times. Dissolve the final precipitate in water and make 4 times the amount of 0.01M.
After adding Tris-HCl buffer (PH7.5) containing EDTA, 0.7M NaCl, and 25% formamide, the mixture was passed through a poly(U) Sepharose 4B column (30 ml volume). After washing the column with 30 ml of the same buffer, 0.01M
EDTA, 0.2% SDS, 90% formamide 0.01M
The mRNA absorbed on the column was eluted with a solution containing potassium phosphate (PH7.5). The eluate was dialyzed against distilled water, then ethanol was added to extract the mRNA.
RNA containing was precipitated. Precipitation occurs 5-20 minutes after dissolution.
After centrifugation at 50,000 xg for 13 hours in a % sucrose density gradient, a fraction containing 120 μg of RNA containing prorennin mRNA and having a molecular weight centered around about 15 g was obtained. This fraction was tested using an in vitro protein synthesis system using rabbit reticulocyte lysate, and was confirmed to have the activity of producing prorennin, which accounts for more than 70% of the total product. (2) Preparation of prorennin cDNA To synthesize single-stranded cDNA using the prorennin mRNA obtained in (1) as a template, 30 μg of mRNA,
AMV reverse transcriptase 140 units, oligo(dT) _12-18 1.6μ
g, each of four types of deoxynucleotide triphosphates
0.5mM, actinomycin D40μg,
A reaction solution containing 60mM NaCl, _6mM MgCl2, 2mM DTT, and 50mM Tris hydrochloride (PH8.3) in 400μ
It was incubated at 42°C for 1 hour. The reaction product was separated by phenol extraction and ethanol precipitation, dissolved in 0.3 ^N NaOH, and heated at 68°C for 15 minutes to degrade the template mRNA.
240ng of cDNA was obtained. Obtained single-stranded cDNA
To synthesize double-stranded DNA using as a template, 200 ng of the above single-stranded cDNA, 18 units of DNA polymerase (fragment A), 1 mM each of four types of deoxynucleotide triphosphates, 4 mM MgCl ₂ , 0.5 mM β-mercaptoethanol, Tris hydrochloride (PH7.5)
A reaction solution containing 30mM in 240μ was incubated at 25°C for 4 hours. Extract the reaction product with phenol,
After separation by ethanol precipitation, sodium acetate 30mM, NaCl 50mM, ZnSO ₄ 1mM (PH4.6)
The hairpin structure was decomposed and removed by treatment with 80 units of nuclease S1 at 40°C for 90 minutes in a 200μ reaction solution containing 200μ of nuclease S1. This was centrifuged at 95,000 xg for 12 hours in a 5-30% sucrose density gradient to obtain 100 ng of prorennin double-stranded cDNA without a hairpin structure and having an average molecular weight of 0.7 megadaltons. (3) Ligation of prorennin cDNA and vector plasmid 0.05 pmole of prorennin cDNA obtained in (2) was mixed with 80 units of terminal deoxynucleotidyl transferase, 0.06 μmole of dGTP, 5 mM MgCl ₂ ,
Incubate in 120μ reaction solution A containing 10mM HEPES-NaOH (PH7.1) at 37℃ for 6 minutes.
(dG) homopolymer was added to the 3′ end of cDNA. Meanwhile, Sal vector plasmid pBR322
Linear DNA obtained by cutting with endonuclease
in the presence of four types of deoxynucleotide triphosphates
0.5 p mole of DNA obtained by incubating with DNA polymerase (fragment A) to repair its terminal single-stranded portion is 24 units of terminal deoxynucleotidyl transferase.
The (dC) homopolymer was incubated at 25°C for 10 minutes in a 120μ reaction solution B containing 0.04μmole of dCTP, 1mM CoCl ₂ , 100mM potassium cacodylate, and 30mM Tris hydrochloride (PH7.6), and the (dC) homopolymer was added to the 3′ end. Added. After terminating the reaction by adding EDTA, 120μ of reaction solution A and 24μ of reaction solution B are mixed at 0°C, and the reaction products are separated by phenol extraction and ethanol precipitation. This was mixed with 1mM EDTA,
100mM Tris Hydrochloride (PH
7.6) at a rate of 0.3 μg/ml, heated at 68°C for 2 hours, and then cooled to room temperature at a cooling rate of 5°C for 30 minutes to add (dG) homopolymer to prorennin.
Added cDNA and (dC) homopolymer
It was ligated to pBR322DNA by annealing. (4) Transformation of E. coli with ligated DNA and acquisition of clones containing prorennin cDNA To transform E. coli with the ligated DNA obtained in (3), a conventional method using calcium treatment was used.
E. After transforming coli C600r ^- _k m ^- _k , transformant strain 804 was selected on an L-broth agar plate medium containing 30 μg/ml of ampicillin, and then transformed with ampicillin resistance and Strains showing tetracycline sensitivity were selected. A total of 622 strains obtained were treated with ampicillin 30μg/
Seedlings were planted in a lattice pattern on a Millipore filter mounted on L-broth agar containing ml of L-broth agar to form colonies, and the filter was then washed, treated with protease, treated with 95% ethanol and chloroform, and then incubated in a vacuum at 80°C for 2 hours. Heat and separate each strain.
DNA was printed onto the filter. Of the three homologous filters prepared at the same time, one was probed with the prorennin mRNA obtained in (1) labeled with 125I, and one was treated with excess rRNA and then the same 125I-mRNA was used as a probe. , and one more
Colony hybridization was performed by a conventional method using 125I-rRNA as a probe. By comparing the obtained hybridization results for every three homologous samples described above, clones containing rDNA were excluded, and 30 clones thought to have prorennin cDNA were obtained. Furthermore, plasmid DNA was extracted from the 30 strains thus obtained by cesium chloride-ethidium bromide equilibrium density centrifugation.
and each of them is the prorennin obtained in (1).
After hybridization with mRNA, it was examined whether prorennin synthesis was inhibited by assaying in an in vitro protein synthesis system using rabbit reticulocyte lysate. A clone clearly determined to contain prorennin cDNA by this hybrid arrested translation method
10 shares were acquired. (5) Characteristics of the cloned prorennin cDNA One of the clones confirmed to contain the prorennin cDNA obtained in (4), strain 102-B, contains it.
When pTACR1 plasmid DNA was extracted, treated with Sal endonuclease, and then subjected to agarose gel electrophoresis, an inserted DNA fragment nearly equivalent to the full length of prorennin cDNA was detected. The base sequence of this inserted DNA fragment is
As shown below, the results determined by the Maxam-Gilbert method clearly demonstrated the existence of a base sequence corresponding to the N-terminal amino acid sequence of prorennin. 5′− GCT GAG ATC ACT AGG ATC
CCT CTG Ala Glu Ile Thr Arg Ile Pro Leu TAC AAA GGC− ³ ′ Tyr Lys Gly Plasmid obtained from a clone determined to contain prorennin cDNA obtained by the above method
One of the results of determining the base sequence of DNA using the Maxam-Gilbert method is
pTACR102B6 is prorennin cDNA full length 1095
Among the bases, pTACR103C2 contained bases from number 1 to number 890, corresponding to the N-terminus of prorennin, and bases from number 771 to number 1095, corresponding to the C-terminus of prorennin. Plasmid pCR1001 containing the full length prorennin cDNA was constructed as follows. That is, pTACR102B6 and pTACR103C2
Each insert obtained by processing with Sal
The DNA fragment was digested with exonuclease to shorten approximately 150 bases from both 3′ ends, and then
The fragment derived from pTACR102B6 is Kpn ;
The fragments derived from pTACR103C2 are Bam H;
After cutting, both were mixed and annealed (a). Separately, pTACR102B6 plasmid DNA
The small DNA fragment (b) obtained by treatment with EcoR + Kpn and pTACR103C2 plasmid DNA were
Large DNA fragment obtained by treatment with R+ Bam H
(c) was prepared, and after mixed annealing of (a), (b), and (c), they were ligated using DNA-ligase. The thus obtained pCR1001 contains a complete cDNA corresponding to the entire amino acid sequence of prorennin,
The base sequence including the stop codon was as shown below. [Table] [Table] pCR2001 was constructed by ligating the complete prorennin cDNA contained in the thus obtained plasmid pCR1001 to the lac promoter region of the E. coli lactose operon in alignment with the reading frame. coli C600-r _k -m _k - by transformation. The above pCR2001 was constructed as follows. (1) lac promoter of E. coli lactose operon,
Plasmid pBH20 derived from pBR322, which contains a ribosome binding site and part of the N-terminus of β-galactosidase, was treated with Eco R to obtain a linear DNA.
Then, DNA polymerase converts the end into a single strand.
After making the DNA part double-stranded, T4 is attached to both ends using a decanucleotide containing a Bam H site as a linker.
- ligated using DNA ligase and further ligated with Bam H
＋ Sal treatment. (2) pTACR102B6 or
pCR1001 was treated with Bam H + Kpn and extracted from the Bam H site of No. 15 in the prorennin cDNA base sequence.
Obtain the inserted DNA fragment up to the Kpn site of No. 253.
(3) pCR1001 was treated with Kpn + Sal to end from the Kpn site of No. 254 in the prorennin cDNA base sequence.
Obtain the inserted DNA fragment up to the Sal site. Above (1)
After ligating each DNA obtained in (2) and (3), further T4−
Circular DNA by DNA ligase treatment
pCR2001 can be obtained. The obtained strain E. ampicillin coli CR1
30μg/ml, tetracycline 10μg/ml and
The cells were cultured with shaking at 37°C in a broth medium containing 1 mM of IPTG, and when the growth reached the maximum, the cells were harvested, and the cells were destroyed by sonication to obtain a cell-free extract. As a result of measuring prorennin protein in this extract by radioimmunoassay using an anti-prorennin antibody labeled with 125I, it was detected that prorennin protein was produced in an amount equivalent to approximately 1000 molecules per host cell. . In addition, the strain E.
coliCR1 has been deposited with the American Type Culture Collection as ATCC39170. The fact that the molecular weight of the product is similar to that of prorennin is demonstrated by passing the same extract through a solid-phase antibody column with an anti-prorennin antibody immobilized on Sepharose 4B, and after eluating the adsorbed proteins, electrophoresis on an SDS-polyacrylamide gel. Confirmed by assaying by electrophoresis.

Claims (1)

[Claims] 1. Microorganism Escherichia coli CR1 carrying plasmid pCR2001
ATCC39170. 2 Escherichia coli CR1
CR1) Escherichia present in ATCC39170
Plasmid pCR2001 consists of the lac promoter region of the E. coli lactose operon and the base sequence excluding the first five codons at the N-terminus encoding prorennin.