JPH0292286A - Production of calmodulin - Google Patents

Abstract

PURPOSE:To produce a polypeptide having calmodulin-activity in high efficiency by transforming E.coli with a plasmid integrated with a DNA chain containing a specific base sequence. CONSTITUTION:A DNA chain containing the base sequence of formula is used as a DNA chain holding the genetic information of calmodulin. A promoter and a terminater are integrated into the DNA chain and the chain is inserted into a plasmid vector such as PBR322. The vector is introduced into E.coli to effect the transformation of the cell. The transformed E.coli is cultured to produce calmodulin protein in the bacterial cell.

Description

BACKGROUND OF THE INVENTION Technical Field The present invention relates to a technology for producing calmodulin by genetic recombination. Specifically, the present invention relates to Escherichia coli transformed with improved rat calmodulin cDNA and a method for producing calmodulin using the same.

Prior Art Calmodulin is a calcium-binding protein that is widely distributed in eukaryotic cells. Calcium-bound calmodulin binds to and activates various inactive enzymes. Enzymes that are known to be activated include adenylate cyclase, bospolylase b kinase, glycogen synthase, tryptophan and tyrosine hydroxylase, cell membrane 2+ Ca, Mg''-ATPase, NAD kinase, phospholipase, and smooth muscle myosin light chain kinase. It is being

The amino acid sequence of calmodulin is known.

That is, calmodulins derived from humans, pigs, rats, and cows are known to have the same amino acid sequence, and they are all proteins with a total molecular weight of 16,000 and consisting of 148 amino acids. Rat calmodulin cDNA has already been obtained (11.NojlII
la and 11.5okabe: J.

Hot, Blol, 193. p, 439-445
．． 1987), production of calmodulin using chicken calmodulin cDNA (A, R, Means et al.:
J, Biol, CheIW, 260.

9.4704-4712, 1985) and expression of the chemically synthesized calmodulin gene in E. coli (D, M
Artin Watterson et al.: Bloche II
istry 24. p, 5090-5098°198
5) has already been reported.

Summary of the Invention Problems to be Solved by the Invention As a method for producing calmodulin, in the case of mammalian calmodulin, extraction and purification methods from organs are usually used. - For example, in the case of bovine brain, 35 per 2 kg
0 mg of calmodulin was obtained (M, Yazava
, M,! 3akuma and Yagl:J.

Biochem, 87. p, l313, 1980)
(S, Kakjuchl, K.

5obue: Pure Biochemistry Experiment Course 6, p, 415-
419.1986.

Tokyo Kagaku Doujin). However, if the removal of cerebral blood vessels and blood components and the use of fresh brain are neglected during purification, the procedure becomes complicated, as it becomes difficult to remove contaminant proteins such as calmodulin. Therefore, it can be said that the genetic recombination method is better for highly purified and efficient production of calmodulin. 30B-40 per 10Ω of medium for production in E. coli using chicken calmodulin cDNA.
Although a method for producing mg has been reported (A, R, Mea
ns et al; J, Biol, Chcm, 200, p, 47
04-4712.1985), not efficient production.

As mentioned above, attempts have been made to express the chemically synthesized calmodulin gene in Escherichia coli, but it can be said that this is not an efficient production (D, Mart).
in Vatterson et al. = B1oehem, 24.
p, 5090-5098.1985).

Therefore, it has been desired to establish a more efficient production technique for polypeptides or proteins having calmodulin activity using genetic recombination techniques.

SUMMARY OF THE INVENTION The present invention aims to solve the above-mentioned problems, and has achieved this purpose by providing a means for efficiently producing polypeptides or proteins having calmodulin activity in Escherichia coli. be. That is, the present invention provides (1) a DNA strand containing the base sequence shown in FIG.

The present invention also provides (2) Escherichia coli (E, c, o 1 i) transformed with a plasmid containing a DNA strand containing the base sequence shown in Figure 1 in a state in which its genetic information can be expressed. It is something.

Furthermore, the present invention provides the following steps: (3) preparing a DNA strand containing the base sequence shown in FIG.
The present invention provides a method for producing calmodulin, which is characterized by producing calmodulin in a culture through a step consisting of transforming E. coli and culturing the resulting transformant.

DETAILED DESCRIPTION OF THE INVENTION DNA Strand of the Invention The DNA strand of the invention contains the base sequence shown in FIG. The fact that the DNA strand of the present invention ``contains the base sequence shown in Figure 1'' means that the upstream and/or downstream side (or in some cases, the strand ``includes any desired base sequence shown in Figure 1'') This means that the DNA strand of the present invention defined in this way may exist as a chain member (details below). In addition to a circular body, it may be in the form of a circular body (or plasmid). In particular, the circular body or plasmid form is the form in which the DNA of the present invention is expressed in Escherichia coli to produce calmodulin.

The base sequence shown in Figure 1 encodes a polypeptide in which Met is added to the N-terminus of the known amino acid sequence of calmodulin, but in order to achieve high expression in E. coli, the 5'
The terminal nucleotide sequence is brought closer to the nucleotide sequence common to the translation initiation site of various E. coli genes, and codons that are preferentially used in E. coli are used as much as possible for the N-terminal 14 amino acids including Met ( A to B in Figure 3)
, which was designed by the present inventors.

Furthermore, the base sequences common to the translation initiation sites of various E. coli genes have been reported by Scherer et al.
rcr et al., Nucl, Aclds, Res, 8.
p, 3895-3905゜1980), and codons preferentially used in E. coli have been reported by Ikemura (Ikemura, Jpn, J. Genet, 56
．． 1), 533-555.1981).

The DNA strand of the present invention is for high expression by ligating it to an appropriate vector and introducing it into E. coli.
In order to achieve high expression of this DNA strand, the following nucleotide sequence TAAGGAGGTATATATT ATTCCTCCCATATAA (Scherer et al., Nucl, Ac1ds Res
, 8, p., 3895-3905°1980). Incidentally, this base sequence is frequently used in the translation initiation site of various genes of E. coli. Furthermore, the DNA strand of the present invention preferably has at least one stop codon (for example, TGA) adjacent to the 3' end of the base sequence shown in FIG. Furthermore, it is preferable to add appropriate restriction enzyme sites to facilitate such genetic recombination operations.

Therefore, the preferred DNA strand of the present invention has the base sequence shown from A to B in FIG.

Production of the DNA strand of the present invention An example of the method for producing the DNA strand of the present invention is as shown below.

(1) A DNA base sequence encoding the N-terminal 14 amino acids of calmodulin containing the Escherichia coli SD sequence and Met, and an 81 base pair oligonucleotide containing several types of restriction enzyme sites so that it can be inserted into an expression vector. , cloning plasmid pU C19 (Messi
ng et al.: Gene 33. p, 110-1151985
) to insert and concatenate. FIG. 2 (c) shows the base sequence and restriction enzyme site of this 81 base pair oligonucleotide. This oligonucleotide is shown in Figure 2 (
It can be obtained by synthesizing the oligonucleotides shown in (a) and (b), respectively, and then associating them.

Specifically, any of the above oligonucleotides can be synthesized by known synthetic methods (for example, solid phase synthesis by phosphoramidite method (Bcaucage et al.: Tctrahcdron Lcttcrs 22.
p, 1859-18 [i2.1981)). It is also possible to use an automatic synthesizer based on this synthesis method. After assembling each oligonucleotide, the 5' end is phosphorylated with polynucleotide kinase as necessary, and then D
It is ligated to the HindIII and BamHI cut fragment of pUC19 using NA ligase to obtain plasmid pCALN1 containing the DNA base sequence shown in FIG. 2 (c). E. coli JM109 is transformed with this plasmid pCALN1. Next, pCALNl is isolated and the base sequence is confirmed by the dideoxy method.

(2) Plasmid pRCM1 (11, Nojima and I
l, 5okabe: J, Mo1. Biol, 193
, p. 439-445.1987) as HindnI
By cutting with
Rat calmodulin cDNA with part of its end deleted
get. The obtained cDNA is inserted into the HindII and Xbal important fragment of plasmid pcALN1 and ligated to obtain pOcAL7, which is used to transform Escherichia coli JM109. Next, pOcAL7 is isolated and the base sequence is confirmed by the dideoxy method. By digesting pOcAL7 thus obtained with AflII and BamHI, the DNA strand of the present invention (A to B in FIG. 5) containing the base sequence, SD sequence, and stop codon shown in FIG. 1 can be obtained. Can be done.

Preparation of transformants The DNA strand of the present invention prepared as described above contains genetic information for producing calmodulin protein, so a plasmid containing this was introduced into Escherichia coli (E, colt) using biotechnological techniques. Calmodulin protein can be produced by introducing the vector, transforming it, and culturing the resulting transformant.

The procedure or method itself for creating a transformant (and thereby producing calmodulin) can be one that is commonly used in the fields of molecular biology, bioengineering, or genetic engineering. Other than what was done previously, it is sufficient to follow these commonly used techniques. In order to express the gene of the DNA strand of the present invention in E. coli, it is first necessary to connect this gene to a plasmid vector that stably exists in E. coli.

As this plasmid vector, any suitable vector such as pB1322 can be used.

On the other hand, in order to express the gene of the DNA strand of the present invention in E. coli, it is necessary to transcribe the DNA into mRNA. For this purpose, a promoter, which is a signal for transcription, may be incorporated into the 5' upstream side of the DNA strand of the present invention. Regarding this promoter, trp, 1acSP
Various types such as L and OmpF are known, and any of these can be used in the present invention.

Furthermore, it is preferable to incorporate a terminator for terminating transcription on the 3' downstream side of the DNA strand of the present invention. Plasmid stability can be increased by inserting a terminator. As a terminator, trp
a,rrnc.

toop etc. can be used.

In addition, at the stage of translating mRNA into protein, a sequence (called an SD sequence) necessary for the liposome, which is the site of protein synthesis, to bind to the tip of the translation initiation site, is inserted before ATG, which is the initiation signal for protein synthesis. It is necessary to put it on. In order to express it more efficiently, this SD sequence and this S
It is preferable that the base sequence between the D sequence and ATG, which is the initiation signal for protein synthesis, be made close to the base sequence common to translation initiation sites in E. coli. For example, it is preferable that this sequence be the following base sequence.

TAAGGAGGTATATT ATTCCTCCATATAA The DNA strand of the present invention (A to B in Figure 5) prepared by the method shown in the above "Production of the DNA strand of the present invention" preferably contains this base sequence and the stop codon TGA. be.

Transformation of E. coli with the plasmid thus prepared can be carried out by any suitable method commonly used in the fields of genetic engineering and biotechnology. Appropriate basics or general notes on the general matters,
For example, Maniatis et al., rMolecular C
loning-A Laboratory Manua
l JCold Spring Harbor Lab
oratory (1982).

The transformant has a new trait (i.e. calmodulin production ability) due to the genetic information introduced by the DNA strand of the present invention, a trait derived from the vector used, and a trait derived from the vector used during genetic recombination that may have occurred in some cases. Except for the lack of some genetic information, its xenotype, phenotype, and mycological properties are the same as the E. coli used.

Production of calmodulin When the transformant clone obtained as described above is cultured, calmodulin protein is produced in the cells of the culture.

The culture or growth conditions for the transformant are essentially the same as those for the E. coli used. In addition, any method suitable for recovering the produced protein from the culture, that is, the bacterial cells and/or the culture solution (for example, the method described in Example 5 below)
It can be done according to the following. Since calmodulin is produced in E. coli, after disrupting the bacterial cells, Tris-
It is preferable to extract the protein having calmodulin activity using HCl (pH 8,0) buffer or the like and purify it according to a conventional method.

Example Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1: Synthesis of oligonucleotides and insertion into cloning vectors The two oligonucleotides shown in FIG.
trahedron Letters 22. p, 18
59-1882.1981) using an automatic DNA synthesizer (Model 380A, M, H manufactured by Applied Biosystems)
Unkapi Iler et al.: Nature 310.
p, 105-111.1984).

After completion of the synthesis, it was treated with concentrated aqueous ammonia at 60° C. for 5 hours to remove the base protecting group and to cut it out from the carrier. The obtained oligonucleotide was subjected to electrophoresis using 12% polyacrylamide containing 8M urea.

After electrophoresis, a thin layer chromatography plate containing a fluorescent dye was placed under the gel, and the presence of the target band was confirmed using a UV lamp. A gel piece containing the desired oligonucleotide was sealed in a dialysis tube, and the DNA was electrically eluted from the gel. The solution in this dialysis tube was filtered through a gel filtration column (φ1) of Sephadex G-25 (manufactured by Pharmacia).
．． 5×43 cm) and eluted with 0.05 M) ethylamine bicarbonate buffer (pH 7.5) to desalt.

The eluate containing the desired oligonucleotide was concentrated under reduced pressure to obtain a pure oligonucleotide.

Two oligonucleotides obtained in this way (one each
nmol) of each 5' end of phosphorylation reaction solution (50mM Tris-HC1 (pH 7,
4) , 10mM Mg CI 2.10+nMDTT,
5mM ATP, 15 units of T4 polynucleotide kinase (Boehringer Mannheim).
Complete phosphorylation was achieved by reacting at ℃ for 30 minutes.

Next, the reaction solution was mixed, heated at 95° C. for 5 minutes, and returned to room temperature over 2 hours to prepare double-stranded DNA.

The cloning vector is E. coli plasmid pUc1.
9 (Pharmacia) was used. 1 μg pUC19
Add 30 μl of DNA to the reaction solution (10IIMT r i 5
-HCl (pH 7,5), 1011 MM g CI
2.1mM DTT, 50MMNaCl, 10 units of Bindm (Takara Shuzo), 10 units of BamHI (
After reacting for 2 hours at 37°C in Takara Shuzo), 0.8%
Large fragments were separated by agarose electrophoresis.

The gel piece was placed in a tube and eluted by electrophoresis in running buffer. The eluate was precipitated by adding ethanol, and 20 μl of a warm TE solution was mixed with 10 mM Tris.

This precipitate was dissolved in 1+nMEDTAS pH 8.0). 81 base pair double-stranded DNA prepared as described above
Add 0.1 μg of A to the above pUc19 DNA fragment TE solution, add 30 μg of the reaction solution (66mM Tris
-HC1 (pH 7,4), 6.6mM g CI
2.10mM DTT, 0.1mM ATP.

The reaction was carried out in 3 units of T4 DNA ligase (Boehringer Mannheim) at 14°C for 6 hours.

Using this reaction solution, Escherichia coli strain JM109 (Takara Shuzo 11!
l) (C, Yanisch-Perron et al.: G
ene 33, p, 103-119.1985) using known methods (D, IIanahan: J, Mol.

BloI, p. 557-580.1980).

At that time, an LB plate containing 50 Mg/ml ampicillin was used as the plate.

When the double-stranded DNA prepared as described above is inserted between HindIII and BamHI of pUc19,
There is a unique Af Im restriction enzyme site due to the inserted DNA. Therefore, plasmid DNA was extracted using the alkaline method (T, Maniatis et al.: Molecular C
Ioning, p! 6g-389, 1982, Cold
It was isolated from an Escherichia coli strain at Spring Harbor) and confirmed to be digested by Af Im (Takara Shuzo).

The DNA inserted into the thus obtained plasmid pcALN1 was tested using the dideoxy method (Hattori).
et al.: Anal, Blocheffi, 152, p.
, 232-238° 1986) to confirm its nucleotide sequence. The strain was named pcALN1/JM109 (see Figure 6).

Example 2: Subcloning of rat calmodulin cDNA Rat calmodulin cDNA is a cDNA constructed from rat brain mRNA by the Okayama-Berg method.
The A library was transformed into a rat calmodulin pseudogene (H,
NojiIla and H, 5okabe: J, M
ol.

Blot, 190. pJ91-400.1986)
can be cloned by screening using as a probe (H, Nojima and H, 5okabe: J.

Hot, Blot, 193. p, 439-445
．． 1987).

E. coli D transformed with plasmid pRcM1 in which rat calmodulin cDNA has been cloned
From HI strain, alkaline method (T, Man-iatis et al.:
Molecular Cloning pJ88-3
89.1982゜Co1d Spring Harbo
pRcMl was isolated using pRcMl (A-B in Figure 4 shows rat calmodulin cDNA in pRcMl).
. 1Fg of pRcMI DNA was added to 30 μl of the reaction solution (10 mM Tris-HC1 (pH 7,5),
10 lIMM g CI 2, IIfiMDTT,
3 in 50mN aCl, 10 units of Hindm (Takara Shuzo), 10 units of Xbal (Takara Shuzo)
After reacting at 7°C for 2 hours, small fragments were separated by 0.8% agarose electrophoresis. The gel piece was placed in a tube and eluted by electrophoresis in running buffer. Add ethanol to the eluate to precipitate it, and add 20u1 of TE warm solution to 10ffiMTris.

This precipitate was dissolved in 1mM EDTA (pH 8.0).

30 μl of plasmid pcALN1 obtained in Example 1
reaction solution (101uMT r i 5-HC1 (pH 7
,5), 10mM g CI 2.1nMDTT.

50ffiMNaCl, 10:L-tHi
ndm (Takara Shuzo), 10 units of XbaI (Takara Shuzo)
) for 2 hours at 37°C, the large fragment was separated by 0.8% agarose electrophoresis.

The gel piece was placed in a tube and eluted by electrophoresis in running buffer. The eluate was precipitated by adding ethanol, and 20 μl of TE warm solution 10 mM Tris, I
This precipitate was dissolved in IIMEDTAS pH 8.0). This TE warm solution was prepared using calmodulin cDNA containing a portion of the 433 base pair N-terminus prepared as described above.
, 2μg was added to 30ul of the reaction solution (66mMT r i
s-HC1 (pH 7, 4), 6.6 IIIMM
g CI 2.10n+MDTT, 0.1mMATP
, 3 units of T4 DNA ligase (Boehringer Mannheim) at 14°C for 6 hours. Using this reaction solution, Escherichia coli JM109 strain (C, Yanisc)
h-Perron et al.: Gene 33, p, 103
-119.1985) by a known method (D, Hanaha
n: J, Mol.

Biol, I), 557-580.1980).

At that time, an LB plate containing 50 ug/tol of ampicillin was used. Plasmid DNA was extracted from ampicillin-resistant colonies using the alkaline method (T, M
anjatis et al.: Molecular Cl
onjng, p, 368-369.1982, Cold
Spring Harbor) was isolated from an E. coli strain, digested with AflII and BamHI, and reduced to about 500
It was confirmed that DNA encoding mature calmodulin containing a base pair SD sequence was inserted. Then, the dideoxy method (Ilattori et al.: ^na1.Bioc
hem, 152, p. 232-238, 1986
) was used to confirm the base sequence (Figure 5). The obtained strain was named pOcAL7/JMI 09 (see Figure 6).

Example 3: Construction of a plasmid for expressing calmodulin A plasmid for expressing human granulocyte-macrophage colony-stimulating factor (hGMC3F) psT, which has already been proposed
A plasmid pTCAL7 for expression of calmodulin was constructed using 6311 (Fig. 6) (see Japanese Patent Application No. 106148/1982).

psT6311 is derived from pBR322-derived pAT153 (
It was constructed based on the E. coli tryptophan operon chemically synthesized between EcORI and Af III, and contains the promoter, operator, and base sequence from the transcription start point to just before the SD sequence. SD chemically synthesized between [ and BamHI
The sequence and structural gene of hGM-C3F were further modified by Bam
This is an hGM-C3F expression vector containing a chemically synthesized t rpa terminator between HI and sph I.

The pOcAL7 obtained in Example 2 was transformed into Af III and B
After digestion with amHI, a 498 base pair DNA fragment was purified by 0.8% agarose electrophoresis. After the above expression vector psT6311 was digested with Af Im and BamHI, calmodulin containing the above 498 base pair SD sequence was ligated and inserted using T4 ligase, and then E. coli strain W3110 (ATCC27325) was inserted.
was transformed. A plasmid was isolated from an ampicillin-resistant colony and digested with Af Im and BamHI to obtain a DNA fragment of about 500 base pairs, into which the gene containing the SD sequence of the present invention (A to B in Figure 5) was inserted. I confirmed that. The obtained strain was named pTCAL7/W3110 (see Figure 6).

Example 4 Calmodulin production method (expression of DNA strand shown in Figure 1) Expression of plasmid pTCAL7/W3110 pTCAL7/W3110 obtained in Example 3 was cultured with shaking in a medium containing ampicillin at 37°C overnight. did. This culture solution 501 was added to 10100O M9 medium (containing 0.8% glucose, 0.4% casamino acids, 10 μg/m Ithiamine, 50 μg/ml ampicillin) and incubated at 37°C.
The mixture was shaken for 3 hours. Indole acrylic acid was added to a final concentration of 20 μg/n+I. The culture was further continued with shaking for 4 hours. A portion of the obtained E. coli was boiled (5 minutes) in sample buffer, and the boiling solution was 5DS
- Polyacrylamide electrophoresis (SDS-PAGE) was performed to examine the calmodulin content. Under these conditions, the calmodulin content was about 25% or more of E. coli cell protein.

This culture solution was centrifuged to obtain bacterial cells. The obtained bacterial cells were 2011IMT r i s -HC1 (pH 8,0)
Lysozyme was added to this suspension to a final concentration of 10 mg/ml, and after standing in water for 1 hour, IIIM dithiothreitol, 1mMEDT
Ultrasonic treatment was carried out in water with adjustment to A.

This suspension was heated at 90°C for 5 minutes, then cooled in water,
Ultracentrifugation was performed at 10,000x for 30 minutes at 4°C, and the supernatant was collected.

50+nM Tri 5-HCl (pH 7,5
The supernatant obtained above was added to a phenyl Sepharose CL-4B column (Pharmacia: φ1 x 19 cm) that had been equilibrated with 150 ml of
0+nMT r i 5-IC1 (pH 7,5, III
It was eluted with 150 ml of IMEDTA).

The flow-through fractions were collected, adjusted to 5IIIMCaC12, and pretreated with 50mM Tri 5-HC1 (pH 7, 5, 1f
It was added to a Phenyl Sephas CL-4B column (Pharmacia: φ1 x 38 cm) that had been equilibrated with 300 ml of f1MCaC12). Initially 50mMTri
5-HCI (pH 7, 5, 1mM CaCl2
) It was eluted at 300 ml. Then 50mM TrisH
Cl (pH 7, 5, 1liM Ca Cl 2, 0, 5
Elution was performed with 300 ml of MNaCl). Finally 50mM
T r i 5-HCl (pH 7, 5, 1a MED
The target calmodulin protein fraction was recovered by elution with 300 ml of TA). The collected solution was repeatedly dialyzed against water three times and freeze-dried to obtain a powdered calmodulin protein (approximately 400 mg).

Example 5: Activity of Calmodulin C prepared from rat brain with various calmodulins
The activation of yclic AMP (cAMP) phosphogesterase was determined.

Reaction liquid 0. 5ml (80mM imidazole hydrochloride buffer (pt(6,9), 3mM g CI 2.0
．． 3mM dithiothreitol, O-2mM CaC
12.1 mg/ml bovine blood blue albumin, phosphogesterase 200 μg/mL calmodulin (calmodulin expressed in E. coli (lyophilized powder obtained in Example 4) or calmodulin prepared from rat brain), 2mMcAMP) 30 After reacting at 100° C. for 30 minutes, the reaction was stopped by heating at 100° C. for 3 minutes. To this was added 0.21 5'-nucleotidase solution (0.5 mg
/m15'-nucleotidase, 30 mM n
Cl 2 ) was added and incubated at 30°C for 20 minutes to perform Pi shadow formation (S, Kakiuchj et al.
19g2): J, Blochcm, 92. p, 10
41).

After stopping the reaction by adding 0.31% of 16.7% perchloric acid,
Proteins were removed by centrifugation at 3000 rpm for 5 minutes, and Pi in the supernatant fraction was determined using the inorganic phosphorus quantitative method (B, N, A
mes: ”Methods in Enzymolo
gyE, P. Ncufeld, V. Ginsburg.
, Acadmic Press.

New York, Vol. 8. p, 115,198
8).

That is, 2.1 ml of reaction solution (0,71 (above supernatant fraction + H20), 1.4 a + 1 (0,211111 O
% ascorbic acid + 1.2 ml 10.42% ammonium molybdate in IN sulfuric acid)) was incubated at 45° C. for 20 minutes, then returned to room temperature and the absorbance at 770 nm was measured to quantify Pi. As a result, the calmodulin protein expressed in E. coli showed almost the same activity as calmodulin prepared from brain. The measurement results are as follows.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing the amino acid sequence of calmodulin and the DNA base sequence according to the present invention encoding the same. FIG. 2 is an explanatory diagram showing the base sequence and restriction enzyme site of a chemically synthesized oligonucleotide. FIG. 3 is an explanatory diagram showing the amino acid sequence encoded by the DNA base sequence of FIG. 2. Figure 4 shows the rat calmodulin cDNA in pRcMl.
FIG. 2 is an explanatory diagram showing the base sequence and restriction enzyme sites of A. Figure 5 shows the base sequence encoding calmodulin, SD
FIG. 2 is an illustration showing a preferred DNA strand of the invention including sequence and stop codon. FIG. 6 is an explanatory diagram showing the cloning of calmodulin into pUc19 and the construction of an expression vector. Applicant's agent Mr. Sato

Claims (1)

[Claims] 1. A DNA strand containing the base sequence shown in FIG. 2. Escherichia coli (E. coli) transformed with a plasmid containing a DNA strand containing the base sequence shown in FIG.
i). 3. Prepare a DNA strand containing the base sequence shown in Figure 1, create a plasmid containing this DNA strand in a state where its genetic information can be expressed, and use this plasmid to infect E. coli (
E. 1. A method for producing calmodulin, which comprises producing calmodulin in a culture through a step consisting of transforming E. coli and culturing the resulting transformant.