JPS61135586A - Plasmid containing luminescent protein aqualin, and biosynthesis of said luminescent protein - Google Patents

Abstract

PURPOSE:To obtain the title plasmid, by separating the aqualine gene specifying the production of aqualine protein from mRNA as cDNA clone by Okayama- Berg process. CONSTITUTION:Aqualine gene essentially specifying the biosynthesis of aqualine which is a jellyfish calcium luminescent protein was separated from mRNA as a cDNA clone by Okayama-Berg process, and the cDNA clone was named as plasmid pAQ440. The plasmid pAQ440 has the nucleotide sequence shown as the figure, has a size of 958bp (base pair) and contains the open reading claim (translated region) of 588bp. The luminescent protein aqualine is useful as a clinical medicine or reagent for the determination of trace Ca<2+>.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Technology] The present invention relates to plasmid pAQ440 and a DNA transport vector containing this as an active ingredient. More specifically, the present invention relates to a novel plasmid pAQ440 constructed from pBR322-8V40 and a portion containing a gene that specifies the biosynthesis of the jellyfish photoprotein aquarin.

[Background of the invention]

Aquarin is a photoprotein of the luminescent jellyfish that grows in the northern part of the west coast of the United States, and one molecule of aquarin specifically binds two
When combined with molecular Ca2+, it reduces the emitter coelenterazine and emits light. On the other hand, since Ca2+ is indispensable for the luminescence of aquarium, it is possible to
a''+ can be specifically quantified, and trace amounts of Ca2+ concentration of about 10''-'M can be measured.

Currently, there are many Ca2+-dependent enzymes in living organisms, and C
Due to the possibility of causing serious diseases due to A2+ deficiency,
This aquarin has the potential to be widely used in clinical tests, and by injecting it into cells, intracellular Ca''+
It can also be applied to things such as concentration measurement and cold light sources.

However, this jellyfish grows only in the northwestern part of the west coast of the United States, only occurs in the summer, and the yield is only about 1 inch per 10,000, which is not enough. Constant supply cannot be guaranteed. Therefore, the present inventor conducted intensive research in order to solve the above-mentioned problem, and used genetic engineering technology to develop the aquarin gene that essentially specifies the production of aquarin protein.
A cDNA clone was isolated and prepared from mRNA by the Yams-Berg method.

That is, a cDNA clone of the jellyfish photoprotein aquarin was created using recombinant DNA procedures, and this cDNA
It was named NA clone lasmid PAQ 440.

[Structure of the invention]

The nucleotide sequence of plasmid pAQ440 of the present invention is shown in FIG. Furthermore, the present invention encompasses a DN human delivery vector comprising the above-mentioned plasmid pAQ44G and/or a functional equivalent of the above-mentioned nucleotide sequence as an active ingredient. Functional equivalents herein refer to nucleotide sequences that can be used in substantially the same manner to obtain substantially the same results in the production of jellyfish photoprotein aquarium by a suitable host.

To further explain the invention, FIG. 2 shows a restriction enzyme map of cloned Grasmid I) AQ440.

In the figure, Loco indicates the part containing the aquarin gene, W part indicates the Aynpr part of pBR322, and a part is
A part of the vector of Okayama-no(-g) is shown.

Table 1 shows the cleavage sites and the number of cleavage sites using restriction enzymes.

Table 1 FIG. 3 shows a detailed restriction enzyme map of the aquarin gene cloned into plasmid pAQ440 and the direction of determination of the base sequence using the Massarum-Gilbert method. In the figure, the cDNA clone plasmid pAQ
440 contains the entire gene for the jellyfish photoprotein aquarin, as well as several hundred nucleotides of 5' and 3' cranking sequences.

The nucleotide sequence of the aquarin gene revealed in Figure 1 above is 958 bp (pace pair), 5as
Contains an open reading claim (translation portion) of bp.

The method for preparing pAQ440, which is a cDNA clone of the present invention, will be explained in detail based on FIG.

Total RNA is extracted from the multiple edges of jellyfish (A@quarea vtctorta) by the guanidine hydrochloride method*. Next, poly A” mRNA was purified by oligo dt cellulose column chromatography, and Oka
A cDN human library was constructed using the Yama-Berg method (Note *H, Okayame et al.
1. Ce11. Biol 2.161 (1982
) ) (Figure 4 ■).

On the other hand, the amino acid sequence near the N-terminus of aquarin protein extracted from jellyfish and the amino acid sequence after bromocyanide degradation were determined, and 14
Nucleotides were chemically synthesized and used as probes for screening the cDNA library described above.

The nucleotide sequence was determined, and the aquarin gene was identified from the amino acid sequence and amino acid composition of the aquarin protein (Fig. 4 ■).

Okayam used in Figure 5 ■ related to the above explanation
a-Berg vector and 01igo (dG) ■
nker was prepared by the method shown in ■ and ■ in FIG.

〔Effect of the invention〕

As mentioned above, the photoprotein aquarin produced by the aquarin cDNA clone pAQ440 of the present invention is
It is clear that it is useful as a clinical medicine or reagent for quantifying trace amounts of Ca2+.

Then, in order to carry out the synthesis of the aquarin, this resin a
A prokaryotic cell host (eg, E. coli) or a eukaryotic cell host (eg, a cultured cell system) can be used as a host for the protein. Such hosts are well known to those skilled in the art. Furthermore, cDNA clone pAQ440 according to the present invention
The nucleotide sequence of the aquarin gene contained in 1c can also be used for cloning into other vectors or hosts by methods well known to those skilled in the art.

〔Example〕

The present invention will be explained below with reference to Examples.

Example ① cDNA DNA library: This will be explained according to FIG.

(Step 1: Synthesis of cDNA) Jellyfish double border 1169 was treated by the guanidine hydrochloride method to obtain total RNA 5115J. This one is 50μ9
5.1 μI of polyA+Metsenger RNA
(mRNA).

Of this, 3.81J of poIyA+RNA was lyophilized and 10μl of 5mM Tris-H (J(
pH 8,3) buffer and incubated at 90°C for 1 minute.

Next, incubate at 37°C for 5 minutes, add 10 μj of reaction solution (described below), and incubate with reverse enzyme (LifeScIen
ce company jilri 1. OU was added and incubated at 37°C for 60 minutes.

The composition of the reaction solution described above was 500 mM Tris-HCI.
(pH 8,3) buffer, 80mM
gC11, 300mMKC; 2 fig, 20
mMdNTP; 2114.1.4/Jg 1 μl
Okayama-Berg primer-DNA
; Iμ1. : a, o; 2μノ.

3000 Ci/Imol α-(”P)-dcTP;
1μm, 6mM dithiothreitol; 1μ! It is.

2 ttl of 250 mM FiDTA after the above reaction;
The reaction was stopped by adding 1/JA' of 10% SDS, the reaction system was extracted with the same amount of phenol-chloroform, and the supernatant of the extract was diluted with 4M Ammonium acetat.
E was added to perform ethanol precipitation, and the precipitate was washed with 75% ethanol and dried in a vacuum desiccator.

(Step 2: DC chain addition) Add 1.4 μl of the reaction solution (described later) to the precipitate obtained in Step 1 and dissolve it, then add 1 μl (15 U) of terminal transferase (P, I+, Biochem).
(manufactured by i-cal Inc) and reacted at 37°C for 3 minutes, EDTA-8D8 was added to stop the reaction, and the DNA was recovered by a conventional method of extraction with phenol/chloroform. The composition of the above reaction solution was 1.4M cacodylic acid-0
, 3M Tris-KOH (pH 6,8) buffer; 1.5 μl, 10 mM cocll; 1.
5 Al, 1 mM dCTP; I Al, C1-
(”P)dcTP (3000Ci 1mmol);l
μ4 Htoys, sμ41mM diteothreitol; 1.5μ! It is.

(Step 3: Hindll digestion) Reaction solution of recovered DNA K 1 ttl obtained in step 2 (described later)
, water 8μ/, Hind m (20U/Ail)
After adding 1 μj and reacting at 37° C. for 60 minutes, DNA was collected by a conventional method. The composition of the above reaction solution was 200
mM Tris-HCl (pH 7,5)t 70
mMgcA'ty600 mM NaCl.

(Step 4: Denaturation from RNA strand to DNA strand) cDNA-prime digested with Hind m in step 3
r Reaction solution of 1 μl of DNA solution and 10 μl of Ilc (described later)
was added, incubated at 65°C for 2 minutes and at 42°C for 30 minutes, and then cooled on ice to anneal the 1inker DNA having a dG strand and the cDNA prior DNA.

The composition of the above reaction solution was 10 mM Tris-HCl
(pH 7,5), 1 mM EDTA, 0.1 MN
aC47rLgOl: 90(dG)tailed t
Inker @ After the above-mentioned annealing, it was left to stand overnight at 12° C. under the following conditions.

Further, the following materials were added in order, and the mixture was reacted at 12°C for 1 hour, and then ink-epated at 25°C for an additional 3 hours.

(Step 5: Transformation) The circular DNA obtained in Step 4 was transformed into competent cells DH1 prepared in a conventional manner to obtain about 15,000 transformants.

(Note* D, Hanakan, J, Mo1. Biol
, 166557 (1983)) above steps 1~
A jellyfish cDNA library was obtained using 51C.

■ Aquarin gene sparing method: The amino acid sequence of the aquarin protein isolated from jellyfish was determined.

The N-terminus of the protein is Val Lys Leu()()Asp Phe As
p AsmPro... Bromosia/Met Asp Pro Ala Cys Gln L after treatment
ys Leu Tyr GlyMet(Phe) As
n(Phe) Leu Asp Val Asn()A
sn Gly...

Next, four types of DNA corresponding to the above amino acids were created using a synthesizer.

The ends of the above four types of DNA are 3tp 5' under the following conditions.
I labeled it and used it as a glove.

Using this probe, a cDNA library was screened by conventional colony hybridization to obtain a (positive) clone pAQ440.

(Note* R, B, Wallace et al.
Nucleic Ac1d Res 9 879
(1881)) This (positive) clone has pAQ
The nucleotide sequence of 440 was determined by the Maxam-Gi 1bert method, and the amino acid sequence and other protein properties were compared with those of the jellyfish photoprotein.

[Brief explanation of drawings]

FIG. 1 shows the nucleotide sequence of plasmid pAQ440 of the invention. FIG. 2 shows a restriction enzyme map of the aquarin gene of the cloned plasmid pAQ440. Figure 3 shows a detailed restriction enzyme map of the cloned aquarin gene and the Maxam-Gilbert method! ='! = Indicates the direction of base sequence determination. Figure 4 (■) is a process diagram showing a method for creating a cDNA Library, and Figure 4 (■) is a process diagram showing a method for screening an aquarin gene from a cDNA Library. Figure 5 ■ shows the preparation method of Okayama-Berg Vectoc, and Figure 5 ■ shows the preparation method of Okayama-Berg Vectoc.
A method for preparing ker is shown. Applicants for the above patents: Chi, So Co., Ltd. Masato Patent Attorney Yatabu Sasai Katsuhiko Ueno Naka Figure 2 Hlndm [1. Indication of the case 1982 Patent Application No. 176125 8
゜2. Name of the invention Plasmid having the photoprotein aquarin gene and biosynthesis method of the photoprotein 3. Relationship with the case of the person making the amendment Patent applicant 3-6-32 Nakanoshima, Kita-ku, Osaka-shi, Osaka Prefecture (530) )
(207) Chisso Corporation Representative Sadao Nogi 4, Agent 2-8-1 Shinjuku, Shinjuku-ku, Tokyo (160) 1982
November 7, 1982 (7th dispatch 1' Amendment related to November 27, 1982)
Claims of the specification to be amended Contents of the amendment The specification (claims) originally attached to the application
As shown in the attached sheet (no changes in content). Attached document attachment (full text of claims) One or more copies - a? Q-

Claims (10)

[Claims] (1) Plasmid pAQ440 carrying a substantially isolated and complete gene specifying the biosynthesis of the jellyfish calcium photoprotein aquarin. (2) Plasmid pAQ440 having the deoxyribonucleotide sequence of the photoprotein aquarin shown below. [There is an amino acid sequence] Here, the 5'-3' strand begins with the amide end and the amino acids indicated by each triplet number,
and in each triplet, A is deoxyadenyl, T is thymidyl, G is deoxyguanyl, C is deoxytosyl, Gly is glycine, Ala is alanine, Val is valine, Leu is leucine, Ilc is isoleucine, Ser is serine, Thr is tyrosine, Trp is tryptophan, Cys is cysteine, Met is methionine, Azp is aspartic acid, and Glu is glutamic acid, Lys is lysine, ARg is arginine, His is histidine, Pro is proline, Gln is glutamine, and Azn is asparagine. (3) A DNA transport vector comprising plasmid pAQ440 having the deoxyribonucleotide sequence of the photoprotein aquarin shown below and/or a functional equivalent of the nucleotide sequence as an organic component. (However, "functional equivalent" refers to a nucleotide sequence that can be used in substantially the same way to obtain substantially the same results in the production of the jellyfish photoprotein aquarin by a suitable host.) [Amino acid sequences available] here The 5'-3' strand begins with an amine terminus and an amino acid with each triflet number indicated;
and in each triplet, A is deoxyadenyl, T is thymidyl, G is deoxyguanyl, C is deoxytosyl, Gly is glycine, Ala is alanine, Val is valine, Leu is leucine, Ilc is isoleucine, Ser is serine, Thr is tyrosine, Trp is tryptophan, Cys is cysteine, Met is methionine, Azp is aspartic acid, and Glu is glutamic acid, Lys is lysine, ARg is arginine, His is histidine, Pro is proline, Gln is glutamine, and Azn is asparagine. (4) The DNA transport vector according to claim (3), which is transported to a microorganism and replicated by the microorganism. (5) The DNA transport vector according to claim (4), wherein the microorganism is a bacterium. (6) the bacteria is Escherichia coli HB101 or DH1,
The plasmid is pBR322-SV40 (Okayama
-Berg's vector)
The DNA transport vector described in Section 1. (7) A method for producing the jellyfish photoprotein aquarin, which comprises culturing a host modified to contain the following deoxyribonucleotide sequence of the photoprotein aquarin or a functional equivalent of this nucleotide sequence. [There is an amino acid sequence] Here, the 5'-3' strand starts with the amine end and the amino acid with each triflet number indicated,
and in each triplet, A is deoxyadenyl, T is thymidyl, G is deoxyguanyl, C is deoxytosyl, Gly is glycine, Ala is alanine, Val is valine, Leu is leucine, Ilc is isoleucine, Ser is serine, Thr is tyrosine, Trp is tryptophan, Cys is cysteine, Met is methionine, Azp is aspartic acid, and Glu is glutamic acid, Lys is lysine, ARg is arginine, His is histidine, Pro is proline, Glu is glutamine, and Azn is asparagine. (8) The method according to claim (7), wherein the host is a eukaryotic cell. (9) The method according to claim (7), wherein the host is a bacterium. (10) The method according to claim (9), wherein the bacterium is Escherichia coli.