JPS6043378A - Reverse transcriptase - Google Patents

Abstract

PURPOSE:To provide a reverse transcriptase having excellent stability and purifiable without causing the problem of safety, by separating from the cultured cell of Drosophila melanogaster. CONSTITUTION:A reverse transcriptase separated from the cultured cell of Drosophila melanogaster, exhibiting the RNA-dependent DNA synthesis activity and DNA-dependent DNA synthesis activity as well as liponuclease H activity. The conventional reverse transcriptase playing an important role as a reagent in the field of biochemical research and genetic engineering is originated from the tumor cell of poultry and mammal, and accordingly, the purification should be carried out with sufficient caution to safety. In contrast to the above, the reverse transcriptase of the present invention is separated from the cultured cell of Drosophila melanogaster absolutely free from the safety problem paying the attention to the reverse transcriptase activity of the retroviral particles in the nucleus of the cell, and accordingly, it has absolute safety and excellent stability.

Description

[Detailed description of the invention] The present invention relates to reverse transcriptase.

From RNA tumor viruses in 1970, RNA-dependent DNA
A reverse transcriptase with A-synthesizing activity was discovered.
Since it is now possible to synthesize DNA having a complementary nucleotide sequence from -R,NA9, reverse transcriptase plays an important role as a reagent in the fields of biochemical research and genetic engineering. However, since conventional reverse transcriptases are derived from tumor viruses of horses and mammals (mouse, humans, monkeys, etc.), sufficient attention must be paid to safety during the purification process.

The present inventors focused on the reverse transcriptase-like activity of retrovirus-like particles (hereinafter referred to as RVTJP) in the nucleus of cultured cells of Drosophila melanogaster, an insect that poses a complete safety problem. The present invention was achieved by successfully isolating a reverse transcriptase with excellent stability. That is, the gist of the present invention is as follows.

Reverse transcriptase (A) obtained from cultured Drosophila melanogaster cells has the following physicochemical properties: (A) exhibits RNA-dependent DNA synthesis activity; C), showing H activity on ribonucleas.

(A) RNA-dependent DNA synthesis activity (a) Using pol, 7A or pol, yc as a template and a deoxynucleotide oligomer complementary to this as a primer, generate a deoxynucleotide homo71 jellymer having the same nucleotide sequence as the primer. Synthesize.

(1)) DNA having complementary nucleotide sequences can be synthesized from cultured cells of Drosophila melanogaster.

(13) DNA-dependent DNA synthesis activity [po], y Using dA as a template and a complementary monodeoxynucleotide oligomer as a primer, a homo-7 deoxynucleotide oligomer having the same nucleotide sequence as the primer is synthesized.

(c) Ribonuclease H activity po1. Using the yA-poly d T hybrid chain as a substrate, only the poly A chain is degraded.

(b) Optimum pH 7.8 (, Q Optimum temperature for action 23.5°C to 32.5°C (b) Isoelectric point pl 5.6 In the following detailed explanation of the present invention, the reverse transcriptase of the present invention is Drosophila melanogaster, Drosophila melanogaster
ter) is collected from cultured cells. Regarding Doronphylla melanogaster, for example, Chromosoma (
Chromosoma), Volume 38.

1; page 9 (1972).

In order to obtain the reverse transcriptase of the present invention (hereinafter simply referred to as enzyme), for example, as shown in the Examples below, Frosophila melanogaster cultured cell line GM2 is prepared in an appropriate culture medium.
The cultured cells are collected and swollen in a buffer containing a surfactant, the cells are disrupted through a syringe needle, and then centrifuged to collect the nucleus inside the cells. The collected nuclei are suspended in a buffer solution, treated with ultrasonic solution at low temperature (4 t) to destroy the nuclei, and then centrifuged to collect the retrovirus-like particles present in the nuclei. Crude R, V T,
P is purified by treatment with sucrose density gradient centrifugation. Purified RV TJP was solubilized with surfactant under high salt concentration,
By treatment with cesium chloride density gradient centrifugation, R
■■-Enzyme in 1P: Fractions are collected and further subjected to column chromatography and dialysis to purify the desired enzyme.

Next, the physicochemical properties of the enzyme of the present invention will be specifically explained. The enzyme activity of the present invention was measured by the methods described in (G), (B), and (C) below.

[Measurement of enzyme activity] (A) Measurement of RNA-dependent DNA synthesis activity ta) -〇 pol and yA as templates, o as primers], i
Method using go dT (oligothymidyl deoxyribonucleotide), composition of reaction solution Nidris hydrochloric acid (pH 7,5) 10 rr+MK, Ctl
QOmM -〇-DTT (dithiothreitol) 2mM MgC720
,5mM MnCt20.5mM pol,y A [1,0101)260 +3ol
i go (rumor T) 12-1 days (chain length 12-18) 0.
010D26o u Distilled water Total volume 50 μl (Note) 5H-aTTp: Deoxythymidine triphosphate (
Methyl-tritium; Measuring method: Add 5μ of the enzyme solution to the reaction solution, hold at 27°C for 1 hour, and then adjust the pH to 5[]mM ETDA (Na0T).
8) Add 10μt of ion-exchanged cellulose F
i paper (DE81, manufactured by Whatman Co., Ltd., diameter 2.4 tM) is impregnated. Next, add this to 035M Na, 2HPO45
After washing at least 7 times with ME, two times with distilled water, then two times with ethanol, and finally with diethyl ether to completely remove free 5H-dTTP, air drying and toluene scintillator. Measure radioactivity.

60 City Enzyme Degree: "-Under the conditions described, 1 mol of "T" per 4 minutes
The standard activity for incorporating T-dTTl) is 1 u (unit).

(a)-■ pol, , yc as template, ol, as primer
Method using igo dG (oligoguanyl deoxyribonucleotide) Composition of reaction solution: In place of pol and yA in the above (a)-■, poly
Using C0,010D26ou, also Oko]go(
dT) instead of 12-18 ol:1g0(dG)+2-
+s O,010D26ou, and further “T-1-
5H-dGTP [(8-)lithium deoxyguanine triphosphate] (5-20Ci/mm) instead of dTTP
o/! , 1 m (''i/m/-) 3-5 μC], respectively, except that the composition was the same as that of the reaction solution in (a)-〇 above.

Measurement method: Same as the measurement method of (a'l-■) above.

(b) Method using total RNA present in RVTJP (mixture of template and primer) as substrate Composition of reaction solution Nidris hydrochloric acid (pT(7,5) 10 mM KCtoo
mM DTT 2 mM MgC220, 5mM MnC1, 20, 5mM RVT, total T (NA in P) 1 tt?dATP (deoxyadesocine triphosphate) 1 μM dGTP (deoxyguanosine triphosphate) 1 μM → 7H cry9 (50-
80Ci/mmo41mCi/mt) 1 pc] Distilled water j50μt (Note) Total RNA in RVLP: In RVT, P,
4S RNA (t-RNA) V4.5S T (NA (
sma]-1nucl, ear RNA), 5S, and Nature (Nature Arrow No. 30)
Volume 2, March 10th issue, pp. 119-124 (1983)
].゛If 3H-dTTP is taken up using this total FtNA as a substrate, this enzyme will be able to absorb the total RNA-dependent D in T'lVJ,P.
This proves that it has NA synthesis activity.

Measurement method: The measurement method is the same as that of (a)-■ above.

(B) Measurement of DNA-dependent DNA synthesis activity Notes: Composition of solution: p used as template in (a)-■ of (Δ) above.
ol, instead of yAO, polydA O, 010D26
The composition of the reaction solution in (g) (a)-〇 is the same except that 0u is used.

Measurement method: The measurement method shall be the same as in (a)-(2) of (g) above.

(C) Measurement of 'J ponuclease H activity Composition of reaction solution Nidris acid (pH 7,5) 10mM K, C/1 [10mM DTT 2mM MgCt2 0.5mM MnCL2 0.5mM -] 〇-'HpolyA-polydT ;0X10'Cl' River distilled water Total amount 101C/Measurement method: Add 5μt of enzyme solution to the reaction solution and keep at 27℃, 20%
90μ of trichloroacetic acid and heat-denatured calf thymus DNA (
After mixing 30μt of 11n9/ml), cool on ice for 20 minutes and centrifuge at 4°C (12[1[1[]XL,
15 minutes). Collect 150μ of the supernatant and add 1
0ml liquid scintillator-[Toluene-Triton X-
100 (manufactured by Rohm and Haas) thread] and measure the radioactivity.

[Physical and chemical properties]

(a) Effect (T) (indicates NA-dependent DNA synthesis activity, (a) p
Using oly A or pocoyc as a template and a deoxynucleotide oligomer complementary thereto as a primer, a homozygous oligomer of deoxynucleotides having the same nucleotide sequence as this primer is synthesized. That is,
Using pOJyA as a template, 011g0(dT)12-18
PolydT (7 lithimidyl deoxyribonucleotide) was synthesized using the primer, polyC was used as the template, and o]ig○dG was used as the primer to synthesize pol-y.
dG (;e liganyldeoxyliponucleotyl-”
).

DNA having the sequence can be synthesized.

(13) Shows DNA-dependent DNA synthesis activity.

Using pol and ydA as templates and a complementary deoxynucleotide oligomer as a primer, a deoxynucleotide homopolymer having the same nucleotide sequence as the primer is synthesized. For example, pol, ydA
was used as a mold, and the weight was 011g.

(dT)12-18 as a primer, poly
Synthesize dT.

(C) Ribonuclease H activity is shown.

Using the pol, yA, -pol, ydT hybrid chain as a substrate, only the pol, yA chain is degraded.

(b) Optimal pH 7,8 The optimal pH for synthesizing pol and ydT in a Tris-HCl buffer using polyA as a template and 0co-igo dT as a primer was Z8.

09 Suitable temperature for action: 235°C to 62.5°C Poly A is used as a template, oljgodT is used as a primer, pol
The optimal temperature for synthesizing ydT is 27°C, and if the activity at this time is 100%, at 235°C it is 80%, 1
46% at 8℃, 84% at 32.5℃, 1 at 67℃
It was 9%.

2) Isoelectric point p15.6 ←) Stable pH, 6.5-8.5 When synthesizing polydT using po17A as a template and oligodT as a primer, adjust the temperature in advance to pH 8, 4°C.
When using enzymes that had been allowed to stand overnight, no decrease in activity was observed.

(f) Stabilized by the addition of a stabilizing nonionic surfactant. For example, it is stabilized by adding the nonionic surfactant Nonidetsu) P-4013- (NP-40, a product of Shell Co., Ltd.).

As detailed above, the enzyme of the present invention is derived from cultured cells of the insect Drosophila melanogaster, which is different from reverse transcriptase derived from avian or mammalian tumor viruses. Since there is no need to consider safety at all, and the stability is good even at relatively low protein concentrations, it is expected to be used as a reagent in the fields of biochemical research and genetic engineering. The method for preparing the enzyme of the present invention will be explained in more detail below with reference to Examples.

Example [Collection of 0M2 cells] Doronphila melanogaster M2 cells, 1 in 4
．． 0 g of potassium glutamate, 2. Of's Grishinkari,
6.99 sodium glutamate, 6.57 sodium glycine, 102 MgC42・6H20,6,72 Mg
SO4・7H20,0,47t of NaH2PO4・2H
20,1,22 CaCA2 ・2H20,0,67f
of malic acid, 0067 of cohelic acid, 0.0257 of sodium acetate/3H20, 34-2.07 of glucose, 0011 of phenol red, 0
．． 02 mg thiamine hydrochloride, o, 02117 Noriboflavin, 0.02 g pyridoxal, 0. [12m
g niacinamide, 0.0211+9 calcium pantothenate, 0.01Q biotin, [ ], 002M
folic acid, [], 051119 choline chloride, 0.02■
inositol, 002■ p-aminobenzoic acid, 20
Culture solution 2 containing 10,000 units of Nijirin and 100 μg of streptomycin at pH 6.7 (prepared with IOM's KOH aqueous solution) was added so that the cell density was 2 x 105/-, and kept at 26°C for 8 days. After stirring and culturing, centrifuge (3000Xr, 10 minutes) to culture cells at 2.5 m
1 was collected.

[Nucle collection]

10 mg of GM2 cells collected by the above method were added to buffer A.
(13.6mM NaCL, 2.7mM KCL'
17.3 mM Na2HPO4.12H20, and 1
．． Wash with 100 m/ml (containing 1mM KH2PO4),
Additionally, buffer BrD, 5M hexylene glycol, o,
05 mM piperazine N,N'-bis(2-ethanesulfonic acid) (pH 6,5) and 1 mM CaCt2
After washing with 100fnl containing [containing] 100 fnl, the cells were suspended in an equal volume of buffer B and incubated at 26°C for 15 minutes. Cells swollen by this treatment were passed through a syringe needle to disrupt the cell membrane, centrifuged (3000×g, 15 minutes) to collect nuclei, and washed with buffer B.

[Collection of RVT, P] 5 ml of nuclei collected by the above method was added to an equal volume of buffer C [0.01 M Tris-HCl (pH 7.2), 0.0 [
] containing 5M MgCl2 and 0.1M NaC7] at 0°C, sonicated in an ice water bath to destroy the nuclei, and then centrifuged at 4°C (10000Xr, 10 minutes).
The supernatant (T (containing VLPs) was collected. The residue was suspended in 2-bound buffer C, and the operation of centrifugation (10,000Xr, 10 minutes) was repeated three times, and each supernatant containing RvLPs was collected. Combine with the previous supernatant, then centrifuge again (8000
0Xl for 2 hours) and the crude RVLP precipitate was collected.

This crude I(VT,P) was further suspended in 2 me buffer C to elute RVT,P, and then centrifuged (1ooooo
xr.

15 minutes), collect the supernatant containing crude RVLP, layer it on a 5-20% (w/w) sucrose-buffer C concentration gradient solution, centrifuge (80,000Xf, 80 minutes) and separate. OD (Optical Density) 2
RVLPs were detected at 60 nm and both fractions of RVLPs were collected. This fraction was further centrifuged (150,000X!, 2 hours) and the precipitate was collected to obtain purified RVLP. Sei 11V
LPs were suspended in buffer C containing 50% (v/v) glycerin and stored at -20°C.

[Separation and purification of reverse transcriptase]

7 The sperm RV1 obtained above. P O,4- to buffer D[1
M KCl, 2% (v/v) NP-40, 5 mM KDTA, 25% (v/v) glycerin, 50 m
M Tris-HCl (pH 7,5) and 50 mM DTT
was dissolved so that the Xv LP concentration containing
C1 solution r50rr+14 Tris-HCl (1) H7,5
), 0.1mM BDTA, 'lmM DTT.

05% (v/v) NP-40 and 25% (v/v)
(using a buffer containing glycerin)] and ultracentrifuged at 2°C (420DDrpm for 140 hours) to fractionate the 2
0-1'i'- fraction), the active sites were collected and treated with buffer Er50mM Tris-HCl (pH 7,5), 100mM KCl,
5mM DTT, 0.5% (V/V) NP-
40 and 50% (V/V) of glycerin].

Phosphocellulose column chromatography - The enzyme fraction obtained above was preliminarily mixed with a buffer containing 100 mM KCl, Fr10mM KH2PO4 (pH 8), 100%
mM KCL, 5 mM DTT, 0.5% (V/
V) NP-40, Q, 1mM EDTA and 20
% (V/V) of glycerin (tr)) was introduced into a column (2-) of phosphocellulose (using Whatman P-11) and washed with buffer F in an amount 10 times the column volume. After that, the enzyme was eluted with buffer F containing 500mM KCl, and the active fraction was collected with buffer E18. Procedural amendment (voluntary) September 2 and 7, 1980 Kazuo Wakasugi, Commissioner of the Japan Patent Office 1. Indication of the incident 1982 Patent Application No. 151675 2
, Title of the invention Reverse transcriptase 6, Relationship with one person making the amendment Patent applicant address 2-2-1-5 Kasumigaseki, Chiyoda-ku, Tokyo Column 6 for detailed description of the invention in the specification to be amended; Contents of the amendment (1) Insert legal text between lines 2 and 3 on page 14 of the specification.

[In addition, for molecular group measurement, this enzyme was separated from Sephadex 0.
200 (manufactured by Pharmacia, crosslinked text rangel)
using a column (diameter 1 ctn, length 46,5 tyn), 0.050 M Tris-HCl (p)I7,'5),
0.400M KCl, 0.0115M DTTlo
, 1mM EDTA, 0.5% (V, /'V
) NP-40 and a buffer containing 20% ('V/V) glycerin.
It eluted between 10,000 and 10,000. ” Above 2-

Claims (1)

[Scope of Claims] Reverse transcriptase (a) obtained from cultured cells of Drosophila melanogaster and having the following physicochemical properties; Te(n), D
NA-dependent DNA synthesis activity and (CIJ RNA-dependent DNA synthesis activity (a) without IJ bonuclease H activity (a) using p017 A (;H lyadenyl liponucleotide) or polyc (J lysitidyl ribonucleotide) as a template; Using a deoxynucleotide oligomer complementary to the primer as a primer, a deoxynucleotide homopolymer having the same nucleotide sequence as the primer is synthesized. (b) Retrovirus-like particles obtained from cultured Drosophila melanogaster cells (D DNA having a nucleotide sequence complementary to RIIJA can be synthesized. (B) DNA-dependent DNA synthesis activity pO]ydA (polyadenyldeoxyribonucleotide) is used as a template, and an oligomer of deoxynucleotides complementary thereto is used as a primer. Synthesize a homopolymer of deoxynucleotides having the same nucleotide sequence as the primer. (C) Ribonuclease H activity po], y A -po:Ly d T (#? lythymidyl deoxyribonucleotide) hybrid chain as a substrate, only po1yA chain (b) Optimum pH 7.8 CI U Action temperature 235℃~32.5℃゛←) Isoelectric point p+ s, s