JPH03153700A - Protein, immobilized on carrier by physical adsorption method and capable of exhibiting aequorin activity - Google Patents

Abstract

PURPOSE:To obtain the subject immobilized protein, readily recoverable and reutilizable after use and useful as biosensor, etc., by physically binding a luminous protein having aequorin activity to an organic or inorganic carrier having the ability to bind to the protein. CONSTITUTION:An organic or inorganic carrier (e.g. activated alumina) having the ability to bind to a protein (e.g. apoaequorin) is swelled in a buffer solution and the swollen carrier is then suspended in a solution containing the protein having aequorin activity and allowed to stand at 37 deg.C for 30min to adsorb the protein thereon. The buffer solution containing the carrier is further subjected to centrifugation and separated into the carrier and a supernatant liquid and the carrier is subsequently washed with the buffer solution to afford the objective immobilized protein having the aequorin activity. Furthermore, a protein having the aequorin activity prepared according to a technique of recombinant DNA is preferably used as the protein having the aequorin activity.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a protein having aequorin activity immobilized on a carrier, a complex of the protein and a luminescent material that can be a substrate for the protein, and a method for preparing them. Regarding.

[Prior art and its problems] Aequorin is a calcium-accepting photoprotein isolated from a luminescent jellyfish that lives near Friday Harbor Island, Washington, USA.

Aequorin contains two (or three) molecules of Ca in one molecule.
When the ions combine, coelenterazine in the activated state contained in the aequorin molecule is oxidized and emits light and 002.Since Ca' ions are essential for this light emission, aequorin is used to oxidize coelenterazine in the activated state and emit light and 002. It is possible to specifically and quantitatively detect °. The detection limit for Ca'' ions is approximately 10-'kl, and is characterized by extremely high sensitivity.

However, the amount of aequorin extracted from natural products is only about 201 mg from 101 mg of luminescent jellyfish, and the amount of production is insufficient, and a constant supply is not guaranteed.

The present inventors used a recombinant DNA technique to clone the cDN^ of apoaequorin from a luminescent jellyfish, and determined its primary structure (Japanese Patent Application Laid-Open No. 81-1355861
．． Next, using this cDN^, they used a large I1g bacterium as a host and succeeded in producing apoaequorin inside and outside the bacterium (Japanese Patent Application Laid-open No. 52-196-Q31).

Furthermore, a purification method for apoaequorin by anion exchange chromatography was also established (JP-A-1-1FF2).
．． 3!17) Furthermore, we created an aequorin gene combined with a functional gene and hoped to successfully produce its fusion protein (Japanese Patent Application Laid-open No. 64-261,942), and also developed a metal detection method using the luminescence of aequorin. Shitaku Tokukai 1986
-281,942), created an aequorin gene combined with a gene for a specific binding protein, and succeeded in producing the fusion protein (Patent Application Sho 6:l-308,424).
).

Furthermore, we established a method for preparing a highly purified sample of the fusion protein for use in the enzyme-linked I2 measurement method (Patent Application No. 1-6).
9.8521. An immunoassay method using a fusion protein with a protein having this specific binding ability was also established (Japanese Patent Application No. 1-74-742).

The above invention has made it possible to stably supply aequorin. The applicability of aequorin itself as a diagnostic agent is well known to those skilled in the art, but in order to further increase its usefulness, immobilization of aequorin is essential. It is possible to produce aequorin that is insoluble in water, i.e., immobilized aequorin, while retaining its activity, and it is also possible to recover only aequorin from the reaction solution without denaturation and reuse it. becomes. If such reuse becomes possible, it can be predicted that it will also become possible to use it for biosensors and the like.

In view of the above-mentioned technical circumstances, as a result of research, the present inventor has
As is clear from one or more explanations in which aequorin could be immobilized on a specific carrier by a physical adsorption method, the object of the present invention is to immobilize aequorin to a specific carrier by a physical adsorption method, and to immobilize the protein and the protein. A method for preparing a complex with a luminescent material that can serve as a substrate for the protein, and a method for preparing a complex with a luminescent material that can serve as a substrate for a protein having aequorin activity immobilized by a physical adsorption method, and a complex of the protein and a luminescent material that can serve as a substrate for the protein. be.

Measures to solve problem E] The present invention has the following configurations (1) to (3).

(1) A method for preparing a protein having aequorin activity immobilized on a carrier, which comprises physically binding a protein having aequorin activity to a preferably inorganic carrier that has the ability to bind to white matter. .

(2) An immobilized protein having aequorin activity prepared by the preparation method described in item (1) above.

(3) A complex of the protein according to item (2) above and a luminescent material that can serve as a substrate for the protein.

The configuration of the present invention will be explained in detail below.

Here, proteins having aequorin activity include, in addition to apoaequorin protein, proteins such as fusion proteins of various functional proteins and apoaequorin, and modified apoaequorin modified with various substances, which have aequorin activity. say.

The present invention is based on aequorin immobilized on a carrier by a physical adsorption method having aequorin activity, and a method for preparing the same, which can be carried out, for example, by the method shown in the Examples below.

The present invention will be explained with reference to the accompanying figures and tables. Figure 1 shows a process diagram (flow sheet) for producing iyA, a protein with aequorin activity immobilized on a carrier.
is-HCI (pH 7,5), lOmkl EDT
A loose'fr? Inflate with lt.
10II+M EDTA: A swollen carrier is mixed with a solution dissolved in a sealing liquid, and apoaequorin is immobilized on the carrier by physical adsorption (hereinafter referred to as an immobilized substance adsorption carrier).

After separating the aboaequorin solution from the adsorption carrier by centrifugation, 20mM T'1s-HCl (pH 7,6
).

By washing the adsorption carrier twice with 1QmM EDTA liquid to remove free apoaequorin, more than 99% of the free apoaequorin was removed, and the apoaequorin immobilized on the neutral carrier by physical adsorption was removed. Aequorin is prepared.

FIG. 2 schematically shows apoaequorin immobilized by the above operation. Apoequorin is physically adsorbed onto the carrier by various physical forces such as intermolecular force and hydrophobic interaction. Accordingly, all of the above operations in which apoaequorin is physically immobilized on the carrier are carried out continuously at 0° C. to 40° C. in an open atmosphere within one night, preferably within 6 hours. As the carrier used, organic powder or inorganic powder (spherical fine particles) as exemplified in the Examples below can be used. Further, whether the bonding state between the carrier and apoaequorin is physical adsorption or chemical adsorption can be determined from the description of Examples below.

Furthermore, the carrier (commercial product) used in the Examples does not have a group that chemically bonds with apoaequorin.

Table 1 is @recorded! The figure shows the adsorption amount of apoaequorin immobilized by the preparation process shown in each carrier. The amount of adsorption is determined from the amount of free apoaequorin. As for adsorption amount by carrier, Glass Powder
is the most common, followed by ^1barliteXAD-2.

Table 2 It was determined by mixing the treated product at 4° C. for 2 hours with a swollen carrier and measuring the luminescence of unadsorbed aequorin.

Table 3 Table 2 shows how much the intensity of light emission decreases due to scattering caused by the carrier.

To measure the photon reduction rate, for example, as illustrated in Example 3 below, the same amount of carrier as that required for immobilization was used.
0mM Tris-H[:1 (pH 7,8), lOv&
Table 3 shows the relative activity of immobilized aequorin with respect to the activity of aequorin not immobilized on a carrier. I'm watching. This (a is the luminescence of aequorin immobilized on a carrier is determined as 1i1), and the actual luminescence amount was determined from the photon reduction rate in Table 2 above.

As a result, the relative activity was determined by the agarose-based 5ephadax.
G-200 was considered to be the best and most advantageous as a carrier. In this way, a method for preparing a protein having aequorin activity immobilized on a carrier was established.

Taking the above results together, apoaequorin was immobilized on a neutral carrier by physical adsorption, and the solidified apoaequorin clearly had aequorin activity. Moreover, considering the high proportion of adsorbed apoaequorin, it can be said that the adsorption power of apoaequorin is very strong, and it is easily predicted that apoaequorin can be immobilized on any carrier.

Furthermore, since apoaequorin is a protein with extremely strong adsorption power, it is thought that immobilization by physical adsorption is possible if the protein has aequorin activity. Furthermore, immobilization means to carry out a kind of modification, and considering that the modified apoaequorin, that is, the immobilized apoaequorin, has aequorin activity, it is possible to increase the aequorin activity of the previously immobilized apoaequorin. It is thought that proteins that exhibit aequorin activity as well. In other words, any protein having aequorin activity is immobilized on a carrier and is considered to exhibit aequorin activity.

In addition, aequorin activity is first shown when a protein with aequorin activity and a luminescent material that can be a substrate for the protein form a complex. It is easy to understand that the potential luminescent complex is immobilized and has aequorin activity.

As described above, the present invention provides an immobilized protein having aequorin activity, a complex of the protein and a luminescent material that can serve as a substrate for the protein, and a method for preparing the same.

[Effects of the Invention] According to the method of the present invention, aequorin, which is a photoprotein, can be immobilized on a carrier, and furthermore, by immobilization, it becomes insoluble in water, and the protein after use can be recovered and reused. It also becomes possible.

Furthermore, due to its high detection sensitivity, it can be expected to be applied to various measurement and detection methods such as biosensors by immobilization, and its range of use is extremely wide.

The utility of the proteins with immobilized aequorin activity of the present invention will be obvious to those skilled in the art, and the above disclosure will enable one skilled in the art to practice the claimed invention. However, in order to increase the understanding of this patent, the procedures used in the preparation of immobilized erifolin, which are important to the present invention, are set forth below.

[Example] Example 1 [Immobilization of aequorin onto a carrier by physical adsorption method] As a carrier, Glass Powder (325ae
sh) (Wako Pure Chemical Industries ■), activated alumina (Wako Pure Chemical Industries ■), Wakogel C-200 (Wako Pure Chemical Industries [), ToyopearlHW-60Fine (
Toyo Soda Kogyo @l), CallulofinaGC
L-300-m (Chisso@), Callulo
fina GCL-1000-+e (Chisso H), 5
ephadex G-10 (Pharsacla Fi
neChemicals), 5ephadex
G-200 (Pharmacia FineChem
icals), 5epharose 4B (Pha
ra+acla Fine (hBicals), ^m
berme ta X^to 2 (Aldrich), Amb
erlite XAD-7 (AIdrich), and 5ephacryl S-1000 (Pharmaci
a Fine Chemicals) was used.

5 to 10 mg of the carrier was measured using a Lumiphotometer (TO-40
Polystyrene cuvette (00゜Lab Science Co., Ltd.)
201M Tris-HC in outer diameter fox 6Smm)
I (pH 7,6).

After swelling with 10mM EDTA kpifi solution, excess 20mM Trls-HCI (pH
7J), 10mM EDTA irritant fluid was removed,
Suspended in a carrier swollen in an apoaequorin solution containing 20mM Tris-IIcI/10mM EDTA,
The reaction was carried out at 37°C for 30 minutes.

Apoequorin used in this step was prepared by the following method. That is, apoaequorin produced by recombinant DNA method (Japanese Patent Application Laid-open No. 102-695-1983)
was treated by an anion exchange chromatography method (JP-A-1-132,397), a reverse phase II P
Purified by LC. Reverse phase 1) As PLc, product name Cosmosil 1Oc4-300 (10x 150s
+o) water/acetonitrile as solvent system (
Using a system (both containing 0.1% trifluoroacetic acid), a gradient was performed from 20 to 80%, and the apoaequorin peak was fractionated. This apoaequorin fraction was freeze-dried to prepare apoaequorin, which was stored at -20°C and used (see Figure 1 for the following explanation).

Next, after adsorption at 37°C for 30 minutes, centrifugation was performed to separate the carrier and supernatant. The supernatant at this time is the supernatant (1).
Then, in order to remove excess apoaequorin not adsorbed to the carrier, 20 MTrls-HCI
(pt17J), the carrier was washed with lomM EDTA buffer, centrifuged, and separated into the carrier and supernatant (2).

Furthermore, the carrier was again treated with 20 m1J Trls-HCI (p
H7, 5), washed with IO++ MED Ding^Tsuiri liquid,
The supernatant (3) and the carrier were separated by centrifugation (0 or more), and the carrier was used as an apoaequorin-immobilized carrier.

Example 2 [Measurement of the amount of apoaequorin adsorbed on the carrier] Next to the above iRs (1) to (3) obtained in Example 1, the aequorin activity was measured, and from this the amount of adsorption on each carrier was determined. 20+*MTris in 100 μL of the determined 0 reaction solution
-1) cI (pH 7, 8), 10mM EDTA
Buffer, 2 μg/ail coelenterazine 5 μm
, 1 μl of 2-mercaptoethanol, including supernatant.

After being left at 4°C for 2 hours, a portion of the reaction solution was transferred into a cuvette of a Lumiphotometer (TD-4000, Labo Science) and diluted with 3hM CaCl2/:10mM T.
100 μl of ris-IIcI (pH 7, a) was injected, and the amount of luminescence was measured.

From these results, the adsorption amount for each carrier was determined, and the proportion of immobilized apoaequorin in the total apoaequorin was determined, as shown in Table 1 above. The adsorption amount is Gla
ss Powder (325mash) and ^■bar
It was considered that the spherical carrier was more advantageous as an immobilization carrier than the gel-like carrier.

Furthermore, looking at the adsorption rate, for all carriers approximately 8
More than 0% of apoaequorin was adsorbed, and the adsorption amount was extremely large, indicating the strength of apoaequorin's adsorption power.

Also, the amount of apoaequorin in the supernatant (3) is the same as that in the supernatant (1).
The amount of apoaequorin in the sample was about 1% or less. This indicates that the excess apoaequorin not adsorbed to the carrier was almost completely removed by the two washings, and that the apoaequorin was completely immobilized by adsorption.

Example 3 [Measurement of the influence of scattering of luminescence by a carrier] When measuring the relative activity of apoaequorin immobilized on a carrier, the measured value is lower than the actual amount of luminescence due to scattering of luminescence by the carrier, etc. It is measured as. Therefore, the following measurements were carried out to examine the effects of scattering of light emitted by the carrier.

The same amount of carrier as used in Example 1 was swollen in the same manner as in Example 1, and the excess 20mM T was swollen by centrifugation.
rls-IIcI(p)17.8), lO+aM ED
TA buffer was removed and 20mM Tris-1
) CI (pH 7,6), 10ml EDTA liquid,
A reaction solution containing 10 μm of 2 μg/ml coelenterazine, 2 μm of 2-mercaptoethanol, and 500 μg of apoaequorin was allowed to stand at 4° C. for 2 hours, and then quickly mixed with the above carrier, and the aequorin activity immediately after mixing was measured.

In this operation, it has been confirmed that almost no adsorption of aequorin occurs during the time required from mixing the carrier and reaction solution to measuring aequorin activity (approximately 10 seconds), so based on the results of this measurement, It was determined to what extent the luminescence of unadsorbed aequorin was measured as a low value by each carrier due to scattering or the like. Table 2 shows the results.

Example 4 [Relative activity measurement of immobilized aequorin 20mM
Trls-HCI (pH 7,5), IOm&l ED
TA&1ifi solution, 2μg/-42 coelenterazine 5
Reaction solution 1 containing 1 μm μ2.2-mercaptoethanol
00 μm was added to the apoaequorin immobilized carrier obtained in Example 1, and after leaving it to stand at 4° C. for 2 hours, the aequorin activity was measured.

The relative activity of immobilized aequorin was determined from this result and the results of the influence of scattering etc. by each carrier obtained in Example 3 above.

The results are shown in Table 3. It was confirmed that all carriers retained aequorin activity even after immobilization, but 5ephadex G-200, a dextran-based carrier, was immobilized due to its high relative activity. It has been confirmed that this is the most advantageous carrier.

[Brief explanation of the drawing]

FIG. 1 is a process diagram (flow sheet) showing a method for producing X15+ of immobilized apoaequorin having aequorin activity according to an example of the present invention. FIG. 2 schematically shows apoaequorin immobilized by physical adsorption. Apoequorin is immobilized on the carrier by physical rJ forces such as intermolecular forces and hydrophobic interactions. that's all

Claims (3)

[Claims] (1) A method for preparing a protein having aequorin activity immobilized on a carrier, which comprises physically binding a protein having aequorin activity to an organic or inorganic carrier capable of binding to a protein. (2) An immobilized protein having aequorin activity prepared by the preparation method described in claim (1). (3) A complex of the protein according to claim (2) and a luminescent substance that can serve as a substrate for the protein.