JP3515139B2 - Lectin purification method using lactitol - Google Patents

Description

Detailed Description of the Invention

[0001]

[Industrial applications]

The present invention relates to a lectin purification method using lactitol.

[0003]

[Prior art]

[0004] Lectin is a protein or glycoprotein that interacts with sugar and has a unique property of aggregating cells such as erythrocytes and precipitating a sugar complex. It is present in most species of.

Lectins have been mainly used for microscopic studies on cell surfaces, but in recent years, they have been found to be deeply involved in the control and differentiation mechanisms of various biological cells, and It has been revealed that its important function is that various kinds of lectins have a function of promoting the synthesis of nerve growth factor, which has been pointed out to be effective for the prevention and treatment of Alzheimer-type dementia.

[0006] Along with such knowledge, studies on the distribution of lectins in biological species and their properties are being actively conducted.

[0007] Among these lectins, a group having a property of binding to a functional group containing a galactopyranosyl structure is known, and when these lectins are used for various purposes, they are not isolated from organisms. If the extracted lectin-containing product is used as it is, the influence of impurities contained therein may be manifested, and in many cases, it needs to be purified.

To obtain a lectin from an organism, a method of extracting it with an appropriate buffer is adopted, but when it is further purified, the fraction by ammonium sulfate or the like and the property that the lectin binds to sugar are usually used. Affinity chromatography and the like used are mainly adopted.

Particularly, when a lectin having a property of binding to a functional group containing a galactopyranosyl structure is adsorbed by affinity chromatography and then eluted from the adsorbent, it has been conventionally used as an eluent. Others are D-galactose, lactose and methyl-β-D
It was an aqueous solution of galactopyranoside.

[0010]

[Problems to be Solved by the Invention]

However, various problems remain in the conventional method.

[0012] For example, methyl-β-D-galactopyranoside is commercially available as a reagent, but it is not produced on an industrial scale because it has no use in which it is consumed in a large amount. However, there is an economic problem that the cost of the object becomes extremely high when the object is obtained by adopting this material.

With respect to D-galactose, the economical problem that it is relatively expensive, and this is aldose, and when it is made into an aqueous solution, it is an anomer, that is, α-form and β-form.
Since it becomes a mixture of bodies, there is a problem that it is often inconvenient in various analyzes such as two peaks in high performance liquid chromatography.

Further, when lactose is used, it is relatively inexpensive, so that the economical problem can be solved, but, for example, the binding with lectin becomes too strong, and the lectin-containing substance after elution is used. There remains a problem that it is sometimes difficult to remove lactose, and a problem that it is inconvenient for various analyzes as with galactose.

Therefore, there has been a demand for the development of a lectin purification method using a substance that is economically advantageous and that is easily removed from the lectin-containing liquid after elution as an eluent.

[0016]

[Means for Solving the Problems]

Means for solving the problems of the present invention are as follows.

In order to solve the above-mentioned problems, the inventors of the present invention have earnestly studied the behavior of lectins and the properties of various adsorbents,
By adopting lactitol, which is produced in large quantities for foods, as an eluent for affinity chromatography used in the purification of lectins, the above problems have been successfully solved and the present invention has been completed.

That is, the present invention is as follows.

In the first aspect of the present invention, when a lectin having a property of binding to a functional group containing a D-galactopyranosyl structure is purified, an aqueous lactitol solution or an aqueous lactitol-containing buffer solution is used as an eluent for column chromatography. A method for purifying a lectin, which is characterized in that it is used.

In a second aspect of the present invention, the functional group containing a D-galactopyranosyl structure is a β-D-galactopyranosyl group,
β-D-galactosaminyl group, N-acetyl-β-D-
One or two selected from the group consisting of galactosaminyl groups
The method for purifying a lectin according to the above-mentioned first, which comprises at least one species.

The contents of the present invention will be described in detail below.

The lectin used in the present invention may be any lectin as long as it has a property of binding to a functional group containing a D-galactopyranosyl structure, but β is preferred.
Adsorption to a chromatographic column when the present invention is carried out by a substance having a property of binding to a -D-galactopyranosyl group, a β-D-galactosaminyl group and / or an N-acetyl-β-D-galactosaminyl group. It can be advantageously used because it has a high rate and is easily eluted from the column by using lactitol, and lactitol can be easily separated by a subsequent operation.

There are many known lectins having the property of binding to a functional group containing a D-galactopyranosyl structure, and examples of their origin include mushrooms (Agaricus bisporus) and gray Shimeji (Clitocybe nebularis), Uramurasaki (Lccar)
ia amethystina) Ischnoderma resinosu
m), Maitake (Grifola frondosa), Tsuriganetake (F
omes fomentarius), Araicularia jellyfish (Auricularia p)
olytricha), peanut, (Arachis hypogaea), soybean (Glycine max), castor bean (Ricinus communis), mokwanju (Bauhinia purpurea) common bean (Phas)
eolus vulgaris), Nepenthes (Gymnothorax javanicu)
s) etc.

The lectin used in the present invention has no particular serious restrictions other than the above-mentioned restrictions and that it is preferable to handle it at a temperature as low as possible.
In most cases, the extract is crushed into small pieces, and physiological saline or phosphate buffer that isotonic with the organism is used to adjust the pH to near neutral.
Since the conditions such as room temperature to 2 ° C. are adopted, after extraction, it is usually used as it is on an affinity column such as an agarose gel column at room temperature to 2 ° C., and the liquid flow rate SV [the packing material volume in the column ( It is possible to selectively adsorb the lectin by passing the liquid at a volume (ml) of about 0.01 to 0.3 per hour (ml).

However, the fact that galactose, galactosamine, N-acetylgalactosamine, etc. are mixed in the lectin used in the column chromatography of the present invention,
It is not preferable because it often reduces the adsorption rate of the lectin to the column.

Further, column chromatography is adopted in the practice of the present invention. As the packing material, those having galactose residues exposed on the surface have a high adsorption rate of the lectin. It can be advantageously adopted for the reason,
For example, there are commercially available agarose gels, those in which a galactopyranosyl structure is added to the surface of various base materials, and the like.

As this agarose gel, for example, Sepharose manufactured by Pharmacia, or Sepharose CL series obtained by further cross-linking it, which is commercially available as a reagent for chromatography, can be used as it is.
Use a commercially available agarose gel in 40-5 with 3-6N hydrochloric acid.
Those treated at 0 ° C. for several hours can also be advantageously used.

After the lectin is adsorbed on the column, the column is usually washed. The washing solution at that time is the same as that used in the extraction, such as physiological saline or phosphate buffer solution that is isotonic with the living body. Etc. are preferably used, and whether or not the washing is completed can be controlled by measuring the ultraviolet absorption (UV) of the liquid coming out of the column.

The lactitol used as the lectin eluent in the present invention has no particular restriction on the quality as long as it has a small amount of unreduced components, and it is sufficient that it is manufactured for industrial use or food use. As for the crystal form, any of anhydrous crystal, monohydrate crystal, dihydrate crystal and trihydrate crystal can be advantageously adopted in the form of an aqueous solution or a buffered aqueous solution.

The lactitol concentration in the aqueous lactitol solution or the aqueous lactitol-containing buffer solution used as the eluent is set to about 10 to 500 mM (mmol).
It is preferable for minimizing the adverse effect on the lectin.

It is preferable that the lactitol concentration be kept constant during the usual elution operation, but if necessary, for example, after adsorbing a plurality of lectins onto the column,
When it is desired to elute sequentially, it is also possible to advantageously change the concentration of lactitol with the elapsed time after the start of elution.

The flow rate of the eluent is set to about SV = 0.01 to 0.3 as in the case of adsorbing the lectin so that the gel is not physically damaged and the flow rate is more than necessary. It is preferable because it is not increased.

The end point of elution can be controlled by measuring the UV or the like of the eluate as in the case of the above washing operation.

The greatest feature of the present invention is that the lectin adsorbed on the adsorbent column such as agarose is eluted with an aqueous lactitol solution or an aqueous lactitol-containing buffer solution. The lactitol is reduced in sugar. One of the hemiacetal rings is open, and the molecular weight is about twice that of galactose, which is relatively large.
It is said that it does not exist in the living body.

It is said that such lactitol has an affinity for lectin, which is a substance in the living body, and that the lectin can be easily eluted from the adsorbent, and that it is easier to remove than lactose by the subsequent operation. It was quite surprising from the general belief that most of the substances that are familiar with biological substances are substances that exist in the living body, and that common knowledge is that small molecules are more likely to pass through the dialysis membrane.

The solution of lectin and lactitol obtained by carrying out the present invention has almost all components derived from organisms other than lectins removed at this point, so depending on the case, it may be used for various purposes as it is. However, if necessary, it is also possible to advantageously carry out removal of contaminated lactitol, phosphoric acid used as a buffer, and the like.

As a method for removing lactitol from the mixture of lectin and lactitol obtained by the method of the present invention, a method usually used for purification of lectin can be adopted, and there is no particular limitation, but for example, dialysis According to such a method, it is possible to remove lactitol almost completely very easily by simply applying the conditions conventionally adopted for removing sugar.

[0039]

【Example】

Hereinafter, the contents of the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these.

In the following examples,% means% by weight unless otherwise specified.

[Example-1]

20 g of raw peanuts (Arachis hypogaea) were finely crushed, taking care not to raise the temperature excessively.
50 ml of phosphate buffer solution (PBS) of 7.4 was added and the mixture was stirred at room temperature for 3 hours to extract soluble components including lectin,
After centrifugation at 10,000 G for 15 minutes, the supernatant was obtained. Next, 50 ml of the chromatographic agarose gel (Pharmacia, Sepharose-4B), which was a reagent, was filled in the column and washed with 10 mM PBS at a temperature of 15
The supernatant at a temperature of 6 ° C. and a flow rate of 6 ml / h.
1 at a flow rate of 30 ml / h until no UV absorption is observed
The column was washed by flushing with 0 mM PBS. Further, the lectin was eluted with 0.2 M lactitol [Towa Kasei Kogyo Co., Ltd., Milchen] aqueous solution until UV absorption of the liquid coming out of the column was no longer observed, and the eluate had an average pore diameter of 2
Put it in a tube of 4 Å commercially available dialysis membrane (Viskase, Dialysis Membrane) and change the water occasionally.
It was dialyzed in pure water at 5 ° C. for about 18 hours. By freeze-drying the dialyzed solution, purified peanut lectin 15 mg
Got This purified peanut lectin was analyzed,
The quality of purification was high, containing no sugar.

[Example-2]

In an atmosphere of 4 ° C., Maitake (Grifola fr
ondosa) fruit body 20g is shredded and 50mM EDTA
And 1 mM 2-mercaptoethanol mixed solution 100 m
The resulting suspension was filtered with gauze, the filtrate was centrifuged at 10,000 G for 15 minutes, and the supernatant was obtained. Ammonium sulfate was added to the supernatant to collect a lectin-containing precipitate formed at 30% to 80% saturation, and the precipitate was dialyzed against deionized water at 4 ° C., freeze-dried, and Maitake mushroom. Crude lectin 46.8
mg was obtained. Use 3-6N hydrochloric acid at 40-50 ° C for 2
Chromatographic agarose gel (Pharmacia, Sepharose) treated with acid for 10 hours and washed with 10 mM PBS
CL-4B) A column in which 50 ml was packed was run with a solution of the lyophilized product in PBS at a temperature of 4 ° C. and a flow rate of 3 ml / h, and then 10 mM at a flow rate of 20 ml / h until no UV absorption was observed. The column was washed by flushing PBS. Further, the lectin was eluted with 50 mM lactitol [Towa Kasei Co., Ltd., Milchen] aqueous solution until UV absorption of the liquid coming out of the column was not observed, and the eluate was concentrated with an ultrafiltration membrane, and then averaged. Commercially available dialysis membrane with a pore diameter of 24Å (Viskase, dialysis membrane)
It was put in a tube of No. 2 and dialyzed in pure water at 3 ° C. for about 12 hours while occasionally changing water. The dialyzed solution was lyophilized to obtain 0.1 mg of purified Lentinus edodes lectin. As a result of analysis, the purified lectin of Maitake was of a quality with a high degree of purification containing no sugar.

[Example-3]

Under an atmosphere of 5 ° C.,
10 g of liver of rax javanicus) was crushed, put in 100 ml of 0.9% sodium chloride aqueous solution and stirred overnight, and the obtained suspension was filtered with gauze, and ammonium sulfate was added to the filtrate. The precipitate formed at 80% saturation,
00 mg of crude lectin was obtained. The crude lectin was dissolved in 10 ml of 10 mM PBS and centrifuged at 13,000 G for 10 minutes to obtain the supernatant. The supernatant is 10 mM
Flow through a column packed with 50 ml of agarose gel for chromatography (Pharmacia, Sepharose CL-4B) washed with PBS at a temperature of 5 ° C and a flow rate of 2 ml / h, and then a flow rate of 30 until UV absorption is no longer observed.
The column was washed by flushing with 10 mM PBS at ml / h. Further, 0.5M lactitol [Towa Kasei Kogyo]
[Milchen Co., Ltd.] lectin was eluted with an aqueous solution, and the eluate was put into a tube of a commercially available dialysis membrane (Viskase, Dialysis Membrane) with an average pore diameter of 24 Å, and pure water at 5 ° C with occasional water exchange. It was dialyzed in water for about 10 hours. The dialyzed solution was freeze-dried to obtain 0.2 mg of purified dokutsubo lectin. As a result of the analysis, the purified doughnut lectin was of a highly purified quality containing no sugar.

[Comparative Example-1]

Lectin was extracted and purified in the same manner as in Example 2 except that lactose was used instead of lactitol as an eluent to obtain a lectin. As a result, sugar was detected and lectin was detected. The degree of purification was insufficient.

[Test Example-1]

In Example-2, a 0-type human erythrocyte treated with pronase was suspended in an acetate buffer isotonic solution at a concentration of 3% using a microtiter U-plate manufactured by Sigma. The minimum agglutination concentration of the obtained crude lectin and purified lectin was measured to calculate the hemagglutination activity per mg of both. As a result, the activity of the purified lectin was 428 when the hemagglutination activity of the crude lectin was set to 1.
Met. From this result, it is understood that the purified lectin has the active ingredient purified and concentrated by 428 times as compared with the crude lectin.

[0052]

【The invention's effect】

By carrying out the present invention, it is possible to obtain a lectin-containing liquid which is economically advantageous and which is easy to remove sugar in the subsequent operation without adding a complicated operation to the conventional operation.

Front page continued (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 1/00 C07K 14/42 CA (STN) BIOSIS / MEDLINE / WPID S (STN)

Claims (2)

(57) [Claims] 1. When purifying a lectin having a property of binding to a functional group containing a D-galactopyranosyl structure, a lactitol aqueous solution or a lactitol-containing buffer aqueous solution is used as an eluent for affinity chromatography. Method for purifying lectin using lactitol. 2. The functional group containing a D-galactopyranosyl structure is selected from the group consisting of a β-D-galactopyranosyl group, a β-D-galactosaminyl group and an N-acetyl-β-D-galactosaminyl group. The method for purifying lectin using lactitol according to claim 1, which is one kind or two or more kinds.