JPH0154993B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an affinity adsorbent for affinity chromatography. Affinity chromatography is an excellent method for specifically purifying specific proteins. Affinity adsorbents with nucleotides as ligands include polyadenylic acid-Sepharose 4B, polyuridylic acid-Sepharose 4B, 5'-adenosine monophosphate-Sepharose 4B, 2',5'-adenosine diphosphate-Sepharose 4B (Cepharose is Pharmacia agarose (trademark) made by the company), but despite the fact that the ligand of the former two adsorbents is a product of polynucleotide phosphorylase, an enzyme whose substrates are adenosine diphosphate and uridine diphosphate. , shows no affinity for this enzyme. Furthermore, the ligands of the latter two adsorbents are analogs of the substrates of polynucleotide phosphorylase, but since all of these ligands are bound to the support with an adenine base, e.g. It shows no affinity with enzymes that recognize and bind to base moieties, such as orilase. These affinity adsorbents are therefore unsuitable as adsorbents for enzymes such as polynucleotide phosphorylase. The present inventors have developed an affinity adsorbent for affinity chromatography that has the potential to be widely applied to the discovery and purification of various enzymes that exhibit the ability to bind adenosine diphosphate, such as polynucleotide phosphorylase. As a result of intensive research into development, we have arrived at the present invention. The gist of the first invention is to oxidatively cleave adenosine diphosphate, couple it to a support, and then reduce the adenosine diphosphate, which is obtained as a ligand. The second aspect of the invention resides in an affinity adsorbent for affinity chromatography characterized in that it is bonded to a support, and the gist of the second invention is that after oxidative cleavage of adenosine diphosphate and coupling with the support, The mixture is purified by affinity chromatography using an affinity adsorbent that uses adenosine diphosphate obtained by reduction as a ligand and is bound to a support at the 2'-C and 3'-C positions of ribose. The invention consists in a method for separating substances capable of binding to adenosine diphosphate. To explain the present invention in more detail, the affinity adsorbent for affinity chromatography of the present invention uses adenosine diphosphate (ordinary 5'-adenosine diphosphate, the same applies hereinafter) as a ligand. This makes it an adsorbent that is widely used for purifying substances (such as enzymes) that have the ability to bind adenosine diphosphate. The second feature is that the ligand adenosine diphosphate is bound to the support at the 2'-C and 3'-C positions of the ribose as shown below (in the following formula, adenosine diphosphate is bound to agarose via a spacer ( _-CH2- ) ₆ . And by joining at this position,
Since the base moiety exists as it is, it has great application value as a ligand for enzymes that recognize and bind to the base moiety. Enzymes that do not bind to adenosine diphosphate have the advantage of being clearly separated as unadsorbed substances, even if they are present in extremely small quantities or have very similar protein properties. Agarose derivatives are commonly used as the support. The type of agarose derivative is selected depending on the reaction used to immobilize adenosine diphosphate on the support. Other supports such as polyacrylamide can also be used. For example, the 2'-C of adenosine diphosphate and
When the hydroxyl group at the 3'-C position is oxidatively cleaved to an aldehyde or carboxyl group and coupled to a support, a support into which an ω-aminoalkyl group has been introduced is selected. As the support, in addition to a functional group that reacts with a ligand, those having a spacer between the functional group and the substrate can be used. However, as such a support, Pharmacia, P.
-Those commercially available from L Biochemicals and Biorats Laboratories can be used. In order to prepare the affinity adsorbent for affinity chromatography of the present invention, a support and a ligand may be coupled together by a conventional method. The coupling reaction may be appropriately selected depending on the type of support. When an agarose derivative having an amino group is used as the support, adenosine diphosphate is cleaved by periodate oxidation as shown in the following formula, and then coupling is performed. After that, it can be reduced with _NaBH4 . The reduction treatment suppresses the reverse reaction and stabilizes the structure, and also prevents the adsorption of contaminant proteins to the hydroxyl groups before reduction. (In the formula, P is a phosphoric acid group, A is an adenyl group,
R-NH ₂ represents an agarose derivative having an amino group. ) Therefore, the amount of adenosine diphosphate bound to the agarose derivative as a ligand is usually 4 to 4.
5 μmole/ml support is sufficient. The affinity adsorbent for affinity chromatography of the present invention obtained as described above can be used to separate a substance capable of binding to adenosine diphosphate from a mixture. For example, Thermus thermophilus
thermophilus) HB8 (ATCC27634). As shown in the Examples below, the polynucleotide phosphorylase in the mixture was specifically adsorbed to the affinity adsorbent of the present invention and desorbed using a buffer containing adenosine diphosphate or NaCl. High purity polynucleotide phosphorylase can be obtained in high yield. Specifically, polynucleotide phosphorylase can be separated and purified by a three-step treatment of 50% ammonium sulfate fractionation of a disrupted bacterial cell solution, diethylaminoethyl cellulose column treatment, and affinity chromatography using the affinity adsorbed solid of the present invention. To explain in more detail, the bacterial cells were ground by alumina grinding in a buffer, centrifuged to obtain a supernatant, ammonium sulfate was added to this supernatant to 30% saturation, and centrifuged.
Obtain the supernatant, add ammonium sulfate again to 50% saturation, collect the precipitate by centrifugation, and dissolve in a buffer. After dialysis in a buffer, it was packed into a diethylaminoethyl cellulose column equilibrated in the same buffer.
Elute with a linear gradient from 0M to 0.5M NaCl. Polynucleotide phosphorylase elutes when the NaCl concentration is around 0.18-0.25M. The active fractions are collected, dialyzed, subjected to the affinity chromatography of the present invention equilibrated with the same buffer, and eluted with a buffer containing adenosine diphosphate to obtain polynucleotide phosphorylase. Column chromatography of diethylaminoethyl cellulose is not necessarily required, but
This may be done to avoid contamination of the affinity chromatography support. Furthermore, since non-specific adsorption may occur depending on the sample, it is preferable to perform gel filtration or ion exchange chromatography once after affinity chromatography. Hereinafter, the present invention will be explained in more detail with reference to Examples. Note that the enzyme activity in the examples was measured as follows. A reaction solution with the following composition was maintained at 70℃ for 20 minutes. Reaction solution composition: Buffer 90μ (Ap) ₃ A, that is, [adenylyl-(3'→5')] Buffer containing ₃ adenosine 100μ Enzyme solution 10μ Total 200μ Color development Solution composition: 6NH ₂ SO ₄ 1 volume H ₂ O 2 2.5% ammonium molybdate 1 10% ascorbic acid 1 Add 1 ml of coloring solution and 0.7 ml of H ₂ O and hold at 37°C for 1 hour. The A ₈₂₀ absorbance of this solution is then measured. One unit of enzyme activity is the amount of enzyme that incorporates 1 μmole of adenosine diphosphate into the polymer in 1 hour. The buffer solution is 50mM Tris-HCl (PH9.0)
with 2mM _MgCl2 and 2mM adenosine diphosphate
10 μM (Ap) ₃ A was added. Example 1 (Preparation of affinity adsorbent) 4.6 g of AH-Sepharose 4B (aminohexyl Cepharose 4B, manufactured by Pharmacia) was mixed with 0.5M
It was suspended in 200 ml of NaCl and left overnight at room temperature. After filtering, it was washed with 200 ml of 0.5M NaCl, and further washed with water until there was no Cl ^- in the supernatant. This was then suspended in 15 ml of 0.1M Borax (sodium tetraborate) and equilibrated. Meanwhile, add 30ml of 9mM adenosine diphosphate and 1ml of saturated Borax to 1.2ml of 0.2M _NaIO4 ,
The pH was adjusted to 9.0 and the mixture was left at 4°C for 1 hour to oxidize the ribose portion of adenosine diphosphate with periodate. The above suspension of AH-Sepharose 4B was added to the obtained solution, and the mixture was reacted overnight at 4°C with stirring.
Coupling of adenosine diphosphate and AH-Sepharose 4B was performed. Then solid NaBH ₄ 45
mg was added and left to stand for 2 hours while stirring. Exactly the same operation was repeated twice. After the reaction was completed, the reaction solution was separated, 5M NaCl was added to the obtained solid, and the solid was washed in batches until absorption of A ₂₆₀ disappeared. Thereafter, it was thoroughly washed with water, 0.1N Borax, and water in that order. Next, this was suspended in 35 ml of 0.7M saturated sodium acetate, kept at 4°C, and 0.2 ml of acetic anhydride was added 4 times, once.
It dripped in time. The mixture was further stirred at 4° C. for 1 hour to acetylate the remaining free amino groups. After separating the reaction solution, water, 5M NaCl, water, 0.1M
Washed with borax and water in that order. The obtained solid was found to be AH-Sepharose 4B in which adenosine diphosphoric acid was bound at a ratio of 4.6 μmole to 1 ml of AH-Sepharose 4B. In the following, the affinity adsorbent obtained here is referred to as ADP-AH-Sepharose 4B. Example 2 (Separation and purification of polynucleotide phosphorylase) (1) Culture of bacteria Thermus Thermophilus HB8 was mixed with 0.3% yeast extract, 0.5% peptone, 0.1% glucose, and 0.2%.
NaCl, 0.05% inorganic salt vitamin solution (in 1 part water)
25g MgCl ₂ 6H ₂ O, 5g CaCl ₂ , 2g
MnSO ₄・6H ₂ O, 0.5g ZnSO ₄・7H ₂ O, 0.5g
_H3BO3 , 5g _FeCl3・_6H2O , _{15mg CuSO4} ,
25mg _{Na2MoO4 ・ 2H2O} , _50mgCoNO3・_6H2O ,
20mg NiNO ₃ 6H ₂ O, 80mg pyridoxane, 10
mg folic acid, 0.5ml _H2SO4 , 10mg thiamine pyrophosphate, 40mg riboflavin, 80mg _nicotinamide , 80mg para-aminobenzoic acid, 10mg biotin,
0.4mg cyanocobalamin, 80mg pantothenic acid, 20
mg lipoic acid, 200 mg inositol, 50 mg choline, 50
Using a medium containing (mg orotic acid, 100 mg spirmin), aerated culture was carried out at 75°C for 5 to 6 hours, and the bacteria were collected at around 300 creets. Approximately 10g per medium
HB8 bacterial cells were obtained. (2) Separation and purification of enzyme (A) Extraction Approximately 1 kg of the wet weight of the HB8 cells obtained as described above was mixed with extraction buffer (50mM Tris-HCl (PH7.5), 14mM Mg ( _CH3COO )). ) ₂ , 1mM
EDTA, 0.14mM KCl, 50mM β-mercaptoethanol) were added, and the mixture was ground by a mill to extract the enzyme. The extract was centrifuged at 10,000g for 20 minutes to separate the precipitate and supernatant, and the precipitate was extracted again with extraction buffer 1 and centrifuged to obtain the supernatant. The supernatants from the two centrifugations were combined to obtain crude enzyme solution 2.7. (B) Diethylaminoethylcellulose (DEAE−
Add ammonium sulfate to the crude enzyme solution obtained by cellulose DE-32 (manufactured by Fumarmacia) column chromatography (A) until 30% saturation. Ammonium sulfate was added until saturation, left to stand, and centrifuged in the same manner to collect the precipitate. Transfer this precipitate to as little buffer as possible (50mM Tris-HClPH7.5, 1mM EDTA)
and dialyzed overnight against the same buffer.
A DEAE cellulose DE32-column (φ5×44
cm) for adsorption, and elution was performed using a linear concentration gradient of sodium chloride in the buffer from 0M to 0.5M. The enzyme is eluted at salt concentrations between 0.18M and 0.25M. (C) ADP-AH Sepharose 4B affinity chromatography The polynucleotide phosphorylase active fraction obtained in (B) was collected and thoroughly dialyzed against 10 mM Tris-HCl (PH7.5) buffer. This dialyzed enzyme solution was applied to an ADP-AH Sepharose 4B column (φ1 x 10 cm) equilibrated with the same buffer solution, adsorbed, and eluted with a buffer solution containing 0.2 M NaCl. The obtained active fraction had a protein content of 8 mg, a total activity of 780 units, and a specific activity of 98 units/mg protein.

Claims (1)

[Scope of Claims] 1. Adenosine diphosphate is oxidatively cleaved, coupled with a support, and then adenosine diphosphate obtained by reduction is used as a ligand, and the 2'-C of ribose is
An affinity adsorbent for affinity chromatography, characterized in that it is bonded to a support at the and 3'-C positions. 2 Adenosine diphosphate is oxidatively cleaved, coupled with a support, and then reduced, and the adenosine diphosphate obtained is used as a ligand to cleave the 2′-C of ribose.
A method of separating a substance capable of binding to adenosine diphosphate from a mixture by affinity chromatography using an affinity adsorbent bound to a support at the and 3'-C positions. 3. The mixture is a mixture containing an enzyme capable of binding to adenosine diphosphate obtained by adsorption separation using an anion exchanger from a disrupted bacterial cell extract of Thermus thermophilus, and the substance to be separated is a polynucleotide phosphoric acid. The method according to claim 2, which is orilase.