JPS63222200A - Production of avidin - Google Patents
Production of avidinInfo
- Publication number
- JPS63222200A JPS63222200A JP5311487A JP5311487A JPS63222200A JP S63222200 A JPS63222200 A JP S63222200A JP 5311487 A JP5311487 A JP 5311487A JP 5311487 A JP5311487 A JP 5311487A JP S63222200 A JPS63222200 A JP S63222200A
- Authority
- JP
- Japan
- Prior art keywords
- avidin
- dextran
- egg white
- proteins
- adsorption
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090001008 Avidin Proteins 0.000 title claims abstract description 48
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 235000014103 egg white Nutrition 0.000 claims abstract description 24
- 210000000969 egg white Anatomy 0.000 claims abstract description 23
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims abstract description 20
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 20
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 20
- 229920002307 Dextran Polymers 0.000 claims abstract description 14
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 claims abstract description 12
- 238000001179 sorption measurement Methods 0.000 claims abstract description 12
- 238000005342 ion exchange Methods 0.000 claims abstract description 7
- 150000003839 salts Chemical class 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 230000007935 neutral effect Effects 0.000 claims abstract 2
- 239000012266 salt solution Substances 0.000 claims description 7
- 150000003384 small molecules Chemical class 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 239000003431 cross linking reagent Substances 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 238000005185 salting out Methods 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- 235000002639 sodium chloride Nutrition 0.000 description 13
- 102000016943 Muramidase Human genes 0.000 description 11
- 108010014251 Muramidase Proteins 0.000 description 11
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 11
- 235000011130 ammonium sulphate Nutrition 0.000 description 11
- 229960000274 lysozyme Drugs 0.000 description 11
- 235000010335 lysozyme Nutrition 0.000 description 11
- 239000004325 lysozyme Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 108010064983 Ovomucin Proteins 0.000 description 5
- 239000003729 cation exchange resin Substances 0.000 description 5
- 239000000356 contaminant Substances 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 108010088751 Albumins Proteins 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 3
- 229910003460 diamond Inorganic materials 0.000 description 3
- 239000010432 diamond Substances 0.000 description 3
- 238000005194 fractionation Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 229920000298 Cellophane Polymers 0.000 description 2
- 229920002125 Sokalan® Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 229940023913 cation exchange resins Drugs 0.000 description 2
- 229920001429 chelating resin Polymers 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000609886 Addax Species 0.000 description 1
- 241001164374 Calyx Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000047703 Nonion Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000991 chicken egg Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 125000000962 organic group Chemical group 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は卵白アビジンの製造方法に関し、更に詳しくは
卵白中のりゾチームやアビジン等の塩基性蛋白質を吸着
させた陽イオン交換樹脂から高純度のアビジンを効率的
に製造する方法に関するものである。[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for producing egg white avidin, and more specifically, it relates to a method for producing egg white avidin, and more specifically, a method for producing egg white avidin, which is produced from a cation exchange resin that adsorbs basic proteins such as lysozyme and avidin in egg white. The present invention relates to a method for efficiently producing avidin.
ここでいう卵白とは生卵白、乾燥卵白、濃縮卵白、冷凍
卵白等をいう。The term "egg white" as used herein refers to raw egg white, dried egg white, concentrated egg white, frozen egg white, etc.
卵白アビジンは、ニワトリ卵白中に０．００３％程度含
まれる分子量６．８００の塩基性蛋白質であ、るが１本
物質はビオチンと強固に結合する性質を有することから
、近年分子生物学の分野における架橋試薬として広く利
用され、特に酵素免疫法における架橋試薬として種々の
診断薬に使用され需要が増大しつつある。Egg white avidin is a basic protein with a molecular weight of 6.800 that is present in chicken egg white at about 0.003%.However, this substance has the property of strongly binding to biotin, so it has recently been studied in the field of molecular biology. It is widely used as a crosslinking reagent in enzyme immunoassays, and is used in various diagnostic reagents, and demand is increasing.
（従来技術）
卵白アビジンはこのように卵白中の微量成分であるため
、精製工程で卵白成分中のアルブミン類やオボムコイド
等の他の蛋白質成分の夾雑が生じやす（高純度のアビジ
ンを得ることは難かしい。(Prior art) Since egg white avidin is a trace component in egg white, contamination with other protein components such as albumins and ovomucoid in the egg white components is likely to occur during the purification process (it is difficult to obtain highly pure avidin). It's difficult.
従って、夾雑蛋白質を除くため塩濃度勾配溶出という工
業的な製造法としては不適当なりロフトグラフィー法を
行っている。Therefore, in order to remove contaminant proteins, the loftography method is used, which is inappropriate as an industrial production method called salt concentration gradient elution.
すなわちこの溶出方法は、まず卵白液の塩基性蛋白質を
吸着し、ついで無機塩を用いた濃度勾配法によるクロマ
トグラフィーでアビジン画分を抽出し、これを濃縮後共
存するりゾチームを結晶化させて除去し、更にＣＭ−セ
ルローズによるクロマトグラムを再度行い、同じ（塩濃
度勾配法によってアビジン画分を溶出して、これを濃縮
後硫安による結晶化を行い分離する方法が行われている
。In other words, this elution method involves first adsorbing basic proteins from egg white fluid, then extracting the avidin fraction by chromatography using a concentration gradient method using inorganic salts, and concentrating this fraction to crystallize the coexisting lysozyme. The avidin fraction is eluted using the same method (salt concentration gradient method), and the avidin fraction is concentrated and then crystallized using ammonium sulfate for separation.
（発明が解決しようとする問題点）
しかしながら前述の従来法においては、一度に少量しか
アビジンを溶出できない上に、溶出に時間と手間を要す
るため、工業的大量生産する製造前として不適当である
という問題があった。(Problems to be Solved by the Invention) However, in the conventional method described above, only a small amount of avidin can be eluted at a time, and elution requires time and effort, so it is not suitable as a pre-production method for industrial mass production. There was a problem.
また、アビジンは現在市販されているが、９３％程度の
純度のものしか得られず、これらは前述したような医薬
的用途にむかないのが現状である。Furthermore, although avidin is currently commercially available, it can only be obtained with a purity of about 93%, and at present it is not suitable for the above-mentioned medical uses.
（問題点を解決するための手段）
そこでアビジンの需要が増大しつつある今日において、
アビジンを高純度でかつ工業的に製造する技術の確立が
望まれていることから９本発明者等は鋭意研究を重ねた
結果、卵白中の塩基性蛋白質に低分子吸着用イオン交換
デキストランに吸着させると、リゾチームが吸着される
とともに、驚１くべきことに高分子のアビジンをも吸着
し、夾雑スルアルブミン類やオボムコイドなどを容易に
除去しうろことを見い出し、高純度のアビジンを効率よ
（容易に製造する本発明を完成するに至った。(Means to solve the problem) Nowadays, the demand for avidin is increasing.
Since it is desired to establish a technology to industrially produce avidin with high purity,9 the present inventors have conducted intensive research and found that basic proteins in egg white can be adsorbed to ion-exchanged dextran for adsorption of small molecules. They found that lysozyme was adsorbed and, surprisingly, the high-molecular weight avidin was also adsorbed, and contaminants such as sulalbumin and ovomucoid were easily removed. We have now completed the present invention, which is easy to manufacture.
すなわち９本発明は卵白中の塩基性蛋白質に低分子吸着
用イオン交換デキストランを使用して。That is, the present invention uses ion exchange dextran for adsorption of low molecules to basic proteins in egg white.
アビジンを吸着させて分離することを特徴とするアビジ
ンの製造方法に関する。The present invention relates to a method for producing avidin, which is characterized by adsorbing and separating avidin.
本発明の実施にあたっては、まず生卵白液、乾燥卵白、
濃縮卵白、冷凍卵白のリゾチーム、アビジン等の塩基性
蛋白を含む原料を用意し、濃縮卵白、乾燥卵白は適宜水
で稀釈するか、あるいは水戻しすることにより適当な固
形分濃度に調整すると共に９次に行う樹脂による吸着を
容易にするため、予めホモゲナイズし。In carrying out the present invention, first, raw egg white liquid, dried egg white,
Prepare concentrated egg whites, frozen egg whites containing basic proteins such as lysozyme and avidin, and adjust the concentrated egg whites and dried egg whites to an appropriate solid concentration by diluting them with water or rehydrating them.9 Homogenize in advance to facilitate the next adsorption with the resin.
そのｐＨを６．５〜７．５に調整しておくことが望まし
い。It is desirable to adjust the pH to 6.5 to 7.5.
次に上記原料に弱酸性陽イオン交換樹脂を添加してリゾ
チームアビジン等の塩基性蛋白を吸着させる。Next, a weakly acidic cation exchange resin is added to the above raw material to adsorb basic proteins such as lysozyme avidin.
本発明で使用する弱酸性陽イオン交換樹脂としては、デ
ュオライト−〇−４６４ （ダイヤモンドシャロックケ
ミカル社製）、アンバーライトＩＲＯ−５０（ロームア
ンドハースター社製）、ダウエックスＭＷＣ−１（ダウ
ケミカル社製）、ダイアイオンＷＫ−１０（三菱化成工
業社製）などの弱酸性陽イオン交換樹脂が使用できる。The weakly acidic cation exchange resins used in the present invention include Duolite-0-464 (manufactured by Diamond Sharrock Chemical Co.), Amberlite IRO-50 (manufactured by Rohm and Haaster), and Dowex MWC-1 (manufactured by Dowex MWC-1). Weakly acidic cation exchange resins such as (manufactured by Chemical Co., Ltd.) and Diaion WK-10 (manufactured by Mitsubishi Chemical Industries, Ltd.) can be used.
次に吸着させた弱酸性陽イオン交換樹脂を水洗して洗浄
した後、塩溶液で脱着する。Next, the adsorbed weakly acidic cation exchange resin is washed with water and then desorbed with a salt solution.
塩溶液としては、塩化ナトリウム、塩化カリウム、硫酸
す） ＋７ウム等強酸・強塩基の塩の水に溶解した時ｐ
Ｈ６〜８．！１度２〜６９６付近のものを使用する。Salt solutions include sodium chloride, potassium chloride, sulfuric acid, etc. When salts of strong acids and bases such as +7um are dissolved in water, p
H6-8. ! Use one around 2 to 696 degrees.
この脱着処理により樹脂に吸着されたリゾチーム・アビ
ジン等の塩基性蛋白が塩溶液中に溶出するが、アルブミ
ン類やオボムコイドなどの夾雑蛋白も共存して溶出する
。そこで電導度２０ｍ５／ｃｍ以下、 ｐＨ５４）〜７
りの条件下で低分子吸着用イオン交換デキストランに吸
着させ、同条件下で充分に洗浄することにより共存する
アルブミン類やオボムコイドなどが充分に除去される。Through this desorption process, basic proteins such as lysozyme and avidin adsorbed on the resin are eluted into the salt solution, but contaminant proteins such as albumins and ovomucoid are also eluted together. Therefore, the electrical conductivity is 20m5/cm or less, and the pH is 54) to 7.
Coexisting albumins, ovomucoid, etc. can be sufficiently removed by adsorbing on ion-exchanged dextran for low-molecular adsorption under the same conditions and thoroughly washing under the same conditions.
低分子吸着用イオン交換デキストランは９分子０、２５
（ファルマシア社製）等を用いる。Ion exchange dextran for small molecule adsorption has 9 molecules 0,25
(manufactured by Pharmacia) etc. is used.
この吸着樹脂の脱着処理は、ｐＨ５〜８．濃度２〜６９
６付近の塩溶液２例えば塩化す）＋Ｊウム、塩化カリウ
ム、硫酸ナトリウム等の強酸６強塩基の塩溶液で行い９
次に該塩溶液に夾雑するリゾチームを食塩による結晶化
法や硫安分画等の塩折あるいはキチンや、非イオン交換
性の吸着剤〔例えばアンバーライトＸＡＤ−７（オルガ
ノ社製）〕等による吸着によって除去する。This adsorption resin desorption treatment is carried out at a pH of 5 to 8. Concentration 2-69
6. Perform with a salt solution around 6.2, for example, chloride) + strong acids such as potassium chloride, sodium sulfate, etc. 6. Perform with a salt solution of a strong base.9
Next, the lysozyme contaminant in the salt solution is removed by crystallization using common salt, salt separation such as ammonium sulfate fractionation, or adsorption using chitin or a non-ion exchange adsorbent (for example, Amberlite XAD-7 (manufactured by Organo)). Remove by.
硫安分画の場合、硫安濃度３０〜８０％飽和、好ましく
は５０〜７５％飽和で行い、ここで得られる両分から夾
雑アルブミン類やオボムコイド、更にはりゾチームなど
を充分にとりのぞいた高純度のアビジンが得られる。In the case of ammonium sulfate fractionation, it is carried out at an ammonium sulfate concentration of 30 to 80% saturation, preferably 50 to 75% saturation, and highly purified avidin is obtained from both of the obtained fractions, from which contaminant albumin, ovomucoid, and alizozyme have been sufficiently removed. can get.
実施例１
生卵白ＩＬをホモジナイズ後２ Ｎ −ＨＣＩを加えｐ
Ｈ６，５とする。次に０．１Ｍリン酸緩衝液（ｐＨ６，
５）で緩衝化したデュオライトＣ−４６４１６０ｇｒ
（ｗｅｔ）（ダイヤモンドシャロックケミカル社製）に
接触せしめアビジンを吸着させた後、°水により充分洗
浄して、３％ＮａＣ１溶液１５０ ｍＬで溶出しアビジ
ンを含む溶出液１５０耐が得られた。Example 1 After homogenizing raw egg white IL, 2N-HCI was added and p
Let it be H6.5. Next, 0.1M phosphate buffer (pH 6,
5) Duolite C-464160gr buffered with
(wet) (manufactured by Diamond Sharrock Chemical Company) to adsorb avidin, and then thoroughly washed with water and eluted with 150 mL of 3% NaCl solution to obtain an eluate containing avidin of 150 mL.
この溶出液を水で希釈し電導度１２ｍ５／ｃｍ、 ｐ）
４６．５にしてからＣＭ−シェフアダツクスＣ，２５５
ｇｒ（ｄｒｙ）（ファルマシア社製）に接触させた。電
導度１２ｍ５／（３のＮａＣ１溶液（ｐＨ６，５）によ
って充分に洗浄し９次に３ ％ ＮａＣ１溶液１００
ｍＬで溶出を行う。得られる溶出液をｐＨ３，５に調整
し、 ＮａＣ１を５％となるように加え４℃で放置し夾
雑するリゾチームを結晶化除去し、アビジン液を得た。This eluate was diluted with water to give an electrical conductivity of 12 m5/cm, p)
46.5 and then CM-Chef Addax C, 255
gr(dry) (manufactured by Pharmacia). Thoroughly wash with conductivity 12m5/(3% NaCl solution (pH 6,5), then 3% NaCl solution 100%
Perform elution in mL. The resulting eluate was adjusted to pH 3.5, NaCl was added to give a concentration of 5%, and the mixture was allowed to stand at 4°C to crystallize and remove contaminating lysozyme to obtain an avidin solution.
このアビジン液に硫安濃度５０９６飽和になるよう硫安
を加え共存リゾチームを除去後、硫安７５％飽和に加え
た。得られる両分を炉別し、溶解後セロファン膜で透析
・脱塩後、凍結乾燥にてアビジン末３００萼を得た。（
収率１０％）実施例２
生卵白１０Ｌをホモジナイズ後、２Ｎ−ＨＣＩを加えｐ
）！ ６．５とし９次に０．１Ｍリン酸緩衝液（ｐＨ６
−５）で緩衝化したデュオライトＣ−４６４１，６００
ｇｒ （ｗｅｔ）（ダイヤモンドシャロックケミカル社
製）に接触せしめアビジンを吸着させた。水により充分
に洗浄し、３９６ＮａＣ１溶液１．５００ｉｄテ溶出し
、アビジンを含む溶出液１．５００１ＬＬが得られた。Ammonium sulfate was added to this avidin solution so that the ammonium sulfate concentration was 5096 saturated, and after removing the coexisting lysozyme, it was added to ammonium sulfate at 75% saturation. Both obtained fractions were separated in a furnace, dissolved, dialyzed with a cellophane membrane, desalted, and freeze-dried to obtain 300 calyxes of avidin powder. (
Yield: 10%) Example 2 After homogenizing 10 L of raw egg white, 2N-HCI was added and p
)! 6.5 and 9 then 0.1M phosphate buffer (pH 6
-5) Duolite C-4641,600 buffered with
gr (wet) (manufactured by Diamond Sharrock Chemical Co.) to adsorb avidin. After thorough washing with water, 1.500 id of 396NaCl solution was eluted to obtain 1.5001 LL of an eluate containing avidin.
この溶出液を限外が過で脱塩し、電導度５ｍｓ ／ Ｃ
１ｌ 、 ｐＨ６，５にしてからＣＭ−シェフアダツク
ｘ Ｃ，２５３５ｇｒ（＆ｙ） （７フル−ｖシｙ社製
）に接触させた。次に電導度５ｍ８／αのＮａα溶液（
ｐＨ６，５）により充分に洗浄し、 ３４ ＮａＣ！
１溶液１００ｍ１で溶出を行う。得られる溶出液をｐＨ
３，５に調整しＮａＣ１を５％となるように加え４℃に
放置し夾雑するリゾチームを結晶しアビジン液を得る。This eluate was desalted by ultraviolet filtration and the conductivity was 5ms/C.
After adjusting the pH to 6.5, the mixture was brought into contact with CM-Chef Addac xC, 2535gr (&Y) (manufactured by 7 Flu-VCY Co., Ltd.). Next, a Naα solution with an electrical conductivity of 5 m8/α (
Wash thoroughly with 34 NaC!
Elution is carried out with 100 ml of one solution. The resulting eluate was adjusted to pH
3.5, add NaCl to 5%, and leave to stand at 4°C to crystallize contaminating lysozyme to obtain an avidin solution.
このアビジン液に硫安濃度５０９６飽和になるよう硫安
を加え沈殿除去後さらに硫安７５９６飽和になるよう加
えて分画を行つた。得られる百分を炉別し、水で溶解し
、セロファン膜で透析・脱塩後凍結乾燥にてアビジン末
２．５ｇｒを得た。（収率８．３９６）
（純度試験）
実施例１で得られたアビジンを次の条件によるゲル浸透
−高速液体クロマトグラフィーによって分析した結果純
度９８．７９６であった。Ammonium sulfate was added to the avidin solution so that the ammonium sulfate concentration became saturated with 5096, and after removing the precipitate, ammonium sulfate was further added so that the ammonium sulfate became saturated with 7596 to perform fractionation. The resulting solution was separated in a furnace, dissolved in water, dialyzed through a cellophane membrane, desalted, and freeze-dried to obtain 2.5 gr of avidin powder. (Yield: 8.396) (Purity Test) The avidin obtained in Example 1 was analyzed by gel permeation-high performance liquid chromatography under the following conditions, and the purity was found to be 98.796.
ゲル浸透−高速液体クロマトグラフィー（ｃｐｃ−ＨＰ
ＬＣ）の条件
カラム： ＴＳＫゲルＧ ３．０００ ＳＷ、 ７５
ｃｍ ＩＤＸ６０α溶 媒： ０．Ｉ Ｍ燐酸緩衝液
、 ０．２ Ｍ ＮａＣ１−流 速＝１．０耐１分
検出波長： ２７５ ｎｍ
（効 果）
以上のごとく、卵白中のりゾチーム・アビジン等の塩基
性蛋白質を低分子吸着用イオン交換デキストランに吸着
させる処理を一度だけ行うだけで。Gel permeation-high performance liquid chromatography (cpc-HP
LC) condition column: TSK gel G 3.000 SW, 75
cm IDX60α solvent: 0. IM phosphate buffer, 0.2 M NaCl - Flow rate = 1.0 1 minute resistance Detection wavelength: 275 nm (Effect) As described above, it is suitable for adsorbing basic proteins such as lysozyme and avidin in egg white with small molecules. All you have to do is adsorb it to ion-exchanged dextran just once.
効率よくしかも高純度のアビジンを溶出することができ
るもので、工業的製造を容易にした。It can elute avidin with high efficiency and high purity, making industrial production easy.
Claims (1)
キストランを使用してアビジンを吸着させて、分離する
ことを特徴とするアビジンの製造方法。 ２、低分子吸着用イオン交換デキストランは、分子量１
０，０００程度の低分子量蛋白質を吸着する架橋デキス
トランあることを特徴とする特許請求の範囲第１項記載
のアビジンの製造方法。 ３、低分子吸着用イオン交換デキストランから吸着した
アビジンを脱着するにあたり３％〜５％の中性塩溶液を
使用することを特徴とする特許請求の範囲第１項記載の
アビジンの製造方法。 ４、低分子吸着用イオン交換デキストランからアビジン
を脱着させた後、塩折またはおよび吸着によって、アビ
ジンを分離することを特徴とする特許請求の範囲第１項
記載のアビジンの製造方法。[Scope of Claims] 1. A method for producing avidin, which comprises adsorbing avidin to basic proteins in egg white using ion-exchange dextran for low-molecular adsorption and separating it. 2. Ion exchange dextran for low molecular adsorption has a molecular weight of 1
2. The method for producing avidin according to claim 1, which comprises crosslinked dextran that adsorbs proteins with a low molecular weight of about 0,000. 3. The method for producing avidin according to claim 1, characterized in that a 3% to 5% neutral salt solution is used to desorb the avidin adsorbed from the ion-exchanged dextran for adsorbing small molecules. 4. The method for producing avidin according to claim 1, which comprises desorbing avidin from ion-exchanged dextran for adsorption of small molecules, and then separating the avidin by salt folding or adsorption.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5311487A JPH07100718B2 (en) | 1987-03-10 | 1987-03-10 | Avidin manufacturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5311487A JPH07100718B2 (en) | 1987-03-10 | 1987-03-10 | Avidin manufacturing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63222200A true JPS63222200A (en) | 1988-09-16 |
| JPH07100718B2 JPH07100718B2 (en) | 1995-11-01 |
Family
ID=12933771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5311487A Expired - Lifetime JPH07100718B2 (en) | 1987-03-10 | 1987-03-10 | Avidin manufacturing method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07100718B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4966851A (en) * | 1986-12-01 | 1990-10-30 | The University Of British Columbia | Process for isolation of lysozyme and avidin from egg white |
| WO2003099035A1 (en) * | 2002-05-23 | 2003-12-04 | Indian Institute Of Technology | Process for the isolation and purification of a glycoprotein avidin |
-
1987
- 1987-03-10 JP JP5311487A patent/JPH07100718B2/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4966851A (en) * | 1986-12-01 | 1990-10-30 | The University Of British Columbia | Process for isolation of lysozyme and avidin from egg white |
| WO2003099035A1 (en) * | 2002-05-23 | 2003-12-04 | Indian Institute Of Technology | Process for the isolation and purification of a glycoprotein avidin |
| US7205393B2 (en) | 2002-05-23 | 2007-04-17 | Indian Institute Of Technology | Process for the isolation and purification of a glycoprotein Avidin |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07100718B2 (en) | 1995-11-01 |
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