JP3213985B2 - Novel protein - Google Patents
Novel proteinInfo
- Publication number
- JP3213985B2 JP3213985B2 JP27136291A JP27136291A JP3213985B2 JP 3213985 B2 JP3213985 B2 JP 3213985B2 JP 27136291 A JP27136291 A JP 27136291A JP 27136291 A JP27136291 A JP 27136291A JP 3213985 B2 JP3213985 B2 JP 3213985B2
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- hhgf
- protein
- xaa
- chain
- 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.)
- Expired - Lifetime
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION
【０００１】[0001]
【産業上の利用分野】本発明は、肝細胞増殖因子（ＨＧ
Ｆ）を特定の位置で切断し、１本鎖型から２本鎖型へ変
換させるプロテアーゼ活性を有する新規なタンパク質に
関する。The present invention relates to hepatocyte growth factor (HG).
The present invention relates to a novel protein having protease activity that cleaves F) at a specific position and converts a single-chain form to a double-chain form.
【０００２】[0002]
【従来の技術および発明が解決しようとする課題】近
年、組換えＤＮＡ技術により、外来の構造遺伝子を宿主
細胞に導入して得られる形質転換体を培養して、該構造
遺伝子を発現させタンパク質を産生させる方法が実施さ
れている。そして、構造遺伝子が動物由来のものである
場合、天然により近いタンパク質を得るために、宿主細
胞として動物細胞を使用する例が増加している。2. Description of the Related Art In recent years, a transformant obtained by introducing a foreign structural gene into a host cell by recombinant DNA technology is cultured, and the protein is expressed by expressing the structural gene. Production methods have been implemented. When the structural gene is derived from an animal, an example of using an animal cell as a host cell to obtain a protein closer to nature is increasing.
【０００３】従来、動物細胞培養物から目的とするタン
パク質を取得する場合に用いられる培養液中には、動物
細胞の増殖に必要な血清が約５〜１０％程度経験的に添
加されている従って、培養物中には、血清由来のタンパ
ク質が混在することにより、目的とするタンパク質が種
々の修飾を受けたり、抑制されたり、また、目的とする
蛋白質を精製するのに手間がかかるという問題点があ
る。Heretofore, about 5 to 10% of serum necessary for the growth of animal cells has been empirically added to a culture solution used for obtaining a target protein from an animal cell culture. However, the presence of serum-derived proteins in the culture causes the target protein to undergo various modifications or is suppressed, and it takes time to purify the target protein. There is.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、ヒトＨＧ
Ｆ（ｈＨＧＦ）遺伝子を組み込んだＣＨＯ細胞を無血清
培地で培養して、ｈＨＧＦを産生させることを試みた。
５〜１０％の血清存在下では、すべてのｈＨＧＦは２本
鎖の型をとっていたが、無血清培養下で産生されたｈＨ
ＧＦの大半は、１本鎖型のｈＨＧＦであり、約２０〜３
０％のみが２本鎖ｈＨＧＦであった。ｈＨＧＦの生理活
性は、ｉｎｖｉｔｒｏ実験において１本鎖では見られ
ず、２本鎖になった場合に発現することが知られてい
る。Means for Solving the Problems The present inventors have developed human HG.
It was attempted to produce hHGF by culturing CHO cells into which F (hHGF) gene had been incorporated in a serum-free medium.
In the presence of 5-10% serum, all hHGFs were in double-stranded form, whereas hHGF produced in serum-free culture
Most of GF is single-chain type hHGF, and is about 20-3
Only 0% was double-stranded hHGF. It is known that the biological activity of hHGF is not observed in a single strand in an in vitro experiment, but is expressed when it becomes double-stranded.
【０００５】本発明者らは、ｈＨＧＦは１本鎖で培養液
中に分泌されて、血清中のプロテアーゼ又はＣＨＯ細胞
由来のプロテアーゼによって２本鎖に切断されるものと
考え、活性型の２本鎖ｈＨＧＦを効率的に調製すること
を目的として、ｈＨＧＦの切断に関与するプロテアーゼ
活性を持つタンパク質の精製を試みた。その結果、非還
元下のＳＤＳ−ポリアクリルアミドゲル電気泳動による
分子量が約３４，０００ダルトンと約３８，０００ダル
トンの、プロテーアゼ活性を有する新規のタンパク質を
見い出し、本発明を完成するに至った。The present inventors believe that hHGF is secreted into the culture medium as a single chain and is cleaved into two chains by a protease in serum or a protease derived from CHO cells. In order to efficiently prepare chain hHGF, purification of a protein having protease activity involved in cleavage of hHGF was attempted. As a result, non-return
Under the present invention, a novel protein having a protease activity having a molecular weight of about 34,000 daltons and about 38,000 daltons by SDS-polyacrylamide gel electrophoresis has been found, and the present invention has been completed.
【０００６】即ち、本発明の第１の要旨は、下記の理化
学的性質を有することを特徴とするヒト血清をカラムク
ロマトグラフィーで精製することにより得られた新規な
タンパク質に存する。非還元下のＳＤＳ−ポリアクリ
ルアミドゲル電気泳動による分子量が約３４，０００ダ
ルトンである。７２８アミノ酸残基より成る１本鎖型
ヒト肝細胞増殖因子をそのアミノ末端から４９４番目の
アミノ酸であるアルギニンと４９５番目のアミノ酸であ
るバリンとの間で特異的に切断して活性型の２本鎖型ヒ
ト肝細胞増殖因子に変換するプロテアーゼ活性を有す
る。非還元下で下記の２つのＮ−末端アミノ酸配列を
有する。 ａ：Ile Ile Gly Gly Ser Ser Ser Leu ｂ：Val Gln Leu Ser Pro Asp Leu Xaa （Xaaは未同定のアミノ酸を表す） また、本発明の第２の要旨は、下記の理化学的性質を有
することを特徴とするウシ胎児血清をカラムクロマトグ
ラフィーで精製することにより得られた新規なタンパク
質に存する。非還元下のＳＤＳ−ポリアクリルアミド
ゲル電気泳動による分子量が約３８，０００ダルトンで
ある。７２８アミノ酸残基より成る１本鎖型ヒト肝細
胞増殖因子をそのアミノ末端から４９４番目のアミノ酸
であるアルギニンと４９５番目のアミノ酸であるバリン
との間で特異的に切断して活性型の２本鎖型ヒト肝細胞
増殖因子に変換するプロテアーゼ活性を有する。非還
元下で下記の２つのＮ−末端アミノ酸配列を有する。 ａ：Ile Ile Gly Gly Ser Ser Ser Leu ｃ：Ile Gln Pro Pro Xaa Xaa Glu Ala （Xaaは未同定のアミノ酸を表す） [0006] That is, a first gist of the present invention is to provide a human serum that has the following physicochemical properties.
It is a novel protein obtained by purification by chromatography . The molecular weight by non-reducing SDS-polyacrylamide gel electrophoresis was about 34,000 daltons.
Luton . 728 specifically cleaves to activity between the valine is arginine and 495 th amino acids is 494 amino acids single stranded form <br/> human hepatocyte growth factor consisting of amino acid residues from the amino terminus Double-stranded type
It has protease activity to convert to hepatocyte growth factor . Under non-reduction, the following two N-terminal amino acid sequences are
Have. a: Ile Ile Gly Gly Ser Ser Ser Leu b: Val Gln Leu Ser Pro Asp Leu Xaa (Xaa represents an unidentified amino acid) The second gist of the present invention has the following physicochemical properties.
Column chromatography using fetal bovine serum
A novel protein obtained by laffy purification
It depends on quality. SDS-polyacrylamide under non-reducing
Gel molecular weight of about 38,000 daltons
is there. Single chain human liver consisting of 728 amino acid residues
Vesicle growth factor is the 494th amino acid from its amino terminus
Arginine and valine 495th amino acid
Activated double-chain human hepatocytes specifically cleaved between
Has protease activity to convert to growth factors. Non-return
It has the following two N-terminal amino acid sequences: a: Ile Ile Gly Gly Ser Ser Ser Leu c: Ile Gln Pro Pro Xaa Xaa Glu Ala (Xaa represents an unidentified amino acid)
【０００７】本タンパク質を無血清培養下のｈＨＧＦ生
産時に適量添加することにより、ほぼ１００％２本鎖の
ｈＨＧＦを得ることができ、また、試験管内で本タンパ
ク質を作用させると、１本鎖ｈＨＧＦを２本鎖ｈＨＧＦ
に変えることができる。本発明をさらに詳細に説明する
に、本発明のプロテアーゼ活性を有する新規なタンパク
質は、以下のような精製段階を経ることにより得られ
る。例えば健常人より採血した血液を、ガラス容器中、
冷蔵庫内で、一晩放置後、生じた血ぺいと細胞を遠心分
離により除去後、０．４μｍの目のフィルターでろ過し
たヒト血清、又は、市販の種々の動物血清（Ｇｉｂｃｏ
社製のウシ胎児血清、ＩｒｖｉｎｅＳｃｉｅｎｔｉｆｉ
ｃ社製のウシ血清、ウマ血清、ブタ血清等）を材料とす
る。これら血清を水で１．５〜２倍に希釈後、Ｈｅｐａ
ｒｉｎ−ｓｅｐｈａｒｏｓｅカラム（ファルマシア社製
等）等に供する。そのカラムクロマトグラフィーより得
られた当該タンパク質を含むフラクションを疎水クロマ
トグラフィー（たとえば、ファルマシア社製Ｐｈｅｎｙ
ｌ−ｓｅｐｈａｒｏｓｅカラム等）にかける。得られた
当該タンパク質を含むフラクションをＡｐｒｏｔｉｎｉ
ｎ固定化アフィニティーカラム（ペンタファーム社製
等）に供し、その後で、ゲルろ過カラムクロマトグラフ
ィー（旭化成社製ＧＳ５２０等）で処理することによ
り、本タンパク質を得ることができる。必要に応じて、
イオン交換カラムクロマトグラフィー、ハイドロキシア
パタイトカラムクロマトグラフィー等を精製のステップ
に組み込むこともできる。[0007] By adding the present protein in an appropriate amount during the production of hHGF in serum-free culture, almost 100% of a double-stranded hHGF can be obtained. Is converted to a double-stranded hHGF
Can be changed to In order to explain the present invention in more detail, the novel protein having the protease activity of the present invention can be obtained through the following purification steps. For example, blood collected from a healthy person is placed in a glass container,
After standing in a refrigerator overnight, the resulting blood cells and cells were removed by centrifugation and then filtered through a 0.4 μm filter, or various commercially available animal sera (Gibco).
Fetal bovine serum, Irvine Scientific
c bovine serum, horse serum, pig serum, etc.). After diluting these sera 1.5 to 2-fold with water, Hepa
A rin-sepharose column (manufactured by Pharmacia) or the like is used. The fraction containing the protein obtained by the column chromatography was subjected to hydrophobic chromatography (for example, Phenyl by Pharmacia).
l-sepharose column). The obtained fraction containing the protein is subjected to Aprotini
The present protein can be obtained by subjecting to an n-immobilized affinity column (Pentafirm Co., Ltd. or the like) and then performing a gel filtration column chromatography (Asahi Kasei GS520 or the like). If necessary,
Ion exchange column chromatography, hydroxyapatite column chromatography and the like can also be incorporated in the purification step.
【０００８】精製された本発明のタンパク質は非還元下
のＳＤＳ−ポリアクリルアミドゲル電気泳動による分子
量が約３４，０００ダルトンと約３８，０００ダルトン
であり、１本鎖ｈＨＧＦを、特定の位置で切断するこに
より、２本鎖ｈＨＧＦを生じさせるプロテアーゼ活性を
有する。また、本発明のタンパク質を、還元カルボキシ
メチル化処理後、逆相カラムクロマトグラフィー（ワイ
エム シー 社製ＹＭＣ ｐａｃｋ Ｃ４カラム
等）で処理することにより得られる主要なペプチドは配
列番号：１に記載のＮ−末端アミノ酸配列を含む。[0008] Proteins of the present invention the purified under non-reducing
Molecular weight by Bruno SDS- polyacrylamide gel electrophoresis of about 34,000 daltons and about 38,000 daltons
, And the single-stranded hHGF, by this to cleave at a specific position, having protease activity to generate double-stranded hHGF. The protein of the present invention is subjected to reductive carboxymethylation treatment, and then subjected to reverse phase column chromatography (YMC pack C4 column manufactured by YMC).
Etc.) comprise the N-terminal amino acid sequence of SEQ ID NO: 1.
【０００９】[0009]
【発明の効果】本発明に係わるプロテアーゼ活性を有す
るタンパク質は１本鎖ｈＨＧＦを活性型の２本鎖ｈＨＧ
Ｆに変換する能力を持つため、ｉｎ ｖｉｖｏまたはｉ
ｎ ｖｉｔｒｏでのｈＨＧＦの活性の調節因子として、
また２本鎖ｈＨＧＦを無血清培養時や試験管内で調製す
るために使用される。According to the protein having protease activity of the present invention, the single-chain hHGF is converted into the active double-chain hHG.
F or in vivo or i
As modulators of hHGF activity in vitro
It is also used for preparing double-stranded hHGF during serum-free culture or in a test tube.
【００１０】[0010]
【実施例】以下の実施例により、本発明をさらにより詳
細に説明するが、本発明は、その要旨を越えない限り、
以下の実施例によって限定されるものではない。 実施例１（ウシ胎児血清を用いての当該タンパク質の精
製とアミノ酸配列解析） ウシ胎児血清（Ｇｉｂｃｏ社製）を水で１．５倍に希釈
し、それをＨｅｐａｒｉｎ−ｓｅｐｈａｒｏｓｅカラム
（ファルマシア社製：１．５倍に水で希釈されたダルベ
ッコのＰＢＳで平衡化されている）に添加し、緩衝液Ａ
（１０mM ＮａＨ2 ＰＯ4 −Ｎａ2 ＨＰＯ4 バッファ
ー，pH７．０，０．１Ｍ ＮａＣｌ含）で洗浄後、この
緩衝液Ａと緩衝液Ｂ（１０mM ＮａＨ2 ＰＯ4 −Ｎａ2
ＨＰＯ4 バッファー，pH７．０，０．７Ｍ ＮａＣｌ
含）を用いて、ＮａＣｌ濃度０．１Ｍから０．７Ｍの濃
度勾配溶出を行った。そして、１本鎖ｈＨＧＦを２本鎖
ｈＨＧＦに変換するプロテアーゼ活性が存在する画分
（約３００〜４５０mM ＮａＣｌ画分）を回収した。こ
の画分を、２Ｍ硫酸アンモニウムと等量混合後、１Ｍ硫
酸アンモニウムで平衡化されたＰｈｅｎｙｌ−ｓｅｐｈ
ａｒｏｓｅカラム（ファルマシア社製）に添加した。１
Ｍ硫酸アンモニウムで洗浄後、硫酸アンモニウム１Ｍか
ら０Ｍへの負の濃度勾配溶出を行い、１本鎖ｈＨＧＦを
２本鎖ｈＨＧＦに変換するプロテアーゼ活性が存在する
画分（約６００〜４００mM硫酸アンモニウム画分）を回
収した。当画分を緩衝液Ｃ（３００mM ＮａＣｌ，５０
mM Ｔｒｉｓ／ＨＣｌ，pH８．０）に一晩透析後、緩衝
液Ｃで平衡化したＡｐｒｏｔｉｎｉｎ固定化カラム（ペ
ンタファーム社製）に添加した。緩衝液Ｃで洗浄後、緩
衝液Ｄ（０．１Ｎ ＨＣｌ，２０mM ＣａＣｌ2 ）で溶
出した。溶出画分を集め、緩衝液Ｅ（１Ｍ Ｔｒｉｓ／
ＨＣｌ，pH８．０）で中和後、限外ろ過膜で濃縮した。
この濃縮液を、緩衝液Ｃを用いて０．５ml／min の流速
で平衡化したＧＳ５２０カラム（旭化成社製）に添加し
た。分子量約３６，０００ダルトンの位置のピークを分
取し、この標品を用いて以下に述べるアミノ酸配列の解
析を行った。EXAMPLES The present invention will be described in further detail with reference to the following Examples, which do not depart from the gist of the present invention.
It is not limited by the following examples. Example 1 (Purification of the Protein Using Fetal Bovine Serum and Amino Acid Sequence Analysis) Fetal bovine serum (manufactured by Gibco) was diluted 1.5-fold with water and then diluted with a Heparin-sepharose column (manufactured by Pharmacia: Buffer A (equilibrated with Dulbecco's PBS diluted 1.5 times with water)
(10 mM NaH 2 PO 4 —Na 2 HPO 4 buffer, pH 7.0, containing 0.1 M NaCl), and then buffer A and buffer B (10 mM NaH 2 PO 4 —Na 2).
HPO 4 buffer, pH 7.0, 0.7 M NaCl
) Was used to perform a concentration gradient elution with a NaCl concentration of 0.1 M to 0.7 M. Then, a fraction having protease activity for converting single-chain hHGF to double-chain hHGF (approximately 300 to 450 mM NaCl fraction) was collected. This fraction was mixed with 2 M ammonium sulfate in an equal amount, and then Phenyl-seph equilibrated with 1 M ammonium sulfate.
It was added to an arose column (Pharmacia). 1
After washing with ammonium sulfate M, a negative concentration gradient elution was performed from ammonium sulfate 1M to 0M, and a fraction having a protease activity for converting single-chain hHGF to double-chain hHGF (about 600 to 400 mM ammonium sulfate fraction) was recovered. did. This fraction was added to buffer C (300 mM NaCl, 50
mM Tris / HCl, pH 8.0) overnight, and then added to an Aprotinin-immobilized column (Pentafirm) equilibrated with buffer C. After washing with buffer C, elution was performed with buffer D (0.1 N HCl, 20 mM CaCl 2 ). The eluted fractions were collected and buffer E (1 M Tris /
HCl, pH 8.0) and concentrated with an ultrafiltration membrane.
This concentrated solution was added to a GS520 column (manufactured by Asahi Kasei Corporation) equilibrated with the buffer C at a flow rate of 0.5 ml / min. A peak at a position of a molecular weight of about 36,000 daltons was collected, and the amino acid sequence described below was analyzed using this sample.
【００１１】尚、上記の各精製工程において、後述実施
例３に記載の方法で、１本鎖ｈＨＧＦを２本鎖ｈＨＧＦ
に変換する活性を指標として、分画精製した。上記の精
製されたプロテアーゼ活性を有するタンパク質を、緩衝
液Ｆ（６Ｍ塩酸グアニジン，０．００２Ｍエチレンジア
ミン４酢酸塩、１Ｍトリス塩酸バッファー，pH８．５）
中で、２−メルカプトエタノールにより、４０℃，２時
間還元した後、等濃度のモノヨード酢酸を加え、窒素ガ
ス下、室温、遮光下で１時間反応させ、カルボキシメチ
ル化を行った。反応後、ＹＭＣ ｐａｃｋ Ｃ４カラム
（ワイ エム シー 社製）に添加し、アセトニトリル
／イソプロピルアルコール（３／７）濃度１０％から７
０％まで３０分間の濃度勾配溶出を行い、１本の主要な
ピークを分取した。このピークを真空状態で乾燥した
後、５０％ＴＦＡ（トリフルオロ酢酸）６０μｌに溶解
し、ポリブレン処理したグラスフィルターに添加し、Ａ
ｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製４７０Ａシー
クエンサーでエドマン分解し、Ｎ末端域のアミノ酸配列
を決定した。フェニルチオヒダントイン（ＰＴＨ）アミ
ノ酸の同定は、三菱化成社製“ＭＣＩ ｇｅｌ ＯＤＳ
ＩＨＵ”（０．４６×１５cm）カラムを用い、酢酸緩
衝液（１０mM酢酸緩衝液，pH４．７，０．０１％ＳＤ
Ｓ，３８％アセトニトリル）による単一溶媒溶出法を流
速１．２ml／分、温度４３℃で行い、ＰＴＨアミノ酸の
検出は２６９nmの吸光度で行った。In each of the above-mentioned purification steps, single-chain hHGF is converted into double-chain hHGF by the method described in Example 3 below.
Fractionation and purification were carried out using the activity of converting to as an index. The purified protein having protease activity was converted to buffer F (6 M guanidine hydrochloride, 0.002 M ethylenediamine tetraacetate, 1 M Tris-HCl buffer, pH 8.5).
After reducing with 2-mercaptoethanol at 40 ° C. for 2 hours, monoiodoacetic acid of an equal concentration was added, and the mixture was reacted under a nitrogen gas at room temperature under light shielding for 1 hour to carry out carboxymethylation. After the reaction, the mixture was added to a YMC pack C4 column (manufactured by YMC) and the concentration of acetonitrile / isopropyl alcohol (3/7) was changed from 10% to 7%.
Gradient elution was performed for 30 minutes to 0%, and one major peak was collected. After drying this peak under vacuum, it was dissolved in 60 μl of 50% TFA (trifluoroacetic acid), and added to a glass filter treated with polybrene.
Edman degradation was performed using a 470A sequencer manufactured by Pplied Biosystems, and the amino acid sequence in the N-terminal region was determined. Identification of phenylthiohydantoin (PTH) amino acid was performed using Mitsubishi Chemical Kagaku's “MCI gel ODS”.
Using an IHU "(0.46 × 15 cm) column, an acetate buffer (10 mM acetate buffer, pH 4.7, 0.01% SD)
(S, 38% acetonitrile) using a single solvent elution method at a flow rate of 1.2 ml / min at a temperature of 43 ° C., and detection of PTH amino acid was performed at an absorbance of 269 nm.
【００１２】この結果、還元カルボキシメチル化された
このタンパク質から得られる１つのペプチドのＮ末端ア
ミノ酸配列は、後述の配列番号：１に示すようであっ
た。また、還元カルボキシメチル化せずに、当該タンパ
ク質をＹＭＣ ｐａｃｋＣ４カラムに添加し、アセトニ
トリル／イソプロピルアルコール（３／７）濃度１０％
から７０％まで３０分間の濃度勾配溶出を行い、１本の
ピークを分取した。このピークを真空状態で乾燥した
後、同様にＮ末端域のアミノ酸配列を分析した。この結
果、カルボキシメチル化されたペプチドから得られたＮ
末端アミノ酸配列を含む、等モルの２個のアミノ酸が各
位置で検出された。アミノ酸配列を下記に示す。 Ｉｌｅ Ｉｌｅ Ｇｌｙ Ｇｌｙ Ｓｅｒ Ｓｅｒ Ｓｅｒ Ｌｅｕ − − − − − − − −・・・ Ｉｌｅ Ｇｌｎ Ｐｒｏ Ｐｒｏ Ｘａａ Ｘａａ Ｇｌｕ Ａｌａ （Ｘａａは未同定のアミノ酸） このことより、当該タンパク質は２本鎖のタンパク質で
ある可能性が示された。As a result, the N-terminal amino acid sequence of one peptide obtained from the reductive carboxymethylated protein was as shown in SEQ ID NO: 1 described below. Also, without subjecting the protein to reductive carboxymethylation, the protein was added to a YMC packC4 column, and the concentration of acetonitrile / isopropyl alcohol (3/7) was 10%.
Gradient eluted from to 30% for 30 minutes, and one peak was collected. After drying this peak under vacuum, the amino acid sequence in the N-terminal region was similarly analyzed. As a result, the N obtained from the carboxymethylated peptide
Equimolar two amino acids, including the terminal amino acid sequence, were detected at each position. The amino acid sequence is shown below. Ile Ile Gly Gly Ser Ser Leu----------Ile Gln Pro Pro Xaa Xaa Glu Ala (Xaa is an unidentified amino acid). Sex was shown.
【００１３】実施例２（ヒト血清を用いての当該タンパ
ク質の精製とアミノ酸配列解析） 常法により得られたヒト血清を水で１．５倍に希釈し、
それを実施例１と同様の方法でＨｅｐａｒｉｎ−ｓｅｐ
ｈａｒｏｓｅカラムを用いて濃度勾配溶出を行った。そ
して、１本鎖ｈＨＧＦを２本鎖ｈＨＧＦに変換するプロ
テアーゼ活性が存在する画分（約３００〜４５０mM Ｎ
ａＣｌ画分）を回収した。この画分を実施例１と同様の
方法でＰｈｅｎｙｌ−ｓｅｐｈａｒｏｓｅカラム（ファ
ルマシア社製）を用いて負の濃度勾配溶出を行い、１本
鎖ｈＨＧＦを２本鎖ｈＨＧＦに変換するプロテアーゼ活
性が存在する画分（約７００〜５００mM硫酸アンモニウ
ム画分）を回収した。以下、実施例１と同様の方法によ
り処理し、分子量約３０，０００ダルトンの位置のピー
クを分取し、この標品を用いて以下に述べるアミノ酸配
列の解析を行った。Example 2 (Purification of the Protein Using Human Serum and Analysis of Amino Acid Sequence) Human serum obtained by a conventional method was diluted 1.5-fold with water.
It was prepared in the same manner as in Example 1 using Heparin-sep
Concentration gradient elution was performed using a harose column. Then, a fraction (about 300 to 450 mM N) having a protease activity for converting single-chain hHGF to double-chain hHGF is present.
aCl fraction) was collected. This fraction was subjected to negative concentration gradient elution using a Phenyl-sepharose column (manufactured by Pharmacia) in the same manner as in Example 1, and a fraction having protease activity for converting single-chain hHGF to double-chain hHGF was present. (About 700-500 mM ammonium sulfate fraction). Thereafter, treatment was carried out in the same manner as in Example 1, a peak at a position of a molecular weight of about 30,000 daltons was collected, and the amino acid sequence described below was analyzed using this sample.
【００１４】尚、上記の各精製工程において、後述実施
例３に記載の方法で、１本鎖ｈＨＧＦを２本鎖ｈＨＧＦ
に変換する活性を指標として、分画精製した。上記の精
製されたプロテアーゼ活性を有するタンパク質のＮ末端
域のアミノ酸配列を実施例１と同様の方法で決定した。
この結果、還元カルボキシメチル化されたこのタンパク
質から得られる１つのペプチドのＮ末端アミノ酸配列
は、後述の配列番号：１に示すようであった。In each of the above-mentioned purification steps, single-chain hHGF was converted to double-chain hHGF by the method described in Example 3 below.
Fractionation and purification were carried out using the activity of converting to as an index. The amino acid sequence in the N-terminal region of the purified protein having protease activity was determined in the same manner as in Example 1.
As a result, the N-terminal amino acid sequence of one peptide obtained from the reductive carboxymethylated protein was as shown in SEQ ID NO: 1 described below.
【００１５】また、還元カルボキシメチル化せずに、当
該タンパク質をＹＭＣ ｐａｃｋＣ４カラムに添加し、
アセトニトリル／イソプロピルアルコール（３／７）濃
度１０％から７０％まで３０分間の濃度勾配溶出を行い
１本のピークを分取した。このピークを真空状態で乾燥
した後、同様にＮ末端域のアミノ酸配列を分析した。こ
の結果、カルボキシメチル化されたペプチドから得られ
たＮ末端アミノ酸配列を含む、等モルの２個のアミノ酸
が各位置で検出された。Ｎ末端アミノ酸配列を下記に示
す。 Ｉｌｅ Ｉｌｅ Ｇｌｙ Ｇｌｙ Ｓｅｒ Ｓｅｒ Ｓｅｒ Ｌｅｕ − − − − − − − −・・・ Ｖａｌ Ｇｌｎ Ｌｅｕ Ｓｅｒ Ｐｒｏ Ａｓｐ Ｌｅｕ Ｘａａ （Ｘａａは未同定のアミノ酸） このことより、当該タンパク質は２本鎖のタンパク質で
ある可能性が示された。Further, the protein is added to a YMC packC4 column without reductive carboxymethylation,
A gradient was eluted from acetonitrile / isopropyl alcohol (3/7) concentration of 10% to 70% for 30 minutes, and one peak was collected. After drying this peak under vacuum, the amino acid sequence in the N-terminal region was similarly analyzed. As a result, equimolar two amino acids including the N-terminal amino acid sequence obtained from the carboxymethylated peptide were detected at each position. The N-terminal amino acid sequence is shown below. Ile Ile Gly Gly Ser Ser Leu------------Val Gln Leu Ser Pro Asp Leu Xaa (Xaa is an unidentified amino acid). Sex was shown.
【００１６】実施例３（当該タンパク質の１本鎖ｈＨＧ
Ｆを２本鎖ｈＨＧＦに変換する活性を測定する方法とそ
の活性） 測定しようとするサンプル１０μｌを、５μｇ１本鎖ｈ
ＨＧＦを含む１００mMＮａＣｌ，５０mMトリス塩酸バッ
ファー，pH８．０溶液４０μｌに添加した。３７℃で３
時間インキュベーション後、この混合液を、還元条件下
でＳＤＳ−ポリアクリルアミドゲル電気泳動に供した。
電気泳動後、クマシーブリリアントブルーＲ２５０（Ｃ
ＢＢ）で染色し、１本鎖ｈＨＧＦと２本鎖ｈＨＧＦの割
合を比較することで、活性を検出した。Example 3 (single-chain hHG of the protein
Method for measuring the activity of converting F into double-stranded hHGF and its activity) 10 μl of the sample to be measured was added to 5 μg of single-stranded hHGF.
It was added to 40 μl of 100 mM NaCl, 50 mM Tris-HCl buffer, pH 8.0 solution containing HGF. 3 at 37 ° C
After incubation for a period of time, the mixture was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions.
After electrophoresis, Coomassie Brilliant Blue R250 (C
BB), and the activity was detected by comparing the ratio of single-chain hHGF to double-chain hHGF.
【００１７】また、最終的に精製された当該タンパク質
を１本鎖ｈＨＧＦと上記条件で反応させた後、反応液を
緩衝液Ｆにバッファー置換させ、実施例１と同様の方法
で還元カルボキシメチル化反応を行った。反応後、ＹＭ
Ｃ ｐａｃｋ Ｃ４カラム（ワイ エム シー 社製）
に添加し、アセトニトリル濃度１０％から７０％まで３
０分間の濃度勾配にて溶出を行い各ピークを分取した。
還元カルボキシメチル化された天然の２本鎖ｈＨＧＦと
同じ位置に溶出したピークについて、実施例１と同様の
方法で、Ｎ末端アミノ酸配列分析を行い、天然のｈＨＧ
Ｆの２本鎖への開裂部位であるＶａｌ−Ｖａｌ−Ａｓｎ
−Ｇｌｙ−Ｉｌｅ−Ｐｒｏ・・・の配列を確認した。従
って当該プロテアーゼ活性を持つタンパク質は、１本鎖
ｈＨＧＦを、天然の開裂部位である配列のところで切断
することが明らかになった。なお、１本鎖のｈＨＧＦは
後述の参考例に示す方法にて調製した。 実施例４（ＳＤＳ−ポリアクリルアミドゲル電気泳動） ウシ胎児血清とヒト血清より実施例１と２に従って精製
されたプロテアーゼ活性を持つ当該タンパク質の見かけ
上の分子量を求めるため、ＳＤＳ−ポリアクリルアミド
ゲル電気泳動を行った。最終的に精製された当該タンパ
ク質を１２．５％のポリアクリルアミド・スラブゲルを
用いたＳＤＳ−ポリアクリルアミドゲル電気泳動に、非
還元下で供した。分子量マーカーとしては、分子量マー
カー「第一」III Ｌａｅｍｍｌｉ法用（第一化学薬品社
製）を用いた。電気泳動後、銀染色試薬（関東化学社
製）を用いて発色させた。当該タンパク質と標準分子量
マーカータンパク質との泳動距離の相対的比較により、
ウシ胎児血清由来のタンパク質はＳＤＳ−ポリアクリル
アミドゲル電気泳動上のみかけの分子量として約３８，
０００ダルトンであり、ヒト血清由来のタンパク質は約
３４，０００ダルトンであった。After reacting the finally purified protein with single-chain hHGF under the above conditions, the reaction solution was replaced with buffer F, and reductive carboxymethylation was carried out in the same manner as in Example 1. The reaction was performed. After the reaction, YM
C pack C4 column (YMC)
To acetonitrile concentration from 10% to 70%.
Elution was performed with a concentration gradient of 0 minutes, and each peak was collected.
The N-terminal amino acid sequence analysis was performed on the peak eluted at the same position as that of the reductive carboxymethylated natural double-stranded hHGF in the same manner as in Example 1, and the natural hHG
Val-Val-Asn which is a cleavage site of F into a double strand
The sequence of -Gly-Ile-Pro ... was confirmed. Therefore, it was revealed that the protein having the protease activity cleaves single-chain hHGF at a sequence that is a natural cleavage site. In addition, single-chain hHGF was prepared by the method described in Reference Examples described later. Example 4 (SDS-polyacrylamide gel electrophoresis) SDS-polyacrylamide gel electrophoresis in order to determine the apparent molecular weight of the protein having protease activity purified from fetal bovine serum and human serum according to Examples 1 and 2 Was done. The protein finally purified was subjected to SDS-polyacrylamide gel electrophoresis using 12.5% polyacrylamide slab gel under non-reducing conditions. As the molecular weight marker, a molecular weight marker “Daiichi” III for Laemmli method (Daiichi Pure Chemicals) was used. After the electrophoresis, the color was developed using a silver staining reagent (manufactured by Kanto Kagaku). By relative comparison of the migration distance between the protein and the standard molecular weight marker protein,
The protein derived from fetal bovine serum has an apparent molecular weight of about 38, on SDS-polyacrylamide gel electrophoresis.
000 daltons and the protein from human serum was about 34,000 daltons.
【００１８】実施例５（当該タンパク質の無血清培養へ
の添加） 欧州公開特許第４１２５５７号に記載された方法に従っ
て作製されたヒトＨＧＦ産生組換えＣＨＯ細胞（１×１
０5 cells／ml）を５％牛胎児血清を含有するｅ−ＲＤ
Ｆ培地１５ml，１０cm ｄｉｓｈ中で、５％ＣＯ2 雰囲
気下、３７℃で培養増殖させた。培養約４日後、コンフ
ルエントな状態に達した時点で、実施例１で得た牛胎児
血清由来の当該タンパク質を含む無血清培地ｅ−ＲＤＦ
１５mlに移し換え、更に２日間培養を続けた。培養上清
を回収して、緩衝液Ｇ（０．３ＭＮａＣｌ，１０mM Ｎ
ａＨ2 ＰＯ4 −Ｎａ2 ＨＰＯ4 バッファー，pH７．５）
で平衡化したＳ−ｓｅｐｈａｒｏｓｅカラム（ファルマ
シア社製）に添加した。緩衝液Ｇで十分洗浄後、１Ｍ
ＮａＣｌを含む１０mM ＮａＨ2 ＰＯ4 −Ｎａ2 ＨＰＯ
4 バッファー（pH７．５）で溶出した。溶出液を集め、
限外ろ過膜により濃縮し、１２．５％ポリアクリルアミ
ド・スラブゲルを用いたＳＤＳ−ポリアクリルアミド電
気泳動に、還元下で供した。電気泳動後、ＣＢＢで染色
し、１本鎖ｈＨＧＦと２本鎖ｈＨＧＦの割合いを比較し
た。結果を図１に示す。図中、１は当該タンパク質無添
加の場合を、２は培地に当該タンパク質を約３０ng／ml
添加した場合の結果を表わす。Example 5 (Addition of the protein to serum-free culture) Human HGF-producing recombinant CHO cells (1 × 1) prepared according to the method described in EP-A-412557.
0 5 cells / ml) containing 5% fetal calf serum e-RD
The cells were cultured and grown at 37 ° C. in a 5% CO 2 atmosphere in 15 ml of F medium and 10 cm dish. Approximately 4 days after the culture, when the cells reach confluence, a serum-free medium e-RDF containing the protein derived from fetal calf serum obtained in Example 1 is obtained.
It was transferred to 15 ml and the culture was continued for another 2 days. The culture supernatant was collected and buffer G (0.3 M NaCl, 10 mM N
aH 2 PO 4 -Na 2 HPO 4 buffer, pH 7.5)
Was added to an S-sepharose column (manufactured by Pharmacia) equilibrated in the above. After washing well with buffer G, 1M
10 mM NaH 2 PO 4 -Na 2 HPO containing NaCl
Elution was performed with 4 buffers (pH 7.5). Collect the eluate,
The mixture was concentrated by an ultrafiltration membrane, and subjected to SDS-polyacrylamide electrophoresis using 12.5% polyacrylamide slab gel under reduction. After electrophoresis, the cells were stained with CBB, and the ratio of single-chain hHGF to double-chain hHGF was compared. The results are shown in FIG. In the figure, 1 is a case where the protein is not added, 2 is a medium containing about 30 ng / ml of the protein.
The results when added are shown.
【００１９】参考例 一本鎖型組換えｈＨＧＦ（ｒ−ｈＨＧＦ）生産方法 培養容器としてＴ−１５０フラスコを用いてｒ−ｈＨＧ
Ｆ産生組換えＣＨＯ細胞を５％牛胎児血清を含む基本合
成培地ｅ−ＲＤＦ６０ml中、３７℃で培養増殖させた。
培養約４日後、コンフルエントな状態に達した時点で、
血清を含む培養液を除去し、新しいｅ−ＲＤＦ培地６０
mlを添加し、さらに一日培養した。次の日、その培養液
を除去し、ｅ−ＲＤＦ培地６０mlで細胞を一度洗浄後、
以下の生産培地を添加し、２日間、３７℃で培養するこ
とにより、一本鎖ｒ−ｈＨＧＦを産生させた。Reference Example Method for producing single-chain recombinant hHGF (r-hHGF) r-hHG using a T-150 flask as a culture vessel
F-produced recombinant CHO cells were grown at 37 ° C. in 60 ml of a basic synthetic medium e-RDF containing 5% fetal bovine serum.
After about 4 days in culture, when confluence is reached,
The culture solution containing serum was removed, and a fresh e-RDF medium 60 was removed.
ml was added and the cells were further cultured for one day. The next day, the culture was removed and the cells were washed once with 60 ml of e-RDF medium.
The following production medium was added, and the cells were cultured at 37 ° C. for 2 days to produce single-stranded r-hHGF.
【００２０】培養後、遠心により細胞を除去した培養上
清を回収した。この培養上清より、２本鎖ｒ−ｈＨＧＦ
精製と同じ条件にて、Ｓ−セファロースカラムにて、一
本鎖ｒ−ｈＨＧＦを精製した。この精製標品は、全ｒ−
ｈＨＧＦ量に対して、一本鎖ｒ−ｈＨＧＦを約９０％含
んでいた。 生産培地 ｅ−ＲＤＦ ６０ml この基本合成培地に以下の物を添加 インスリン １０μｇ／ml トランスフェリン １０μｇ／ml セレナイト １０-8Ｍ エタノールアミン １０-5Ｍ アプロチニン ５０ＫＩＵ／ml メシル酸ナファモスタット（６−アミジノ−２ナフチル
ｐ−グアニジノベンゾアートジメタンスルフォネート）
５０μＭAfter the culture, the culture supernatant from which the cells were removed by centrifugation was recovered. From this culture supernatant, double-stranded r-hHGF
Under the same conditions as for the purification, single-stranded r-hHGF was purified on an S-Sepharose column. This purified sample was completely r-
It contained about 90% of single-chain r-hHGF based on the amount of hHGF. Production medium e-RDF 60 ml The following were added to this basic synthetic medium: insulin 10 μg / ml transferrin 10 μg / ml selenite 10 −8 M ethanolamine 10 −5 M aprotinin 50 KIU / ml nafamostat mesilate (6-amidino-2naphthyl) p-guanidinobenzoate dimethanesulfonate)
50 μM
【００２１】［配列表］ 配列番号：１ 配列の長さ：８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 フラグメント型：Ｎ末端フラグメント （２本鎖の内の１本のＮ末端フラグメント） 起源：生物名：ヒト，ウシ 組織の種類：血清 配列 Ｉｌｅ Ｉｌｅ Ｇｌｙ Ｇｌｙ Ｓｅｒ Ｓｅｒ Ｓｅｒ Ｌｅｕ １ ５ ８[Sequence Table] SEQ ID NO: 1 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: protein Fragment type: N-terminal fragment (N-terminal of one of the two chains) Fragment) Origin: Organism name: Human, bovine Tissue type: Serum Sequence Ile Ile Gly Gly Ser Ser Ser Leu 158
【００２２】 配列番号：２ 配列の長さ：８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 フラグメント型：Ｎ末端フラグメント （２本鎖の内の１本のＮ末端フラグメント） 起源：生物名：ウシ 組織の種類：血清 配列 Ｉｌｅ Ｇｌｎ Ｐｒｏ Ｐｒｏ Ｘａａ Ｘａａ Ｇｌｕ Ａｌａ １ ５ ８ （Ｘａａは未同定のアミノ酸）SEQ ID NO: 2 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: protein Fragment type: N-terminal fragment (N-terminal fragment of one of the two chains) Origin: Organism name: Bovine Tissue type: Serum Sequence Ile Gln Pro Pro Xaa Xaa Glu Ala 158 (Xaa is an unidentified amino acid)
【００２３】 配列番号：３ 配列の長さ：８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 フラグメント型：Ｎ末端フラグメント （２本鎖の内の１本のＮ末端フラグメント） 起源：生物名：ヒト 組織の種類：血清 配列 Ｖａｌ Ｇｌｎ Ｌｅｕ Ｓｅｒ Ｐｒｏ Ａｓｐ Ｌｅｕ Ｘａａ １ ５ ８ （Ｘａａは未同定のアミノ酸）SEQ ID NO: 3 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: protein Fragment type: N-terminal fragment (N-terminal fragment of one of the two chains) Origin: Organism name: Human Tissue type: Serum Sequence Val Gln Leu Ser Pro Asp Leu Xaa 158 (Xaa is an unidentified amino acid)
【図１】本発明のタンパク質の存在の有無による１本鎖
ｈＨＧＦおよび２本鎖ｈＨＧＦの生成割合を示す図面で
ある。FIG. 1 is a drawing showing the production ratio of single-chain hHGF and double-chain hHGF depending on the presence or absence of the protein of the present invention.
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.7，ＤＢ名) C12N 9/64 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＷＰＩ（ＤＩＡＬＯＧ) ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. 7 , DB name) C12N 9/64 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)
Claims (2)
とするヒト血清をカラムクロマトグラフィーで精製する
ことにより得られた新規なタンパク質。非還元下のＳ
ＤＳ−ポリアクリルアミドゲル電気泳動による分子量が
約３４，０００ダルトンである。７２８アミノ酸残基
より成る１本鎖型ヒト肝細胞増殖因子をそのアミノ末端
から４９４番目のアミノ酸であるアルギニンと４９５番
目のアミノ酸であるバリンとの間で特異的に切断して活
性型の２本鎖型ヒト肝細胞増殖因子に変換するプロテア
ーゼ活性を有する。非還元下で下記の２つのＮ−末端
アミノ酸配列を有する。 ａ：Ile Ile Gly Gly Ser Ser Ser Leu ｂ：Val Gln Leu Ser Pro Asp Leu Xaa （Xaaは未同定のアミノ酸を表す） 1. A human serum having the following physicochemical properties is purified by column chromatography.
A novel protein obtained by this . S under non-reduction
Molecular weight by DS-polyacrylamide gel electrophoresis
It is about 34,000 daltons . 728 active and cleaves specifically between the valine is arginine and 495 th amino acids is 494 amino acids single stranded human hepatocyte growth factor from the amino terminus consisting of amino acid residues
It has a protease activity that converts it into a double-stranded human hepatocyte growth factor in the sexual form . The following two N-termini under non-reducing
Has an amino acid sequence. a: Ile Ile Gly Gly Ser Ser Ser Leu b: Val Gln Leu Ser Pro Asp Leu Xaa (Xaa represents an unidentified amino acid)
とするウシ胎児血清をカラムクロマトグラフィーで精製Fetal bovine serum purified by column chromatography
することにより得られた新規なタンパク質。非還元下Novel protein obtained by performing Under non-reducing
のＳＤＳ−ポリアクリルアミドゲル電気泳動による分子By SDS-polyacrylamide gel electrophoresis
量が約３８，０００ダルトンである。７２８アミノ酸The amount is about 38,000 daltons. 728 amino acids
残基より成る１本鎖型ヒト肝細胞増殖因子をそのアミノSingle-chain human hepatocyte growth factor consisting of
末端から４９４番目のアミノ酸であるアルギニンと４９Arginine, which is the 494th amino acid from the terminal, and 49
５番目のアミノ酸であるバリンとの間で特異的に切断しCleavage specifically with the fifth amino acid, valine
て活性型の２本鎖型ヒト肝細胞増殖因子に変換するプロTo convert to active double-chain human hepatocyte growth factor
テアーゼ活性を有する。非還元下で下記の２つのＮ−Has thease activity. The following two N-
末端アミノ酸配列を有する。It has a terminal amino acid sequence. ａ：Ile Ile Gly Gly Ser Ser Ser Leua: Ile Ile Gly Gly Ser Ser Ser Leu ｃ：Ile Gln Pro Pro Xaa Xaa Glu Alac: Ile Gln Pro Pro Xaa Xaa Glu Ala （Xaaは未同定のアミノ酸を表す）(Xaa represents an unidentified amino acid)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27136291A JP3213985B2 (en) | 1991-10-18 | 1991-10-18 | Novel protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27136291A JP3213985B2 (en) | 1991-10-18 | 1991-10-18 | Novel protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05103670A JPH05103670A (en) | 1993-04-27 |
| JP3213985B2 true JP3213985B2 (en) | 2001-10-02 |
Family
ID=17499018
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27136291A Expired - Lifetime JP3213985B2 (en) | 1991-10-18 | 1991-10-18 | Novel protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3213985B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11547743B2 (en) | 2014-04-28 | 2023-01-10 | Eisai R&D Management Co., Ltd. | Lyophilized formulation of HGF |
| US11548926B2 (en) | 2016-03-17 | 2023-01-10 | Eisai R&D Management Co., Ltd. | Method for producing an active hepatocyte growth factor (HGF) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2102463A1 (en) * | 1992-11-05 | 1994-05-06 | Mitsubishi Chemical Corporation | Protein and gene encoding said protein |
| US7045602B2 (en) | 2000-12-05 | 2006-05-16 | Mitsubishi Chemical Corporation | Specific antibody directed to active hepatocyte growth factor activator and method for using the same |
-
1991
- 1991-10-18 JP JP27136291A patent/JP3213985B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11547743B2 (en) | 2014-04-28 | 2023-01-10 | Eisai R&D Management Co., Ltd. | Lyophilized formulation of HGF |
| US11548926B2 (en) | 2016-03-17 | 2023-01-10 | Eisai R&D Management Co., Ltd. | Method for producing an active hepatocyte growth factor (HGF) |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH05103670A (en) | 1993-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fett et al. | Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells | |
| Roberts et al. | Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction. | |
| CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| KR100205078B1 (en) | Basic fibroblast growth factor and the method of preparing the same | |
| KR0184235B1 (en) | A megakaryocytopoietic factor | |
| EP0458959B1 (en) | Novel platelet-derived growth factor b chain analogs and method for homogeneous production thereof | |
| US5149792A (en) | Platelet-derived growth factor B chain analogs | |
| RU2093519C1 (en) | Method of enzymatic preparing fibroblast basic growth factor b fgf (10-155) and pharmaceutical composition based on thereof | |
| CZ287810B6 (en) | DNA sequence, DNA recombinant molecule containing thereof, process for preparing a protein from the TGF-beta substance group, pharmaceutical preparation in which the protein is comprised, antibody or a fragment thereof and use of the antibody | |
| CZ292703B6 (en) | Mutant proteins of human interleukin-4 | |
| CN116535520A (en) | Extracellular matrix protein fusion protein and preparation method and application thereof | |
| EP0621285B1 (en) | Novel megakaryocyte potentiator | |
| JP3213985B2 (en) | Novel protein | |
| Caccia et al. | Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues | |
| JP2000508532A (en) | Α2 subunit of prolyl-4-hydroxylase, nucleic acid sequence of prolyl-4-hydroxylase encoding the subunit, and method for producing the same | |
| Cai et al. | Purification and characterization of an endothelial cell-viability maintaining factor from fetal bovine serum | |
| US5656458A (en) | Expression and processing of amino terminus acetylated FGF's in yeast | |
| RU2278688C1 (en) | Human endostatin preparation and method for production thereof | |
| Li et al. | Cancer-associated lactate dehydrogenase is a tyrosylphosphorylated form of human LDH-A, skeletal muscle isoenzyme | |
| US5661000A (en) | Serine protease and serine protease gene | |
| JPH04501666A (en) | Expression and processing of authentic FGF in yeast | |
| Danley et al. | Identification and characterization of a C-terminally extended form of recombinant murine IL-6 | |
| US5807711A (en) | Parenchymal hepatocyte growth factors | |
| JP2623807B2 (en) | Serine protease and serine protease gene | |
| JP3610395B2 (en) | Mature hepatocyte growth factor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080727 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080727 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090727 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090727 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100727 Year of fee payment: 9 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100727 Year of fee payment: 9 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110727 Year of fee payment: 10 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20120727 Year of fee payment: 11 |
|
| EXPY | Cancellation because of completion of term | ||
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20120727 Year of fee payment: 11 |