JPH06503962A - Analogs of acidic fibroblast growth factor with enhanced stability and biological activity - Google Patents
Analogs of acidic fibroblast growth factor with enhanced stability and biological activityInfo
- Publication number
- JPH06503962A JPH06503962A JP4503720A JP50372092A JPH06503962A JP H06503962 A JPH06503962 A JP H06503962A JP 4503720 A JP4503720 A JP 4503720A JP 50372092 A JP50372092 A JP 50372092A JP H06503962 A JPH06503962 A JP H06503962A
- Authority
- JP
- Japan
- Prior art keywords
- analog
- amino acid
- fibroblast growth
- growth factor
- acidic fibroblast
- 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.)
- Pending
Links
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Abstract
(57)【要約】本公報は電子出願前の出願データであるため要約のデータは記録されません。 (57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】 増強された安定性及び生物学的活性を有する酸性繊維芽細胞成長因子の類似体 １１へ１１ 例えば、怪我または火傷により、組織まで負傷した後の複雑な治癒過程は、往々 にして柔組織成長因子と称する多くの蛋白質因子により仲介される。これらの因 子は、破壊された組織を取り替えるために新しい細胞を成長及び分化させるのに 必要である。柔組織成長因子のこの群に含まれるものとしては、線維芽細胞成長 因子（ＦにＦ）が挙げられる６ＦＧＦは、上皮、開光組織及び神経由来の種々の 細胞に対して有糸***促進性及び化学走化性である。さらに、ＦＧＦは脈管形成 性、即ち、血管の形成を刺激し得る。[Detailed description of the invention] Analogs of acidic fibroblast growth factor with enhanced stability and biological activity 11 to 11 For example, the complex healing process after injury or burn injury to tissues is often It is mediated by a number of protein factors called soft tissue growth factors. These factors The offspring grow and differentiate new cells to replace the destroyed tissue. is necessary. Included in this group of soft tissue growth factors include fibroblast growth 6FGF, which is a factor (F in F), is expressed in various types derived from epithelium, light-emitting tissues, and nerves. It is mitogenic and chemotactic for cells. In addition, FGF is associated with angiogenesis. can stimulate sex, ie, blood vessel formation.
酸性ＦＧＦ（ａＦＧＦ）及び塩基性ＦＧＦ（ｂＦＧＦ）は、ＦＧＦファミリーの ２つのｒオリジナル％１楕成員であると考えられている。Acidic FGF (aFGF) and basic FGF (bFGF) are members of the FGF family. It is believed that there are two r original %1 elliptic members.
ａＦＧＦ及びｂＦＧＦは、同一先祖の遺伝子から誘導されたと考えられており、 同一イントロン／エキジン構造の他に約５５ｚの配列同一性を有する。酸性ＦＧ Ｆ及びｂＦＧＦは同一レセプターに結合することが知られているが、特異的なａ ＦＧＦ及びｂＦＧＦレセプターの存在は除外されていなかった。 ａＦＧＦ及び ｂＦＧＦの幾つかの分子量形が、異なる組織中に知見されている。aFGF and bFGF are thought to be derived from the same ancestral gene, In addition to the same intron/exin structure, there is approximately 55z sequence identity. acidic FG Although F and bFGF are known to bind to the same receptor, specific a The presence of FGF and bFGF receptors was not excluded. aFGF and Several molecular weight forms of bFGF are found in different tissues.
しかしながらサザンブロノティング実験から、ａＦＧＦとｂＦＧＦには各々１ｆ ｌｉの遺伝子しがなく、これらの分子間の違いは翻訳後プロセシングによるだろ うと示唆されている。酸性及び塩基性ＦＧＦは両方共、中胚葉及び神経外胚葉起 源の広範囲の細胞型に関する有糸***促進因子であり、ｉｎνｉｔｒ。However, Southern bronoting experiments showed that aFGF and bFGF each have 1f There is no gene for li, and the differences between these molecules may be due to post-translational processing. It has been suggested that Both acidic and basic FGFs have mesodermal and neuroectodermal origins. It is a mitogen for a wide range of cell types and is an inνitr.
文びｉｎ ｖｉｖｏの両方で脈管形成を誘導し得る［例えば、Ｇｏｓｐｏｄａｒ ｏｗｉｃｚら、Ｅｘｐ、Ｅｙｅ Ｒｅｓ、、２８，５０１−５１４（１９７９） 参照］にの２種の生物学的活性範囲は殆ど同一であるが、ｂＦＧＦは殆どのバイ オアッセイ系でａＦＧＦよりも約１０倍以上も強力である。can induce angiogenesis both in vitro and in vivo [e.g. Owicz et al., Exp, Eye Res, 28, 501-514 (1979) The range of biological activity of the two species is almost the same; It is about 10 times more potent than aFGF in the optical assay system.
ａＦＧＦ及びｂＦＧＦの共通の際立つ特徴は、これらの因子がヘパリンに強く結 合する性質を持つという、二とである。ヘパリンに対するａＦＧＦの親和性は、 ｂＦ［；Ｆよりも弱いだろうが、ａＦＧＦはアニオ〉性の等電点を有する［Ｔｈ ｏｍａｓら、 Ｐｒｏｃ 、Ｎａｔ 。A common distinguishing feature of aFGF and bFGF is that these factors bind strongly to heparin. The two have the property of being compatible. The affinity of aFGF for heparin is Although probably weaker than bF[;F, aFGF has an anionic isoelectric point [Th omas et al., Proc, Nat.
＾ｃａｄ、ｓｃｉ、Ｕｓ＾、８２．６４０９−６４１３（１９８４）：ｌ。ａＦ ＧＦ及びｂＦＧＦの特徴的なヘパリン結合能により、これらの因子の精製が非常 に容易になった。^cad, sci, Us^, 82.6409-6413 (1984):l. aF The unique heparin-binding ability of GF and bFGF makes purification of these factors extremely difficult. became easier.
ＦＧＦが固定化ヘパリンに対して強い親和性を持つという発見は、ＦＣＦのｉｎ ｖｉｖｏ生物学に於けるヘパリン櫟分子の調節的役割の研究にも拍車とかけた 。ヘパリンに対する機能の全スペクトルは未だ確認されていないが、ヘパリンが 幾つかの方法でＦＣＦ機能を調節し得ることは公知である［１．ｏｂｂ。The discovery that FGF has a strong affinity for immobilized heparin suggests that FGF has a strong affinity for immobilized heparin. It also spurred research into the regulatory role of heparin molecules in vivo biology. . Although the full spectrum of functions for heparin has not yet been identified, heparin It is known that FCF function can be modulated in several ways [1. obb.
Ｅｕｒ、Ｊ、Ｃｌ１ｎ、Ｉｎｖｅｓｔ、、１８，３２１−３２６（１９８８）］ 、例えば、ヘパリン様分子は、ａＦＧＦの活性化または増強を含むＦＧＦ機能に 於いて直接の役割を果たし得る［ｔＩｈｌｒｉｅｈら、Ｂｉｏｃｈｅｍ、Ｂｉｏ ｐｈｙｓ。Eur, J, Cl1n, Invest, 18, 321-326 (1988)] , for example, heparin-like molecules affect FGF function, including activation or enhancement of aFGF. [tIhlrieh et al., Biochem, Bio phys.
Ｒｅｓ、Ｃｏｍｍ、、１３７．１２０５−１２１３（１９８６）］。Res, Comm, 137.1205-1213 (1986)].
しかしながら、固定化ヘパリンに対するＦＧＦの親和性と溶解性ヘパリンにより 増強されるべきその能力との間には直接の相関間係がない。この点に於いて、ヘ パリンの増強力は、ａＦＧＦに対して選択的のようである８例えば、Ｕｈｌｒｉ ｅｈら（同上）は、純ａＦＧＦの増強度が純ｂＦＧＦの増強度の約１０倍以上も 太き（、ａＦＧＦの効能はｂＦＧＦの効能と殆ど同レベルであることを知見した 。しがしながら、ウシ胎児血清の存在下では、ヘパリンの増強効果がかなり減少 することが知見された［Ｕｈｌｉｃｈら、同上］。However, due to the affinity of FGF for immobilized heparin and soluble heparin, There is no direct correlation with the ability to be enhanced. In this regard, The potentiation of palin appears to be selective for aFGF8, e.g., Uhlri et al. eh et al. (ibid.) found that the degree of enhancement of pure aFGF was about 10 times or more that of pure bFGF. It was found that the efficacy of aFGF is almost at the same level as that of bFGF. . However, in the presence of fetal bovine serum, the enhancing effect of heparin is significantly reduced. [Uhlich et al., supra].
ＦＧＦ蛋白質を使用することは、外傷を受けた組織の治癒を促進するのに有効で あると考えられている。 ＦＧＦの特徴的な脈管形成の特性は、深い傷の治癒に 於いてこれらの因子を特に有用にする。 ｂＦＧＦ天然蛋白質は、心筋梗塞の治 療に有用であることが主張された［米国特許第４，２９６，１００号及び同第４ ，３７８．３４７号］。さらに、ヒトｂＦＧＦは、ラット胎児海鳥ニューロンに 於いて神経の生存及び神経突起伸長を増大させることが知見され、従ってこの因 子は変性神経障害（例えば、アルツハイマー病及びパーキンソン病〉の治療にも 有用であることを示唆している［Ｎａ１ｌｉｃｋｅら、Ｐｒｏｃ、Ｎａｔｌ。Using FGF protein is effective in promoting healing of traumatized tissues. It is thought that there is. The characteristic angiogenic properties of FGF contribute to the healing of deep wounds. This makes these factors particularly useful. bFGF natural protein is effective in treating myocardial infarction. [U.S. Pat. No. 4,296,100 and U.S. Pat. , No. 378.347]. Furthermore, human bFGF has been shown to stimulate rat fetal seabird neurons. was found to increase neuronal survival and neurite outgrowth, and therefore this factor Children may also be used to treat degenerative neurological disorders (e.g. Alzheimer's disease and Parkinson's disease). [Nallicke et al., Proc. Natl.
＾ｃａｄ、ｓｃｉ、Ｕｓ＾、８３．３０１２−３０１６（１９８６）］。^cad, sci, Us^, 83.3012-3016 (1986)].
治療用途でａＦＧＦの効果的な使用に対する主な障害は。The main obstacles to the effective use of aFGF in therapeutic applications are:
ｂＦＧＦと比較してそのかなり低い生物学的活性に関連するようである。ヘパリ ンに関する研究から、ａＦＧＦとｂＦｃＦとの閏の効能に於いて知見された違い は、ｂＦＧＦの活性と匹敵し得るレベルにａＦＧＦの活性を増強するためにヘパ リンを使用することによりかなり縮小し得るが、医薬製剤に於いてヘパリンを使 用することは必ずしも望ましいわけではない、この点に於いて、ヘパリン、即ち 、異種構造の硫酸化度が高いグリコサミノグリカンは、抗−トロンビン■がホメ オスタシスのプロテアーゼを不活化する際の速度を速めることにより機能する抗 凝血薬であることが知られていることは、特記すべきである［Ｊａｃｑｕｅｓ、 Ｐｈａｒｍａｃｏｌ Ｒｅｖ、３１．９９−Ｉ６６の （１９８０）］、適正に治癒させるためにある程ｉ固が望ましい場合、深い傷の 治療用の医薬製剤にヘパリンを使用することは有害であるかどうかは知られてい ない。This seems to be related to its considerably lower biological activity compared to bFGF. Hepari Differences found in the efficacy of aFGF and bFcF from studies on Heparin to enhance the activity of aFGF to a level comparable to that of bFGF. Although it can be significantly reduced by using phosphorus, the use of heparin in pharmaceutical formulations In this respect it is not always desirable to use heparin, i.e. Glycosaminoglycans with a heterogeneous structure and a high degree of sulfation are homely treated with anti-thrombin. Antibiotics that work by speeding up the inactivation of ostasis proteases. It should be noted that it is known to be a blood clotting agent [Jacques, Pharmacol Rev, 31.99-I66 (1980)], for deep wounds where some stiffness is desirable for proper healing. It is not known whether the use of heparin in therapeutic pharmaceutical preparations is harmful. do not have.
さらに、ヘパリンが傷の治癒用の医薬製剤に含まれると、実際的に問題が発生す ると予想される。ドラッグデリバリ−に関しては、患者の体に入れる際には（ａ ＦＧＦとヘパリンの組み合わせを含む）医薬製剤の組成を制御するという問題が 挙げられる。さらに、Ｕｈｌｒｉｅｈらにより観察されたａＦＧＦに対するヘパ リンの増強効果に於けるウシ胎児血清の負の効果から、ａＦＧＦの活性化または 増強因子として医薬製剤中にヘパリンを配合することにより得られる長所はいず れも完全に否定されるか、または一度患者の血清と接触すると失われるというこ とを示唆している。Furthermore, practical problems arise when heparin is included in pharmaceutical preparations for wound healing. It is expected that Regarding drug delivery, when entering the patient's body (a The problem of controlling the composition of pharmaceutical formulations (including combinations of FGF and heparin) Can be mentioned. Furthermore, the hepatocellular carcinoma against aFGF observed by Uhlrieh et al. From the negative effect of fetal bovine serum on the enhancement effect of phosphorus, activation of aFGF or There are no advantages to be gained by incorporating heparin into pharmaceutical formulations as an enhancer. It is either completely ruled out, or that it is lost once in contact with patient serum. It suggests that.
本発明の目的は、ヘパリンの非存在下で増強された安定性及び生物学的活性を示 すａＦＧＦ類似体を提供することである０本発明のもう一つの目的は、治療的に 使用するためのａＰＧＦ類似体を提供することである。It is an object of the present invention to show enhanced stability and biological activity in the absence of heparin. Another object of the present invention is to provide aFGF analogues that can be used therapeutically. An object of the present invention is to provide aPGF analogs for use.
１１へ１１ 本発明は、天然のａＦＧＦよりもヘパリンの非存在下でより安定で、より大きな 生物学的活性を示すａＦＧＦの新規類似体を提供する。安定性は、天然のａＦＧ Ｆ分子中碕アミノ酸約９０〜９７の領域に於いてループ形成能力の低いアミノ酸 残基を、ループ形成能力の高い少なくとも１個のアミノ酸と置換することにより 強化される３本発明の好ましい類似体は、天然のａＦにＦ中のアミノ酸位置９３ でヒスチジン残基をループ形成能力の高いアミノ酸で置換することを含む。11 to 11 The present invention shows that aFGF is more stable in the absence of heparin and has a larger Novel analogs of aFGF that exhibit biological activity are provided. Stability is natural aFG Amino acids with low loop-forming ability in the region of approximately 90 to 97 amino acids in the F molecule By replacing the residue with at least one amino acid with high loop-forming ability. 3 preferred analogs of the invention that are enriched at amino acid position 93 in F to native aF. This involves replacing the histidine residue with an amino acid with high loop-forming ability.
図面の簡単な説明 図１は、組換えウシ［八１ａ４７．Ｇｌ、９）コａＦＧＦの核酸及びアミノ酸配 列を示す、 図２は、組換えヒトＵ＾Ｉａ”、ＧＩｙ９）コａＦＧＦのアミノ酸配列を示す。Brief description of the drawing Figure 1 shows recombinant bovine [81a47. Gl, 9) Nucleic acid and amino acid sequence of core aFGF indicates a column, Figure 2 shows the amino acid sequence of recombinant human U^Ia'', GIy9) core aFGF.
図３は、疎水性相互作用クロマトグラフィーを使用する、ウシ［＾ｌａ”］及び ［＾１ａ４７Ｊｌ、！：ｌ］の溶離プロフィールを示す。Figure 3 shows bovine [^la”] and [^1a47Jl,! :l] is shown.
図４Ａ及び４Ｂは、ウシ［＾１ａ４７コ及び［＾！ａ”、Ｇｌｙ”］ａＦＧＦ類 似体の円二色性スペクトルを示す。Figures 4A and 4B show cows [^1a47ko and [^! a”, Gly”] aFGFs The circular dichroism spectrum of the analog is shown.
図５は、アミドＩ’（変性蛋白質に於けるＣ−Ｏ伸縮）領域に於けるウシ［＾１ ａイア″Ｊ及びＣ＾ｌａ′７ 、Ｇ１，９３コａＦにＦ［似体の二次導関数ＦＴ ＩＲスペクトルを示す。Figure 5 shows the effect of bovine [^1 aia″J and C^la′7, G1, 93 core aF to F [second derivative of analog FT The IR spectrum is shown.
図６は、最大刺激の２に対するウシ［＾ｌａ”］及び［＾ｌ ａ ４７゜ＧＩｙ ”ｆｆ］ａＦＧＦｉ似体並びにヒト［Ｓｅｒ”、Ｓｅｒ”］ｂＦＧＦの濃度の対 数プロットを示すグラフである。Figure 6 shows the cow [^la''] and [^l a 47゜GIy for the maximum stimulation of 2. Concentration pair of “ff]aFGFi analog and human [Ser”, Ser”]bFGF FIG. 2 is a graph showing a number plot.
［１７は、ヒトＪＳｅｒ”、Ｓｅｒ”コｂＦＧＦと比較したヘパリンの非存在下 に於けるウシ［＾ｌａ”］及び［＾１，４７ 、にｌ、！３コａｌ（：Ｆ類似体 の経時的活性損失を示すグラフである。[17 In the absence of heparin compared to human JSer”, Ser” co-bFGF Cattle [^la”] and [^1,47, Nil,!3 coal (:F analogues) in 1 is a graph showing activity loss over time.
図８は、Ｘ線結晶学により測定しな、本発明のウシ［ＡＩａ４７Ｊｌ、！コ］ａ ＦにＦ類似体の構造を示す。FIG. 8 shows that the bovine [AIa47Jl,!] of the invention was determined by X-ray crystallography. [ko]a F shows the structure of the F analogue.
尺朋！」す［ｒ１朋一 本発明によりａＦＧＦの新規類似体を提供する。これらの類似体は、ヘパリンの 非存在下で天然のＩＬＦにＦと比較して改良安定性及び強い生物学的活性を示す ０本発明のａＦＧＦｇｉ似体は、アミノ酸残基的９０〜９７の領域（図１に示さ れているように、ウシａＦにＦの公知のアミノ酸配列の番号付けに基づく）に於 いて天然のａＦＧＦと異なる少なくとも１個のアミノ酸残基を有する。異なるア ミノ酸は、ａＦＧＦ分子のこの領域を安定化するためにそのループ形成能力が高 くなるように選択される６比較的ループ形成能力の高いアミノ酸としては、グリ シン、プロリン、千ロジン、アスパラギン酸、アスパラギン及びセリンが挙げら れる［Ｌｅｓｚｃｙｎｓｋ ｉら、５ｃｉｅｎｃｅ、２３４．８４９−８５５（ １９８６） （天然分子のループ構造に現れる頻度を基にして決定したループ形 成能力の相対値）］。ループ形成能力の高い異なるアミノ酸で、天然のａＦＧＦ のアミノ酸位置９３のヒスチジン残基を１き換えるのが好ましい。さらに、アミ ノ酸位置９３のヒスチジン残基分、グリシン残基と置き換えるのがより好ましい 。Shakuho! ”S [r1 Tomoichi The present invention provides novel analogs of aFGF. These analogs are heparin In the absence of native ILF, it exhibits improved stability and stronger biological activity compared to F. 0 The aFGFgi analog of the present invention has a region of amino acid residues 90 to 97 (shown in Figure 1). (based on the known amino acid sequence numbering of F) in bovine aF, as shown in and has at least one amino acid residue different from natural aFGF. different a Mino acids have a high loop-forming ability to stabilize this region of the aFGF molecule. Among the amino acids with relatively high loop-forming ability, Syn, proline, 1,000 rosin, aspartic acid, asparagine and serine are listed. [Leszcynsk et al., 5science, 234.849-855 ( 1986) (Loop shape determined based on the frequency of appearance in loop structures of natural molecules) relative value of ability to develop)]. Natural aFGF with different amino acids with high loop-forming ability Preferably, the histidine residue at amino acid position 93 is replaced by one. Furthermore, Ami It is more preferable to replace the histidine residue at amino acid position 93 with a glycine residue. .
本発明のａＦＧＦ類似体には、他に、付加、置換及び／または欠失も実施し得る 。例えば、ａＦＧＦ類似体としては、場合Ｇ′ニーより、非保護システィン残基 く即ち、ウシａＦＧＦ分子の位置４７に於けるシスティン残基及びヒトａＦＧＦ 分子の位置１６に於けるシスティン残基）に関するアミノ酸置換も挙げられる。Other additions, substitutions and/or deletions may also be made to the aFGF analogs of the invention. . For example, as an aFGF analogue, if the G'knee is less than the unprotected cysteine residue namely, the cysteine residue at position 47 of the bovine aFGF molecule and the cysteine residue at position 47 of the bovine aFGF molecule. Also included are amino acid substitutions for the cysteine residue at position 16 of the molecule.
さらに、Ｅ、ｃｏｌｉ宿主細胞から発現され得る本発明のａＦＧＦ類似体として は、開始メチオニンアミノ酸残基く即ち、図１に見られるように位置−１の部分 ）も含み得る。あるいは、末端アミノ酸残基の１個以上は、当業者に公知の如く ＤＮＡ配列から削除され得るが、ａＦＧＦ類似体の強い生物学的活性は実質的に 保持している。Additionally, as an aFGF analogue of the invention that can be expressed from E. coli host cells, is the starting methionine amino acid residue, i.e. the part at position -1 as seen in Figure 1. ) may also be included. Alternatively, one or more of the terminal amino acid residues may be Although deleted from the DNA sequence, the strong biological activity of aFGF analogs is substantially keeping.
本発明は、ａＦＧＦ類似体の全体または一部をコードするＤＮＡ配列も提供する 。このような配列は、好ましくは、選択されたＥ、ｅｏｌｉ宿主株により発現す るための“好ましい”コドン（゛Ｅ、ｅｏｌｉ発現コドン′°）の取り込み、制 限エンドヌクレアーゼ酵素による切断部位の提供、及び／または、発現容易なベ クターを構築し易くする追加の開始、末端若しくは中間ＤＮＡ配列の提供も含み 得る。これらの新規ＤＮＡ配４目よ、真核細胞及び原核細胞宿主（例えば、Ｅ、 ｃｏｌｉ）の両方に於いて、本発明のａＦＧＦ類似体を確実に発現させるのに有 用な配列を含む。The present invention also provides DNA sequences encoding all or part of an aFGF analog. . Such sequences are preferably expressed by the selected E. eoli host strain. Incorporation of “preferred” codons (゛E, eoli expression codon ´°) to control Providing a cleavage site with a limited endonuclease enzyme and/or providing a vector for easy expression. including the provision of additional starting, terminal, or intermediate DNA sequences to facilitate vector construction. obtain. These novel DNA sequences can be used in eukaryotic and prokaryotic hosts (e.g., E. This is useful for ensuring the expression of the aFGF analog of the present invention in both E. coli and E. coli. Contains a useful array.
より具体的には、本発明のＤＮＡ配列は、約アミノ酸９０〜９７の領域に於ける アミノ酸残基をコードする少なくとも１個のコドンが、ループ形成能力の高い異 なるアミノ酸残基をコードするコドン（以後、“類似体配列”と称する）により 置き換えられる図１に記載のＤＮＡ配列、並びに類似体配列の一方またはそのフ ラグメントにハイブリダイズするＤＮＡ配列及び遺伝コードの縮重に関するが、 類似体配列の一つにハイブリダイズするＤＮＡ配列を含み得る。More specifically, the DNA sequence of the invention comprises a region of approximately amino acids 90-97. At least one codon encoding an amino acid residue has a high loop-forming ability. by a codon encoding an amino acid residue (hereinafter referred to as an “analog sequence”). The DNA sequence shown in Figure 1 to be replaced, as well as one of the analog sequences or its flanks. Regarding the degeneracy of the DNA sequence and genetic code that hybridizes to the fragment, It may include a DNA sequence that hybridizes to one of the analog sequences.
本発明のａＦＧＦ類似体は、当業者には公知の種々の組換え技術の任意の一方法 によりコード、発現及び精製され得る。The aFGF analogs of the invention can be prepared by any one of a variety of recombinant techniques known to those skilled in the art. can be encoded, expressed and purified by.
好ましい製造方法は、材料のコスト及び入手性並びに他の経済問題を含む多くの 因子及び問題に広く依存する。所与の場合の最適製造方法は、最小実験により当 業者には明らかである０本発明の類似体は、Ｅ、ｃｏｌｉ宿主細胞を使用して特 に高いレベルで発現でき、得られた発現産物は続いて同業者には公知の方法を使 用して殆ど均質にまで精製し得る。The preferred manufacturing method is subject to a number of considerations, including cost and availability of materials and other economic issues. It depends widely on the factors and the problem. The optimal manufacturing method for a given case is determined by a minimum of experiments. It will be apparent to those skilled in the art that the analogues of the present invention can be characterized using E. coli host cells. can be expressed at high levels, and the resulting expression product can be subsequently expressed using methods well known to those skilled in the art. can be purified to near homogeneity using
典型的な精製方法で、ａＦＧＦ類似体を含む包含物を溶解し、次いでイオン交換 クロマトグラフィーにかけ、蛋白質を再び折り畳み（ｒｅｆｏｌｄｉｎｇ＞、 １に終的に疎水性相互作用クロマ１−グラフィーにかける。A typical purification method involves dissolving the inclusions containing the aFGF analog, followed by ion exchange. Proteins are refolded by chromatography. 1 is finally subjected to hydrophobic interaction chromatography.
本発明のａＦＧＦ類似体は、ヘパリン非存在下で、驚くほど増強された生物学的 活性を示す。より安定なりＦＧＦ類似体は、特異的なシスティン残基く例えば、 ＰＣＴ特許出願公開第８８７０４１８９号に記載）の代わりにセリンまたは他の 中性アミノ酸の置換を介して得られることは公知であるが、天然のウシａＦＧＦ の位置４７の非保存システィン残基のみの置換は、生物学的活性及び／またはａ ＦＧＦ類似体の安定性の強化に重要であると考えられていない。これは、以下の 実施例に記載するウシ［＾ｌａ” 、Ｇｌｙ９３］ａＦＧＦ類似体（ａＦＧＦ分 子の残基９０〜９７の領域に於ける所望のアミノ酸置換を有する）と比較してウ シ［＾１ａ１７３ａＦＣ：Ｆ類似体（システィン置換）が低い活性を示すことに より立証される。特に、ウシ［＾ｌａ” 、Ｇｌｙ”３類似体は、ｂＦＧＦと比 較してやや能力が低いが、ウシ［＾ｌａ”コａＦＧＦ想似体よりも約１０倍強力 であることが知見された。４５μｇ／ｗｅヘパリンを添加すると、ＦＧＦの全部 で３つの型、即ち、ウシ［＾ｌａ’テ、Ｇｌｙ９３］Ｗ似体、ウシ［＾ｌａ’） 、Ｇｌｙ’す類似体及び実質的に同一能力を有しているヒト［５ｅｔＴＯ，Ｓｅ ｒ’δコｂＦｃＦ類似体の生物活性が増強された。The aFGF analogs of the present invention have surprisingly enhanced biological effects in the absence of heparin. Shows activity. More stable FGF analogs contain specific cysteine residues, e.g. Serine or other Although it is known that it can be obtained through substitution of neutral amino acids, natural bovine aFGF Substitution of only the non-conserved cysteine residue at position 47 of It is not thought to be important for enhancing the stability of FGF analogs. This is the following Bovine [^la”, Gly93]aFGF analogue (aFGF portion) described in Examples with the desired amino acid substitutions in the region of residues 90-97). Cy[^1a173aFC:F analogue (cysteine substitution) shows lower activity. more proven. In particular, bovine [^la”,Gly”3 analogues are compared to bFGF. Although its ability is slightly lower than that of the cow [^la” core aFGF analogue, it is about 10 times more powerful. It was found that When adding 45 μg/we heparin, all of the FGF and three types, namely bovine [^la'te, Gly93] W analogue, bovine [^la') , Gly' and human [5etTO, Se The biological activity of r'δ cobFcF analogs was enhanced.
ヘパリンの非存在下に於けるウシ［＾ｌａ”］ａＦＧＦに対するウシ［＾ｌａ” 、Ｇｌｙ９ｆｆ］ａＦＧＦ類似体の増強された有糸***促進活性及び安定性の理 由は現在の処明らかではない。位置９３に於けるヒスチジン残基のグリシンへの 置換は、ａＦＧＦ分子と幾らか疎水性にするようであったが、円二色性及びＦＴ ＩＲ分光学により測定されるように、その三次構造を大きくは変えないようであ った。しかしながら、ウシ［＾１ａ４７．に１，１１］ａＦＧＦ類似体及びウシ ［＾１ａ４７］ａＦＧＦ類似体く位置４７だけに１換がある〉に間するｉｎ ｖ ｉｔｒｏバイオアッセイで知見される活性の相対的な差から、ウシ［＾Ｉａ”、 Ｇｌｙ”］ａＦＧＦ類似体のグリシン−置換アミノ酸９３位置は、レセプター結 合に関与する領域内であるか、またはその近傍であることが示唆された。ａＦＧ Ｆに於けるレセプター結合領域は確定されていなかったが、ａＦにＦ内の位！９ ３は、レセプター結合ドメイン内またはその近傍であると報告されるｂＦＧＦ内 の領域に相当する［Ｂａ１ｒｄら、Ｐｒｏｃ、Ｎａｔ、＾ｃａｄ、ｓｃｉ、Ｕｓ ＾、８５．２３２４−２３２８（１９８８）コ。Bovine [^la”] versus bovine [^la”]aFGF in the absence of heparin , Gly9ff] Reason for the enhanced mitogenic activity and stability of aFGF analogs The reason for this is currently unclear. Histidine residue at position 93 to glycine The substitutions appeared to make the aFGF molecule somewhat hydrophobic, but circular dichroism and FT It does not appear to significantly alter its tertiary structure, as determined by IR spectroscopy. It was. However, cow [^1a47. 1,11] aFGF analogs and bovine [＾1a47] Inv between aFGF analogs with one substitution only at position 47 The relative differences in activity found in intro bioassays indicate that bovine [^Ia'', The glycine-substituted amino acid position 93 of the aFGF analog It was suggested that the area was located in or near the area involved in the process. aFG The receptor binding region in F has not been determined, but aF has a position within F! 9 3 within bFGF, which is reported to be within or near the receptor binding domain. [Ba1rd et al., Proc, Nat, ^cad, sci, Us ^, 85.2324-2328 (1988).
さらに、ウシ［＾１ａ４７］類似体と異なり、本発明のウシ［＾１ａ°”、ＧＩ ｙ”］ａＦｃＦ類似体は、増強された安定性を示し、２５０時を保持したが、ウ シ［＾ｌａ”］類似体はすぐに活性を失った。Furthermore, unlike the bovine [^1a47] analog, the bovine [^1a°'', GI y'']aFcF analog showed enhanced stability and retained 250 hours, but The shi[^la”] analogue quickly lost activity.
ウシ［＾’ｌａ４′、（：Ｉｙ’コ］ａＦｃ：Ｆ類似体を結晶化し、得られた結 晶をＸ線結晶学により検討した。これらの結晶の調査力）ら得られたＸ線結晶学 的データは、残基９３が溶媒Ｇこ暴露されたこと、即ち、位置９３でのヒスチジ ンのグリシンの置換力（分子の親水性をより低下させるとｌｖ）うことを支持し てｌＬ）る。The crystallization of bovine [^’la4′, (:Iy’co]aFc:F analogue) The crystals were examined by X-ray crystallography. X-ray crystallography obtained from the investigation of these crystals The data indicate that residue 93 was exposed to solvent G, i.e., histidyl at position 93. It is supported that the substitutional power of glycine (lv to further reduce the hydrophilicity of the molecule) te lL).
残基９３の回りのウシ［＾ｌａ” 、にｌｙ”］ａＦＧＦ類似体配列の詳細な検 討から、位置的９０のグルタミン酸〜位置約９７のチロシン残基の領域に於いて ループ形成能力の高１１’ｌ約８個のアミノ酸のクラスタリング（ｃｌｕｓｔｅ ｒｉｎｇ）であること力（判明した。Detailed examination of the bovine [^la", nily"] aFGF analog sequence around residue 93. From the discussion, it was found that in the region from glutamic acid at position 90 to tyrosine residue at approximately position 97, Clustering of about 8 amino acids with high loop-forming ability It turned out to be a power (ring).
アミノ酸の関連するループ形成能力は報告されており、グリシンは、全アミノ酸 の中でも最高のル−プ形成能力を有するアミノ酸残基として同定された［Ｌｅｓ ｚｃｙｎｓｋｉらコロ従って、ヒスチジンと比較してグリシン残基番よずつとル −プ形成能力が高いので、グリシンとヒスチジンとを置換すると仮定のループを 安定化すると考えられる。The related loop-forming ability of amino acids has been reported, and glycine [Les Therefore, compared to histidine, the number of glycine residues and - Since it has a high ability to form loops, replacing glycine and histidine creates a hypothetical loop. It is thought that it will stabilize.
本明細書中に特異的に記載した好まし−１［にｌｙ”］類類似体外の他のａＦｃ Ｆ類似体も本発明により意図される。これらの他の類似体は、本明細書中に提供 される教示に従って当業者により容易に作製され得るだろう。例え４ｆ、ヒスチ ジン以外、の高いループ形成能力を有すると報告されているアミノ酸は１５個以 上もある［Ｌｅｓｚｃｙｎｓｋｉら］。これらのアミノ酸は、ループ形成能力が 減少する順で、グリシン、プロリンまたは千ロジン、アスパラギン酸またはアス パラギン、セリン、システィン、グルタミン酸、トレオニン、リシン、シスチン 、グルタミン、アルギニン、フェニルアラニン及びトリプトファンである。これ らの残基の任意のもので天然のａＦｃ：Ｆの位置９３のヒスチジン残基を置換す ると、増強された生物学的活性を有する本発明のａＦＧＦ類似体となると予想で きた。無論、潜在的に負の作用（例えば、追加のシスティンまたはシスチン残基 を挿入することによる望ましくないジスルフィド結合の形成）を全く生み出さず に、最高のループ形成能力を有するアミノ酸で位置９３でのヒスチジン残基を置 換することが好ましい、従って、（即ち、グリは、プロリン、チロシン、アスパ ラギン酸、アスパラギン、セリン、グルタミン酸、トレオニン、リジン、グルタ ミン、アルギニン、フェニルアラニン及びトリプトファンが挙げられる。Other aFcs other than the preferred-1 analogues specifically mentioned herein. F analogs are also contemplated by this invention. Other analogs of these are provided herein. can be easily made by one skilled in the art according to the teachings provided herein. For example, 4f, histi There are more than 15 amino acids reported to have high loop-forming ability other than gin. Also available [Leszcynski et al.]. These amino acids have the ability to form loops. In order of decreasing glycine, proline or 1,000 rosin, aspartic acid or as paragine, serine, cysteine, glutamic acid, threonine, lysine, cystine , glutamine, arginine, phenylalanine and tryptophan. this Replace the histidine residue at position 93 of native aFc:F with any of the residues from It is expected that the aFGF analogue of the present invention will have enhanced biological activity. came. Of course, potentially negative effects (e.g. additional cysteine or cystine residues) (formation of undesirable disulfide bonds due to the insertion of The histidine residue at position 93 was placed with the amino acid with the highest loop-forming ability. Therefore, it is preferable to replace proline, tyrosine, asparagus (i.e., lagic acid, asparagine, serine, glutamic acid, threonine, lysine, gluta amine, arginine, phenylalanine and tryptophan.
本発明は、天然のａＦｃＦのアミノ酸９０〜９７領域（即ち、アミノ酸９０〜９ ２及び９４〜９７）内でループ形成能力の高いアミノ酸で他のアミノ酸を置換す ることも意図する。本発明のａＦＧＦ類似体としては、好ましくは、例えば天然 ａＦＧＦの位置９６のトレオニン残基が、グリシン、プロリンまたはチロシン、 アスパラギン酸またはアスパラギン、セリンまたはグルタミン酸と１換されてい るａＦｃ：Ｆ類似体が挙げられるが、グルタミン酸とトレオニンの２個のアミノ 酸のループ形成能力は似ているため、トレオニンをグルタミン酸で置換しても安 定性及び／または生物学的活性の増強は最小であることが予想される。同様に、 位ｆｉ９０及び９１のグルタミン酸残基は、グリシン、プロリンまたは千ロジン 、アスパラギン酸またはアスパラギン、またはセリンと１換されるのが好ましい 。The present invention relates to the amino acid 90-97 region of natural aFcF (i.e., amino acids 90-97). 2 and 94-97), replacing other amino acids with amino acids with high loop-forming ability. It is also intended that The aFGF analogs of the present invention preferably include, for example, natural The threonine residue at position 96 of aFGF is glycine, proline or tyrosine, Aspartic acid or asparagine, monoconverted to serine or glutamic acid aFc:F analogues, including the two amino acids glutamic acid and threonine. Because the acids have similar ability to form loops, it is safe to replace threonine with glutamic acid. Enhancement of qualitative and/or biological activity is expected to be minimal. Similarly, Glutamic acid residues at positions fi90 and 91 are glycine, proline or 1,000 rosin. , preferably monoconstituted with aspartic acid or asparagine, or serine .
天然のａＦＧＦの位置９２．９４．９５及び９７のアミノ酸残基（各々、アスパ ラギン、チロシン、アスパラギン及び千ロジン）は、これらの特定の残基を置換 することによる効果は最小であると考えられるが、十分に高いループ形成能力を 有する。Amino acid residues at positions 92, 94, 95 and 97 of native aFGF (aspa lagine, tyrosine, asparagine and 1,000 rosine) substitute these specific residues Although the effect of have
本発明のａＦＧＦｉ似体は、アミノ酸９０〜９７の以下のアミノ酸配列。The aFGFi analog of the invention has the following amino acid sequence of amino acids 90-97.
９０’ ９１ ９２ ９３ ９４ ９５ ９６ ９７− Ｇ Ｉ ｕ −Ｃ１ｕ −＾ｓｎ −ｔ（ｉｓ −Ｔｙｒ−＾ｓｎ −Ｔｈｒ −Ｔｙｒ −を有するａ ＦＧＦの全型並びにヒト及びウシａＦＧＦの類似体を包含する。90' 91 92 93 94 95 96 97- G I u - C1u -＾sn -t(is -Tyr-＾sn -Thr -Tyr - a Includes all types of FGF as well as analogs of human and bovine aFGF.
ａＦＧ：Ｆのヒト及びウシ型の両方は公知であり、位１９０〜９７で同一アミノ 酸配列（上記）を有するものと同定された。さらに、ヒト及びウシａＦＧＦの間 には約９２ｚの配列同一性及び９７ｚの゛類似性”（即ち、２つのａＦにＦ形の 間の全部で８２の違いの内の５ｚは保存的である）を有する。天然ａＦＧＦのヒ ト及びウシ型の両方は、実質的に同一のｉｎ ｖｉｔｒｏ有糸***促進活性を示 す。Both the human and bovine forms of aFG:F are known and contain the same amino acids at positions 190-97. It was identified as having the acid sequence (described above). Additionally, between human and bovine aFGF have approximately 92z sequence identity and 97z 'similarity' (i.e., the two aFs have an F-form Of the total 82 differences between them, 5z are conservative). natural aFGF Both the genotypic and bovine forms exhibit virtually identical in vitro mitogenic activity. vinegar.
ヘパリンの非存在下に於いて、その増強された安定性及び生物学的活性により、 本発明の新規の生物学的に活性なａＦＧＦ類似体は特に、補乳類種の種々の型の 傷の医師及び／または獣医師による治療用医薬配合物に於いて使用するのに非常 に適している。このような治療に於いて使用する生物学的に活性なａＦＧＦ類似 体の量は、無論、治療する傷のひどさ１選択した投与経路及びａＦにＦＷ似体の 比活性または純度に依存し、担当の医師または獣医師により決定され得る。Due to its enhanced stability and biological activity in the absence of heparin, The novel biologically active aFGF analogs of the present invention are particularly suitable for use in various types of complement species. Very suitable for use in pharmaceutical formulations for the treatment of wounds by physicians and/or veterinarians. suitable for Biologically active aFGF analogs for use in such treatments The amount of FW mimetics in the body will, of course, depend on the severity of the wound being treated, the chosen route of administration, and the amount of FW mimetic in the aF. Depends on specific activity or purity, which can be determined by the attending physician or veterinarian.
“治療的に有効なａＦＧＦ類似体”量という用語は、補乳類に於いて治療的応答 を生ずるように決定１．たａＦＧＦ類似体量を指す。このような１ｆ９ｆ的に有 効な巣は、当業者（、こより容易に確認される。The term "therapeutically effective aFGF analog" amount refers to the therapeutic response in a supplement. 1. refers to the amount of aFGF analogue. This kind of 1f9f-like Effective nests are easily identified by those skilled in the art.
本発明のａＦＧＦｆｆｌ似体は、治療する傷または状態に対して任意の好適な経 路により投与し得る。本発明のａＦＧＦＷ似体を治療的適用して好都合に治療し 得る状態としては、これに限定されないが、表面の傷の癒合、骨の癒合、血管形 成、神経再生及び臓器生成及び／または再生が挙げられる。The aFGFffl analogs of the invention can be used in any suitable manner for the wound or condition being treated. It can be administered by route. Therapeutic application of the aFGFW analogs of the invention to advantageously treat Conditions that can be obtained include, but are not limited to, superficial wound healing, bone healing, and blood vessel formation. neurogenesis, neural regeneration, and organ generation and/or regeneration.
獣医用途及びヒト用途の両方の本発明の処方物は、１種以上の医不的に許容可能 なキャリヤと場合により他の治療は、処方物の他の成分と混和性であるという点 で“受容可能”でなければならず、且つそのレシピエンドに対し有害であ−）で はならない。本処方物は、単位投与形で都合よく配合でき、従来公知の任意の方 法により製造し得る。総ての方法には、活性成分を１種以上の付属成分からなる キャリヤと組み合わせる段階を含む０通常、処方物は、液体キャリヤ若しくは微 粉砕固体キャリヤまたはこの両方とａＦＧＦとを均−且つ完全に混合することに より製造する。The formulations of the invention for both veterinary and human use contain one or more non-medically acceptable that the carrier and optionally other treatments are compatible with the other ingredients of the formulation; must be “acceptable” and harmful to the recipe end). Must not be. The formulation can be conveniently formulated in unit dosage form and can be prepared in any manner known in the art. It can be manufactured by the method. All methods include the active ingredient comprising one or more accessory ingredients. Typically, the formulation includes the step of bringing into association with a liquid carrier or microcarrier. homogeneously and thoroughly mixing the aFGF with the ground solid carrier or both; Manufacture more.
以下の実施例は、本発明を説明するために記載するが、その真の範囲は付記請求 の範囲に記載づ゛る。本発明の趣旨を逸脱せずに上記の方法に於ける変形が可能 である。The following examples are set forth to illustrate the invention, but the true scope thereof is set forth in the appended claims. Described within the scope of. Variations in the above method are possible without departing from the spirit of the invention. It is.
痣肛１文差ＬＩｌ 鉦」 本発明のウシａＦＧＦｉ似体を以下の実施例で合成且つ考察した。この類似体、 ウシ［Δｌａ” 、Ｇｌｙ”］ａＦにｔ４ｒ、図１．に記載の如（、ａＦＧＦ分 子の残基９０〜９７領域の所望のアミノ酸置換〈位置９３のヒスチジンをグリシ ンで置換）及び位置４７の非保存システィン残基のアラニンによる追加のアミノ 酸置換を含むように構築した。システィンのアラニンによるアミノ酸置換だけを 有するウシ［＾ｌａ”］ａＦＧＦ類似体も、所望のウシ［＾Ｉａ”、Ｇｌｙ”］ ａＦＧＦ類似体の対照として使用するために調製した。これらの実施例は本発明 のウシａＦＧＦ類似体を示すが、相同性の高いヒトａＦＧＦ類似体に関しても同 一の結果が得られる０例えば、本発明の対応するヒ１− ［八１ａ”、Ｇｌｙ” ］ａＦｆ；Ｆ類似体のアミノ酸配列を図２に示す。Bruise and anus 1 sentence difference LIl gong” Bovine aFGFi analogs of the present invention were synthesized and discussed in the following examples. This analog, Bovine [Δla”, Gly”] t4r in aF, Figure 1. As described in Desired amino acid substitutions in the child residues 90-97 region <histidine at position 93 with glycysilyl ) and an additional amino acid substitution by alanine of the non-conserved cysteine residue at position 47. Constructed to include acid substitutions. Only amino acid substitution of cysteine with alanine The bovine [^la"] aFGF analog with the desired bovine [^Ia", Gly"] Prepared for use as aFGF analog control. These examples are based on the present invention. The same results are shown for the highly homologous human aFGF analogs. For example, the corresponding H1-[81a", Gly" of the present invention ]aFf; The amino acid sequence of the F analog is shown in FIG.
ウシａＦｃＦの［＾Ｉａ４７．Ｇｌｙ”］類似体をコードする合成遺伝子は、全 部で２８個のオリゴヌクレオチド成分から２つの区分に組み立てられた。 にｉ ａ＋ｅｎｅｚ−ｆ；ａｌｌｅｇｏらのアミノ酸配列を、Ｅ、ｃｏｌｉ中での類似 体の発現を最適化するために選択したコドンを使用して、この遺伝子のベースと して使用した［Ｇｉ＋＊ｅｎｚ−Ｇａｌｌｅｇｏら、５ｃｉｅｎｃｅ、２３０． １３８５−１３８８（１９８５）］。区分１１６（１Ｎのオリゴヌクレオチドか ら組み立てて２８７ヌクレオチドフラグメントを作り、これを、Ｘｂａ １及び Ｘｈｏ Ｉ制限エンドヌクレアーゼ部位でプラスミドベクターに挿入し得た０区 分■を１２個のオリゴヌクレオチドから組み立てて、Ｘｈｏ Ｉ及びＢａＩＩ旧 適合末端により結合した１７０個のヌクレオチドフラグメントを産生した。２つ の区分を、予め３成分の連結に於いてＸｂａ Ｉ及びＢａｇ Ｈｌで消化した発 現プラスミドｐｃＦＭ１１５６に挿入し、λｐＬプロモーターの制御下で完全ａ ＦＧＦ遺伝子を産生じた。[^Ia47. Synthetic genes encoding analogs of The oligonucleotide was assembled into two sections from 28 oligonucleotide components. ni i a+enez-f; The amino acid sequence of Allego et al. base of this gene using selected codons to optimize body expression. [Gi+*enz-Gallego et al., 5science, 230. 1385-1388 (1985)]. Category 116 (1N oligonucleotide? were assembled to create a 287 nucleotide fragment, which was then combined with Xba1 and Section 0 that could be inserted into the plasmid vector at the Xho I restriction endonuclease site XhoI and BaII old were assembled from 12 oligonucleotides. A 170 nucleotide fragment joined by compatible ends was produced. two The fraction of into the current plasmid pcFM1156 to create a complete a produced the FGF gene.
プラスミドｐｃＦＨ１１５６は公知のプラスミドｐｃＦＭ８３６から製造する。Plasmid pcFH1156 is produced from the known plasmid pcFM836.
プラスミドｐＣＦ１４８３６の合成は米国特許出願第４．７１０゜４７３号に記 載されており、従って、その明細書の関連部分、特に実施例１〜７は本明細書中 に参照として含まれる。The synthesis of plasmid pCF14836 is described in U.S. Patent Application No. 4.710°473. Accordingly, relevant portions of that specification, particularly Examples 1 to 7, are herein incorporated by reference. included as a reference.
ｐＣＦＭ８３６からｐｃＦ８１１５６を製造するために、２つの内因性Ｎｄｅ■ 制限部位を切断し、露出末端をＴ４ポリメラーゼで充填し、充填した末端を平滑 末端と連結した。To produce pcF81156 from pCFM836, two endogenous Nde Cut the restriction sites, fill in the exposed ends with T4 polymerase, and blunt the filled ends. connected to the ends.
得られたプラスミドをＣ１ａ Ｉ及びＫｐｎ Ｉで消化し、切断したＤＮＡフラ グメントを以下の配列。The obtained plasmid was digested with C1a I and Kpn I, and the cut DNA fragment was Arrange the following elements.
のＤＮＡオリゴヌクレオチドと！き換える。With DNA oligonucleotides! Change.
このプラスミドで形質転換したＥ、ｃｏｌｉ細胞を、Ｆｏｘら、Ｊ。E. coli cells transformed with this plasmid were transformed with the method described by Fox et al., J.
Ｂｉｏｌ 、Ｃｈｅｍ、 、２６３ 、１８４５２−１８４５８（１９８８）に 記載の如く、１６リツトルの発酵容器で増殖させた。Biol, Chem, 263, 18452-18458 (1988). Grown in 16 liter fermenters as described.
ウシ［に１，９３．＾１ａ４７］ａＦＧＦをコードする遺伝子を、オリゴ部位特 異的突然変異誘発を使用して［＾Ｉａ４７］形に転換した。Cattle [to 1,93. ^1a47] The gene encoding aFGF was It was converted to the [^Ia47] form using aberrant mutagenesis.
ａＦＧＦ遺伝子をまずファージベクターＭ１３ｍｐ１８にトランスファーし、突 然変異誘発反応用の鋳型として作用させるために一本ｇＤＮＡを製造した。この ＤＮＡ約０，５μｇを、ＤＮＡシークエンシングに使用するための８１３ユニバ ーサルプライマー及び突然変異誘発プライマー（５゛Ｇ＾＾Ｇ＾＾＾＾ＣＣＡＴ ＴＡＣ＾＾ＣＡＣ３’）の各５ピコモルと混合し、３分間６５℃で加熱し、ゆっ くり放冷した。アニールした鋳型−プライマーを、＾ＴＰ、ｃｌＮＴＰ混合物、 ＤＮＡポリメラーゼＩ大フラグメント及びＴ４ＤＮＡリガーゼと混合し、１５℃ で４時間インキュベートした。この反応混合物のアリコートをコンピテントＥ、 ｃｏｌｉＪＭＩＯＩ細胞に添加し、０．７ＳＬ−寒天にプレートした。得られた プラークをニトロセルロース膜上に写し、膜を３２Ｐ−Ｗ識突然変異誘発プライ マーでハイブリダイズした。ハイブリダイズしたファージから製造したＤＮＡを 、所望の突然変異誘発事象がうまく完了したかを確認するためにシーフェンシン グした。得られた遺伝子を、組換え蛋白質と発現するためにｐｃＦＭ１１５６ベ クターに再度組み込んだ。The aFGF gene was first transferred to phage vector M13mp18, and then A single gDNA was prepared to serve as a template for the mutagenesis reaction. this Approximately 0.5 μg of DNA was transferred to an 813 universal tube for use in DNA sequencing. - Monkey primer and mutagenesis primer (5゛G^^G^^^^CCAT Mix with 5 pmol each of TAC^^CAC3'), heat at 65°C for 3 minutes, and slowly incubate. The chestnuts were left to cool. The annealed template-primer was mixed with ^TP, clNTP mixture, Mix with DNA polymerase I large fragment and T4 DNA ligase and incubate at 15°C. and incubated for 4 hours. An aliquot of this reaction mixture was designated as competent E. coli JMIOI cells and plated on 0.7SL-agar. obtained The plaques were transferred onto a nitrocellulose membrane and the membrane was coated with 32P-W mutagenic ply. hybridized with mar. DNA produced from hybridized phages , Shifensin to confirm that the desired mutagenic event was successfully completed. I clicked. The obtained gene was transferred to the pcFM1156 vector to express the recombinant protein. re-installed into the vector.
ウシ［［；１，１）、＾Ｉａ’フコ及び［＾ｌａ”］ａＦＧＦ類似体の両方を、 組換え蛋白質を発現する機械的に溶解したＥ、ｃｏｌｉ細胞を遠心分離して得ら れた不溶画分から精製した。ベレット画分を８Ｍ尿素、０．１Ｍグリシン（ｐ） １２．５）に可溶化し、不溶性物質を除去するために遠心分離した。上清を、６ Ｈ尿素、１０５Ｍグリシン（ｐＨ３，０＞で平衡化したＳ−セファロース（登録 商標：Ｐｈａｒｅ＋ａｃｉａ、Ｕｐｐｓａｌａ、Ｓｗｅｄｅｎ）カラムに載置し 、次いで６Ｍ尿素、２０ｍＮクエン酸ナトリウム（ｐＨ６，５）で洗浄した。カ ラムに結合した蛋白質を、２０−一クエン酸ナトリウム（ｐＨ６，５＞中のＯ〜 ０．５Ｍ塩化ナトリウムの直線勾配液で溶離した。ａＦＧＦを含む画分をプール し、２０１１Ｍクエン酸ナトリウム、０．１Ｍ硫酸アンモニウムで２０倍に希釈 し、沈澱を除去するために遠心分離した。上清を１容量の２０１１Ｎクエン酸ナ トリウム、２Ｍ硫酸アンモニウムと混合し、２０一台クエン酸ナトリウム、ＩＭ ［酸アンモニウム（ｐＨ６，５）で平衡化したフェニル−セファロース（登録商 標）カラムに載置した。結合蛋白質を、硫酸アンモニウムの直線減少勾配液（Ｉ Ｍ〜ＯＮ＞でカラムから溶離した。 ａＦＧＦを含む画分をプールし、２０−８ クエン酸ナトリウム（ｐＨ６，５）で透析した。この生成物は、図４に示される ようにＳＤＳゲルで現れたクーマシーブルーに他のバンドが本質的にないことか ら、本質的に均質であった。Bovine [[;1,1), both ^Ia'fuco and [^la'']aFGF analogs, Obtained by centrifugation of mechanically lysed E. coli cells expressing recombinant proteins. It was purified from the insoluble fraction. The pellet fraction was mixed with 8M urea and 0.1M glycine (p). 12.5) and centrifuged to remove insoluble material. Supernatant, 6 S-Sepharose (registered) equilibrated with H urea, 105M glycine (pH 3.0> Trademark: Phare+acia, Uppsala, Sweden) , followed by washing with 6M urea, 20mN sodium citrate (pH 6,5). mosquito Proteins bound to rum were dissolved in 20-mono-sodium citrate (pH 6.5>O~). Elution was with a linear gradient of 0.5M sodium chloride. Pool fractions containing aFGF and diluted 20 times with 2011M sodium citrate and 0.1M ammonium sulfate. and centrifuged to remove the precipitate. Transfer the supernatant to 1 volume of 2011N sodium citrate. Thorium, mixed with 2M ammonium sulfate, 20 units of sodium citrate, IM [Phenyl-Sepharose (registered trademark) equilibrated with ammonium acid (pH 6,5)] (marked) column. Bound proteins were purified in a linear decreasing gradient of ammonium sulfate (I eluted from the column at M~ON>. Pool the fractions containing aFGF and add 20-8 Dialyzed against sodium citrate (pH 6.5). This product is shown in Figure 4 So, the Coomassie blue that appeared in the SDS gel is essentially free of other bands. were essentially homogeneous.
実」１ｈ」− ゛ルー゛クロマトグラフィー ５ｕｐｅｒｏｓｅ（登録商標）−１２カラムを持つＰｈａｒｗ＋ａｃｉａ ＦＰ ＬＣ系（Ｐｈａｒｍａｃｉａ、Ｕｐｐｓａｌａ、Ｓｗｅｄｅｎ）を使用して室温 でゲルー過を実施した。カラムは−２０ｍＭクエン酸ナトリウム、０．２Ｍ塩化 ナトリウム（ｐＨ６，５）中、０．５ｉ１／分で実施した。Fruit "1h"- Blue chromatography 5uperose (registered trademark) - Pharw+acia FP with 12 columns at room temperature using an LC system (Pharmacia, Uppsala, Sweden). Gel filtration was performed. Column -20mM sodium citrate, 0.2M chloride It was carried out in sodium (pH 6.5) at 0.5i1/min.
ゲルｒ過クロマトグラフィーから、精製ウシ［＾１ａ４７］及び［＾ｌａ４？Ｊ ｌｙ”］ａＦＧＦ類似体は、リボヌクレアーゼＡ（Ｍｒ＝１３．７００）と同一 溶離位置で一本のピークとして溶離したことが知見された。これは、両方の蛋白 質が単量体であり、同一の流体力学的半径を持つことを示しているが、蛋白質の 両方の型がカラムマトリックスと相互作用し、カラムから遅延溶層する可能性も ある。From gel r perchromatography, purified bovine [^1a47] and [^la4? J ly”]aFGF analog is identical to ribonuclease A (Mr=13.700) It was found that it eluted as a single peak at the elution position. This means that both proteins This shows that the protein is monomeric and has the same hydrodynamic radius, but the Both types can also interact with the column matrix and phase out the column late. be.
実」ｌ殊１− １に１皿五１ クロマトグラフィー 疎水性相互作用クロマトグラフィーを、フェニル−５ｕｐｅｒｏｓｅ＜登録商標 ）カラム（Ｐｈａｒｍａｃｉａ ＦＰＬＣ系）を使用して室温で実施した。２Ｍ ［酸アンモニウム、２０ｍＭクエン酸ナトリウム（ｐＨ６，５＞中のサンプル を、２８ ［酸アンモニウムで平衡化させたカラムに載置した。 ２Ｍ塩化ナト リウムで洗浄後、残りの蛋白質を硫酸アンモニウムの２Ｍ〜ＯＮに減少勾配液で 溶離し、２０ＩＩＭクエン酸ナトリウム（ｐＨ６，５）で最終洗浄した。Real 1- 1 in 1 dish 51 Chromatography Hydrophobic interaction chromatography was performed using phenyl-5uperose<registered trademark ) column (Pharmacia FPLC system) at room temperature. 2M [Sample in ammonium acid, 20mM sodium citrate (pH 6,5> was loaded onto a column equilibrated with 28 ammonium chloride. 2M sodium chloride After washing with sodium hydroxide, the remaining protein was washed with a gradient solution decreasing from 2M to ON in ammonium sulfate. Elution and final wash with 20 IIM sodium citrate (pH 6,5).
疎水性相互作用クロマトグラフィー （ＨＩＣ）で蛋白質の溶離位置は、折り畳 み状態の疎水性領域の露出に強く依存するため、この方法は、類似の蛋白質のコ ンフォメーション的均隻性に間する高感度の精査法を提供する。図３は、ウシ［ ＾ｌａ’）］及び［＾ｌａ’）、Ｇ１３１’すａＦＧＦ類似体の溶離プロフィー ルを示す、［＾ｌａ”］ａＦＧＦは、０．２５Ｍ ［酸アンモニウムのとコロテ 溶離する主ピークを示したが、［＾１ａ４７．Ｇｌｙ”］ａＦＧＦ類似体は０． １３８ ｆｆｌ酸アンモニウムのところで単一ピークを示している。このことか ら、両方の蛋白質は、単一の独特なコンフォメーションで主に存在することが示 唆される。In hydrophobic interaction chromatography (HIC), the elution position of proteins is This method is highly dependent on the exposure of hydrophobic regions in the state of Provides a highly sensitive examination method for informational uniformity. Figure 3 shows the cow [ ^la’)] and [^la’), elution profiles of G131’aFGF analogues [^la”]aFGF is 0.25M [ammonium acid The main peak eluting was shown, but [^1a47. Gly”]aFGF analogue is 0. A single peak is shown at ammonium 138ffl acid. Is this about it? showed that both proteins exist primarily in a single unique conformation. be suggested.
低い塩濃度で［＾１ａ１７．に１，９３コａＦＧＦ ｆ！：溶離すると、［＾Ｉ ａ”］形よりもやや疎水性が高いことを示している。この知見は、蛋白質のコン フォメーションが、この残基が溶媒に暴露されているようである場合、位置９３ でグリシンによりヒスチジン残基を置き換えることと一致している。あるいは、 この残基に於ける変化は、分子のコンフォメーションに於いて全体の変化を誘発 し得、より疎水性の構造を形成した。At low salt concentration [^1a17. 1,93 pieces aFGF f! : When eluted, [^I a”] form. This finding indicates that the protein composition is slightly more hydrophobic than the If the formation is such that this residue is exposed to solvent, position 93 is consistent with replacing the histidine residue by glycine in . or, Changes in this residue induce global changes in the conformation of the molecule. and formed a more hydrophobic structure.
塞」１区」１ 円二色性スペクトルを、Ｏｋｉ Ｉｆ ８００ Ｍｏｄｅｌ ３０コンピユータ ー（Ｏｋｉ、Ｔｏｋｙｏｊａｐａｎ＞を装備したＪａｓｃｏ Ｍｏｄｅｌ Ｊ− ５００Ｃ分光計（Ｊａｓｃｏ、Ｔｏｋｙｏｊａｐａｎ＞で室温で測定した。近及 び遠紫外線範囲で各々１及び０．０２ｃｚのキュベラＩ・を使用して１１−のバ ンド幅で測定した。データは、ａＦＧＦの両方の型に関して平均残基重量１１３ を使用して計算した平均残基楕円率［θ］として表した。Block 1 Ward 1 The circular dichroism spectrum was measured using an Oki If 800 Model 30 computer. - Jasco Model J- equipped with (Oki, Tokyojapan) Measured at room temperature with a 500C spectrometer (Jasco, Tokyo Japan). 11 - 100 nm using 1 and 0.02 cz of Cubela I in the deep and deep UV ranges, respectively. Measured in terms of width. The data show an average residue weight of 113 for both types of aFGF. It was expressed as the average residue ellipticity [θ] calculated using .
ウシ［＾Ｉａ”、Ｇｌｙ’コ］及び［＾１ａ４？コａＦｃＦＷｉ似体の円二色性 （ＣＤ）スペクトルは、図、４ Ａ及び４Ｂに各々示すように、近及び遠紫外線 領域の両方で殆ど同一であった。類似体のＣＤは、ヒトｂＦＧＦに関して報告さ れたスペクトルとも非常に酷似していた［＾ｒａｋａｗａら、ＢＢＲＣ，１６１ ，３３５−３４１（１９８９）］、近近紫外領域でのスペクトルの類似性は、Ｆ にＦ［の類似の三次構造と一致した。Circular dichroism of bovine [^Ia”, Gly’co] and [^1a4?co aFcFWi analogues (CD) spectra are shown in the near and far ultraviolet rays as shown in Figures 4A and 4B, respectively. It was almost the same in both areas. The CD of analogues has been reported for human bFGF. The spectra were very similar to those obtained [^rakawa et al., BBRC, 161 , 335-341 (1989)], the spectral similarity in the near-ultraviolet region is was consistent with a similar tertiary structure of F[.
蛋白質の熱転移を、熱プログラム計及び熱キュベツトホルダを装備したＲｅ５ｐ ｏｎｓｅ ＩＩ分光光度計（Ｇｉｌｆｏｒｄ、Ｍｅｄｆｉｅｌｄ。Thermal transition of proteins can be carried out using the Re5p equipped with a thermal programmer and thermal cuvette holder. once II spectrophotometer (Gilford, Medfield).
Ｍａｓｓａｃｈｕｓｅｔｔｓ）で測定した。サンプルを０．１℃／分または０． ５℃／分の上昇速度で加熱し、その吸収を２８７ｎｍでモニターした。蛋白質濃 度は、０１ｚ蛋白質に関して２８０ｎ−で、両方のウシａＦにＦ類似体に関して は１．０４、ｂＦＧＦに関してはｏ、９８の吸光係数を使用して分光光学的に測 定した。Massachusetts). Samples were heated at 0.1°C/min or 0.5°C/min. Heating was performed at a rate of increase of 5° C./min and the absorption was monitored at 287 nm. Protein-rich degree is 280n- for the 01z protein and for the F analog in both bovine aF. was measured spectrophotometrically using an extinction coefficient of 1.04 and o, 98 for bFGF. Established.
ａＦＧＦ類似体の熱変性を、２０ｍＭクエン酸ナトリウムのＩ）１１６．５及び ７．０で、ヘパリンの存在下及び非存在下で測定した。Thermal denaturation of aFGF analogs was performed using 20 mM sodium citrate I) 116.5 and 7.0, measured in the presence and absence of heparin.
総ての場合に於いて、温度が上昇するにつれて蛋白質が沈澱した。急激な吸収増 加が起きる温度を変性温度とした。In all cases, the protein precipitated as the temperature increased. Rapid increase in absorption The temperature at which the addition occurred was defined as the denaturation temperature.
ヘパリン非存在下では、この温度は、ウシ［＾Ｉａ４７］ａＦＧＦ類似体に関し てよりもウシ［＾！ａ４７．Ｇｌｙ！コ］ａＦＧＦ類似体に関して約１０℃高か った。１．４倍または８倍（ｗ／ｗ）過剰のヘパリンを添加すると、使用した温 度上昇速度に依存して、両方の型の変性温度は１４〜２０’Ｃ上昇した。二種の 型の変性温度の間の違いは、約１０℃のままであった。２４０〜３４０ｎｍ領域 のどの蛋白質のＣＤスペクトルに於いても、１．４倍または８倍（ｗ／ｗ）過剰 のヘパリンのはっきりした効果はながったが、８倍過剰のヘパリンの場合には、 ２４０〜２６０ｎｍ領域に於けるａＦＧＦスペクトルは、ヘパリン自身の吸収に より遮蔽された。In the absence of heparin, this temperature is It's more like a cow [^! a47. Gly! ] Approximately 10°C higher than aFGF analogues It was. Adding a 1.4-fold or 8-fold (w/w) excess of heparin will reduce the temperature used. Depending on the temperature rise rate, the denaturation temperature for both types increased by 14-20'C. two kinds The difference between the denaturation temperatures of the molds remained approximately 10°C. 240-340nm region A 1.4-fold or 8-fold (w/w) excess in the CD spectrum of any protein. The clear effect of heparin was diminished, but in the case of an 8-fold excess of heparin, The aFGF spectrum in the 240-260 nm region is due to the absorption of heparin itself. More shielded.
ツー１工 、ＦＴＩＲ’ 両方のａＦＧＦのコンフォメーションの類似性を調べるためにフーリエ変換赤外 線（ＦＴＩＲ）スペクトルを測定した。ＦＴＩＲ分光学にかけるために、蛋白質 を水で十分に透析した。各蛋白質を、Ｄ２０（Ｓｉｇ＋ｓａ Ｃｈｅｓｉｅａｌ Ｃｏ、、９９．９＄同位体純度）中に作製した２０ｍＭイミダゾール緩衝液中 の２ｚ溶液として調製した。溶液を、ＣａＦ ２ウインドウ及び１００μｌスペ ーサーを備えたｒＲセルに入れた。各スペクトルに関し、ゲルマニウム被覆ＫＢ ｒビームスプリッタ−及びＤＴＣＳ検出器を備えたＮ１ｃｏｌｅｔ ８００ Ｆ ＴｒＲシステムで１５００の光学干渉像を集め、コードした。光学台を連続して 乾燥窒素ガスでパージした。1 tool, FTIR' Fourier transform infrared to examine the conformational similarity of both aFGFs. A line (FTIR) spectrum was measured. Proteins are subjected to FTIR spectroscopy. was thoroughly dialyzed against water. Each protein was converted to D20 (Sig+sa Chesiel In 20mM imidazole buffer made in Co, 99.9$ isotopic purity) It was prepared as a 2z solution of Pour the solution into two windows of CaF and a 100 μl The cells were placed in an rR cell equipped with a server. For each spectrum, germanium-coated KB N1colet 800F with r beam splitter and DTCS detector 1500 optical interference images were collected and coded with the TrR system. Optical bench in succession Purged with dry nitrogen gas.
二次導関数スペクトルを、Ｓｕｓ ｉら、Ｂｉｏｃｈｅｍ、Ｂｉｏｐｈｙｓ、Ｒ ｅｓ。The second derivative spectrum was obtained from Sus i et al., Biochem, Biophys, R es.
Ｃｏ！１１１．．１１５，３９１−３９７（１９８３）に記載の方法の如く計算 した。Co! 111. ．． 115, 391-397 (1983). did.
水蒸気−減数スペクトルに９点平滑化関数（ｓｍｏｏｔｈｉｎｇ ｆｕｎｃｔｉ ｏｎ）３適用した。A 9-point smoothing function (smoothing function) is applied to the water vapor-reduced spectrum. on) 3 was applied.
図５は、アミトビ（重水素化蛋白質中のＣ＝Ｏ伸縮）領域に於ける［＾１ａ４７ ］及び［＾１ａ４７．（、ｌ、９）］ウシａＦｃＦ類似体の二次導関数スペクト ルを示している。ポリペプチド及び蛋白質に間しては、この領域に於ける成分バ ンドの振動数は二次構造内容に関連している［Ｓｕｒｅｗｉｃｚら、Ｂｉｏｃｈ ｅｍ、Ｂｉｏｐｈｙｓ。Figure 5 shows the [^1a47 ] and [^1a47. (, l, 9)] second derivative spectrum of bovine aFcF analogue It shows the For polypeptides and proteins, the component balance in this area is The frequency of vibrations is related to the secondary structure content [Surewicz et al., Bioch. em, Biophys.
＾ｃｔａ、９５２，１１５−１３０（１９８８）］、スペクトルは、１６３０及 び１６８５ｃｍ−’に強いバンドを示しており、これは２種の蛋白質中にかなり の量のβ−構造があることを示す。１６４７ｃｍ−’近傍の強いバンドは、不規 則なまたは無秩序構造の存在を示している。 １８６６及び１６７３ｃｍ−’近 傍の弱いピークは、ターン構造から発生している。小さなピークが、両方の蛋白 質のスペクトル中１６５１ｃ■−１近傍にある。この振動数近くのアミド■。^cta, 952, 115-130 (1988)], the spectrum is 1630 and It shows a strong band at 1,685 cm-' and 1,685 cm-', which indicates that the This shows that there is an amount of β-structure. The strong band near 1647 cm-' is irregular. Indicates the presence of a regular or disordered structure. 1866 and 1673cm-'near The nearby weak peaks originate from the turn structure. Small peaks represent both proteins. It is near 1651c-1 in the quality spectrum. Amide ■ near this frequency.
成分は、通常α−へリックスに帰属される。しかし、近年、このバンドはループ 構造から発生することが知見された［Ｗ ｉ ｌ ｄｅｒら 、＾ｂｓｔｒａｃ ｔｓ ｏｆ ｔｈｅ Ｆｏｕｒｔｈ Ｓｙｍｐｏｓｉｕ鴫 ｏｆ ｔｈｅＰｒｏ ｔｅｉｎ ５ｏｃｉｅｔｙ、Ｓａｎ Ｄｉｅｇｏ（１９９０）］、図５に示され ているように、ＣＤと異なり、高解像度ＦＴＩＲスペクトルは、β−構造及びタ ーンの存在を示しており、ウシ［＾１ａ“’］ａＦ（：Ｆ類似体及びウシ［＾１ ａ″’ 、Ｇｌｙ”］ａＦＧＦｉ似体は、殆ど重ねることができ、こ五らの２種 の蛋白質が似た構造を持つことを示唆している。The components are usually assigned to α-helices. However, in recent years this band has become a loop It was found that it occurs from the structure [W i l der et al., ^bstrac ts of the Fourth Symposiu of the Pro tein5ociety, San Diego (1990)], shown in Figure 5. Unlike CD, high-resolution FTIR spectra contain β-structures and Ta bovine [^1a"'] aF (:F analogue and bovine [^1 a″’, Gly”] aFGFi analogs can be almost stacked, and these two types This suggests that the proteins have similar structures.
二次導関数スペクトルは、２種のａＦＧＦ類似体の間のコンフォメーションには 殆ど違いがないことを示した。しかしながら、Ｄ２０溶液で凍結乾燥した蛋白質 を平衡化させる時、［＾１ａ４７．Ｇｌ、９Ｊ類似体よりもウシ［＾Ｉａ”］類 似体の方が、交換可能なプロトンの重水素化が速いことは明白であった。The second derivative spectrum shows that the conformation between the two aFGF analogs is showed that there was almost no difference. However, proteins lyophilized in D20 solution When equilibrating [^1a47. Gl, bovine [^Ia”] than 9J analogues It was clear that the analogue deuterated the exchangeable protons faster.
２種の蛋白質のコンフォメーションは似ていたので、Ｈ−Ｄ交換速度で知見され た違いは、これらの間の交換可能なプロトンの露出量の違いからは説明できない 、［＾１ａ４７］ａＦＧＦ類似体はより柔軟な構造を有するので、アミドプロト ンを溶媒に対してより接近可能にさせるようである。Since the conformations of the two proteins were similar, it was determined by the HD exchange rate. The difference cannot be explained by the difference in the exposure of exchangeable protons between them. , [^1a47]aFGF analogs have a more flexible structure, so the amide proto- It appears to make the particles more accessible to solvents.
丸１隨ｉ ヘパリンクロマトグラフィー ヘパリン−セフＴｏ−ス（登録商標： Ｐｈａｒ＠ａｃｉａ）を、１×８ｃ１１ カラムに詰め、１０−ＭＴｒｉｓ−ｔｌｃ＆（ｐＨ７，２）で平衡化させた。1 circle heparin chromatography Heparin-Sefu Tosu (registered trademark: Phar@acia) was added to 1×8c11 It was packed into a column and equilibrated with 10-MTris-tlc& (pH 7,2).
カラムに載置し、カラムを１０ｍＮ Ｔｒｉｓ−ＨＣｌ（ｐＨ７，２）で洗浄し 、同一緩衝液中０〜２．８Ｍ塩化ナトリウムの直線勾配液で、Ｐｈａｒｍａｃ １ａＦＰＬＣシステムを使用して流速０．５ｚ１／分で溶離した。Place it on the column and wash the column with 10 mN Tris-HCl (pH 7,2). , with a linear gradient of 0-2.8 M sodium chloride in the same buffer, Pharmac. Elution was performed using a 1aFPLC system at a flow rate of 0.5z1/min.
酸性及び塩゛基性ＦＧＦを、ヘパリン及びヘパリン様分子との強い結合性により 識別する。ウシ［＾ｌａ”Ｊｌｙ”］及び［＾１ｍ”］ａＦにＦ類似体は両方と も、１０ｍＭ Ｔｒｉｓ−ＨＣ＆（ｐＨ７，２）中１．５４Ｍ塩化ナトリウムの ところで溶離する１本のピークを実施例５からのａＦＧＦ類似体の旧Ｈ３Ｔ３細 胞に於ける有糸***促進活性を、以下に記載の如く測定した。さらに、ＰＣＴ特 許出願公開第８８１０４１８９号に記載の如く製造したヒト［Ｓｅｒ” 、Ｓｅ ｒ”］ｂＦＧＦ類似体も、ａＦＧＦ類似体と共に生物活性アッセイにより調べた 。Acidic and salt-based FGFs have strong binding properties with heparin and heparin-like molecules. identify Both F analogs in bovine [^la"Jly"] and [^1m"]aF Also, 1.54M sodium chloride in 10mM Tris-HC & (pH 7,2) By the way, one peak eluting is the old H3T3 sample of the aFGF analogue from Example 5. Mitogenic activity in cells was measured as described below. In addition, PCT Human [Ser”, Se produced as described in Patent Application Publication No. 88104189 r”] bFGF analogs were also examined in bioactivity assays along with aFGF analogs. .
ＮＩＨ３Ｔ３細胞は、＾ＴＣＣより得た。細胞を、１０１子ウシ血清、１０単位 ７ｍｌペニシリン、２ｍＭグルタミン及び１０単位７ｍｌストレプトマイシンを 補ったＤＭＥ中で増殖させた。１週間当たり２回、■＝４０の比で細胞を継代接 種した。アッセイの１日目にやや融合性の（ｓｕｂｃｏｎｆ １ｕｎｔ）培養物 とトリプシンで分散させ、ｚｏ、ｏｏｏ細胞細胞７ｍ浪度で、上記培地中ウェル １個当たり１１１で、２４−ウェルにプレートした。５日目に、培地を血清を含 まないが、ペニシリン、ストレプトマイシン及びグルタミンを上記濃度で含む１ １／ウ工ルＤＮＥＭと置き換えな、６日目に、実験サンプルを１００μｌを超え ない容量で培地に添加した。１８時間後、細胞を、トリチウム化したチミジンを ２１０μＣｉを含む上記培地１１１で３７℃で１時間パルスにかけた。パルスに かけた後、細胞を培地で一度洗浄し、次いで２５０ｍＭ蔗糖、１０輪Ｈリン酸ナ トリウム、１ｍＭ ＥＤＴ＾（ｐＨ８）を添加し、細胞を放出させるためにプレ ートを３７℃で１０分間インギュベートした。細胞を５ｋａｔｒｏｎハーベスタ −で収穫した［５ｋａｔｒｏｎ、Ｉｎｃ、、Ｓｔｅｒｌｉｎｇ、Ｖｉｒｇｉｎｉ ａ］、 ７４ルターを乾燥し、シンチレーション液に入れ、Ｂｅｃｋｍａｎシン チレーションカウンターで数えた［Ｂｅｅｋｍａｎ Ｉｎｓｔｒｕｍｅｎｔｓ。NIH3T3 cells were obtained from TCC. Add cells to 101 calf serum, 10 units 7ml penicillin, 2mM glutamine and 10 units 7ml streptomycin Grown in supplemented DME. Passage cells twice per week at a ratio of ■ = 40. I planted a seed. Slightly confluent (subconf 1 unit) culture on day 1 of assay Disperse the zo,ooo cells with trypsin and add them to the wells in the above medium at 7 mL. 111 per plate in 24-wells. On day 5, the medium was added with serum. 1 containing penicillin, streptomycin and glutamine at the above concentrations. On the 6th day, add more than 100 μl of the experimental sample to DNEM. No volume was added to the medium. After 18 hours, cells were treated with tritiated thymidine. Pulsed with medium 111 as described above containing 210 μCi for 1 hour at 37°C. to the pulse After plating, cells were washed once with culture medium, and then treated with 250mM sucrose and 10-ring H phosphate sodium chloride. Add thorium, 1mM EDT^ (pH 8) and pre-prepare to release cells. The cells were incubated at 37°C for 10 minutes. 5katron harvester cells - Harvested at [5katron, Inc., Sterling, Virgini a], 74 Luther was dried, placed in scintillation liquid, and Beckman syn Counted with a cooling counter [Beekman Instruments.
Ｉｎｃ、、Ｆｕｌ Ｉｅｒｔｏｎ、Ｃａｌ １ｆｏｒｎｉａ］。Inc., Ful Ierton, Cal 1fornia].
ＮＩＨ３Ｔ３細胞に於けるウシ［＾Ｉａ４ｆｆ、にｌｙ’コ］及び［＾Ｉａ’） ］ａＦｃＦ類似体の有糸***促進活性を、図６に記載の如く調べた。ヘパリンの 非存在下では、［＾１１１？］ＩＬＦにＦ類似体は、１〜艮取の用量依存性刺激 を産生じた。同一アッセイ条件に於いて、［＾ｌａ”、Ｇｌｙ’すａＦＧＦ類似 体は、ずっと低い蛋白質濃度で同一の有糸***促進活性と産生でき、５０１Ｍｔ 大用量は約１ｎｇ、ｌｚ１であった７組換えｂＦＧＦは、［＾ｌａ”、Ｇｌｙ” ］ａＦＧＦよりも４〜・５倍も強力であり、５ｏｚ最大用量は２２０ｐｇ／ｚ１ であった。４．５μｇｌｚ＆ヘパリンを両方の類似体に添加すると、そのｉｎ ｖｉｖｏ活性は、強力であった［＾Ｉａ”、Ｇｌｙ”］ａＦＧＦ類似体に関して 増加した。４５μｇ７ｍｌヘパリンの存在下では、３種想全部の分子の用量応答 が殆ど同一であるように活性が増強した。５ｏｚ最大用量は９０μｇ／ｚ１であ った。Bovine [^Ia4ff, nily'co] and [^Ia') in NIH3T3 cells ] The mitogenic activity of aFcF analogs was examined as described in FIG. heparin In the absence, [^111? ]F analogues to ILF have a dose-dependent stimulation of 1 to 1 was produced. Under the same assay conditions, [^la'', Gly'aFGF-like The body can produce the same mitogenic activity and 501 Mt at much lower protein concentrations. The large dose was approximately 1 ng, lz1.7 Recombinant bFGF was [^la”, Gly” ] 4-5 times more potent than aFGF, with a maximum dose of 5oz of 220pg/z1 Met. Adding 4.5 μg lz & heparin to both analogs results in its in The in vivo activity was strong for [^Ia”,Gly”]aFGF analogs. increased. In the presence of 45 μg 7 ml heparin, the dose response of all three molecules The activity was enhanced so that the values were almost the same. 5oz maximum dose is 90μg/z1 It was.
その個々の有糸***促進活性の保持により決定されるａＦＧＦ類似体の安定性は 、ＬＨ／ｍｌヘパリンの存在下及び非存在下の両方に於いて、２０Ｊクエン酸ナ トリウム（ｐＨ７）、３７℃で各ＦＧＦ類似体の０．１ｘｇ／ｘ１溶液をインキ ュベーションすることにより調べた。ヘパリン非存在下では、図７に示すように 、ウシ［＾ｌａ”１ａＦＧＦ類似体は活性を急激に失い、半減期は約１３時間で あった。しかしながら、ヘパリン存在下では、ウシ［＾ｌａ”］ａＦｃＦは、実 験の２５０時間の期間で全く生物学的活性と失わなかった。対照的に、ウシ［＾ Ｉａ４７．（：！ｙ”］ａＦＧＦｆｆｌ似体もヒト［ＳｅｒテＯ，Ｓｅｒ”］ｂ ＦＧＦ類似体も、ヘパリンが存在するかどうかに拘わらず、２５０時間にわたっ て全く活性損失を示さなかった７ １１九二 杭！ ウシ［ＡＩａ４７．Ｇｌｙ”］ａＦｃ：Ｆ類似体の結晶を、０．２８ ＮＨ，Ｓ Ｏ，，２８８ａＣ１，０，０９９Ｍクエン酸ナトリウム及び０．０２Ｍ リン酸 ナトリウムカリウム（ｐＨ５，６）に対して蒸気拡散により成長させた。蛋白質 の液滴は、等量のリザーバー溶液と１ｏｚｙ／ｘｉ蛋白質溶液を含んでいた。結 晶は三方晶系（空間群Ｐ３１２１ 。The stability of an aFGF analogue determined by the retention of its individual mitogenic activity is , 20J sodium citrate both in the presence and absence of LH/ml heparin. Thorium (pH 7), ink 0.1xg/x1 solution of each FGF analog at 37°C. This was investigated by nu- vation. In the absence of heparin, as shown in Figure 7 , the bovine [^la"1aFGF analogue rapidly loses activity, with a half-life of approximately 13 hours. there were. However, in the presence of heparin, bovine [^la”]aFcF It did not lose any biological activity during the 250 hour period of the experiment. In contrast, cows [^ Ia47. (:!y”]aFGFffl analog is also human [SerteO,Ser”]b FGF analogues also showed significant effects over 250 hours, regardless of the presence or absence of heparin. showed no loss of activity7 1192 Pile! Bovine [AIa47. Gly"] aFc:F analog crystals at 0.28 NH,S O,,288aC1,0,099M sodium citrate and 0.02M phosphoric acid It was grown by vapor diffusion on sodium potassium (pH 5,6). protein The droplets contained equal volumes of reservoir solution and 1ozy/xi protein solution. Conclusion The crystal is trigonal (space group P3121).
ａ・７８．６人、Ｃ・１１５．９人）及び２．５人の分解能で回折した１強度デ ータを、１８ｋｍ回転アノードジェネレーター上に据え付けたＳｉｅｍｅｎｓ（ Ｍａｄｉｓｏｎ Ｊｉ Ｉｃｏｎｓｉｎ）マルチワイヤエリア検出器で集めた。A, 78.6 people, C, 115.9 people) and 1 intensity data diffracted with a resolution of 2.5 people. The Siemens ( Collected with a Madison Ji Iconsin multi-wire area detector.
データ整理にＳｉｅｍｅｎｓの処理プログラムを使用した。多重同形置換（ｍｕ ｌｔｉｐｌｅ ｉｓｏｍｏｒｐｈｏｕｓ ｒｅｐｌａｃｅｍｅｎｔ；１Ｉｉｒ） 相は、２種の誘導体から３人の分解能で計算し、その数値は０．６８であった。A Siemens processing program was used for data reduction. Multiple isomorphic substitution (mu ltiple isomorphous replacement; 1Iir) The phase was calculated from two types of derivatives with a resolution of three people, and the value was 0.68.
溶媒平坦化（ｓｏｌｖｅｎｔ Ｂａｔｔｅｎｉｎｇ）後、非対称単位中の２種の 個々のａＦＧＦ分子に対応する領域を同定した。これらの分子の間の一般的な非 結晶学的対称関係５ｐａｃｅ ｔｒａｎｌａｔｉｏｎ ｆｕｎｃｔｉｏｎ）及び 密度相関研究（ｄｅｎｓｉｔｙｃｏｒｒｅｌａｔｉｏｎ ５ｔｕｄｉｅｓ）から 決定した９分子エンベロープは、改良Ｂ、ＣＪａｎｇアルゴリズムを使用して平 均化ａＦＧＦ分子の周囲として定義した。相を、分子平均化及び溶媒平坦化によ り相互作用的に純化（ｒｅｆ １ｎｅ） した。After solvent battening, the two types in the asymmetric unit Regions corresponding to individual aFGF molecules were identified. A common non-conformity between these molecules Crystallographic symmetry relationship (5 pace transcription function) and From density correlation studies (density correlation 5 studies) The determined nine-molecule envelope was flattened using the modified B, CJang algorithm. It was defined as the surroundings of the equated aFGF molecule. phase by molecular averaging and solvent flattening. It was then interactively purified (ref 1ne).
図８に示されているように、最初のマツプは、小さな分子エンベロープであるた め、ループのところで切断したβシート構造の伸長領域を示した。モデルを作る ための最終マツプは、［Ｒｏｕｌｄら、５ｃｉｅｎｃｅ、２４６．１１３５−１ １４２（１９８９）に記載の如く、平均比相に対して再純化した重原子のパラメ ーターから］ｓｉｒ相を用いて計算し、最初のモデルの原子位置付近に６人球を 置くことにより作られた分子エンベロープで相互的に平均化した。３人の分解能 で平均化すると、観察された構造因子と、平均化及び溶媒平坦化マツプから計算 した構造因子との間の最終Ｒ−因子は１７．８１に収斂した。ＣＣａ＋５ｂｉｌ ｌａｕによる５ｉｌｉｃｏｎ Ｇｒａｐｈｉｃｓ ４０８０用に供給されたグラ フィックプログラムＴＯＨ／ＦＲＯＤＯを使用して、ａＦにＦ配列の１０〜１３ ６残基を平均化電子密度マツプに構築した。As shown in Figure 8, the first map is a small molecular envelope. Therefore, the extended region of the β-sheet structure cut at the loop is shown. make a model The final map for [Rould et al., 5science, 246.1135-1 142 (1989), the parameters of the repurified heavy atoms relative to the average ratio phase. Calculate using the sir phase] and place six spheres near the atomic positions of the first model. The molecular envelopes created by placing the molecules were mutually averaged. resolution of 3 people calculated from the observed structure factors and the averaged and solvent flattened map. The final R-factor between the obtained structure factors converged to 17.81. CCa+5bil Graphics supplied for 5ilicon Graphics 4080 by lau 10 to 13 of the F sequence in aF using the Fic program TOH/FRODO. The six residues were constructed into an averaged electron density map.
結晶学解析の結果は、９０〜９７領域がループ構造に含まれるという本出願人の 仮説を支持した。この領域が実際にＢａ ｉ ｒｄらに示唆されたようにレセプ ター結合に含まれるならば、ループを安定化する任意のアミノ酸買換により分子 の生物学的活性が安定化及び／または増強するだろう。これは、多分ウシ［＾１ ａ４７Ｊｌｙ”］ａＦＧＦにより達成され知見さくコト メＯＱ ■０ ７−肖 ＩＴ’Ｓ −−１−Ｉ Ｆ−１１”Ｐ’１図３ − ［Ａｌａ４７． Ｇｌｙ９３］ ａＦＧＦ＋＋＋＋＋＋＋＋＋＋＋ ［Ａｌ ａ” ］ ａＦＧＦ［○］ （ｄｅｇｌｌｃｍ ／ｄｅｃ＋ｍｏｌｅ）［０Ｊｘ １０ （ｄｅｇ・ｃｍ ／ｄｅｃ＋ｍｏｌｅ）図５ 呂 呂 ８ ♀ ８ ロ （イ乙寝護￥等）ＷＮΩ−（工９ 耐１５％ 国際調査報告 フロントページの続き （５１） Ｉｎｔ、 Ｃ１，５識別記号 庁内整理番号／／（Ｃ１２Ｐ ２１１ ０２ Ｃ１２Ｒ１：１９） ＩThe applicant's crystallographic analysis results indicate that the 90-97 region is included in the loop structure. The hypothesis was supported. This region is actually a receptor as suggested by Baird et al. molecule by replacing any amino acid that stabilizes the loop if it is included in the ter bond. biological activity will be stabilized and/or enhanced. This is probably a cow [^1 a47Jly"] Achieved by aFGF and summarized the knowledge OQ ■0 7- Portrait IT'S --1-I F-11"P'1 Figure 3 - [Ala47. Gly93] aFGF++++++++++ [Al a''] aFGF[○] (degllcm/dec+mole) [0Jx 10 (deg・cm/dec+mole) Figure 5 ro ro 8 ♀ 8 ro (Iotunego ¥ etc.) WNΩ- (Eng. 9 15% resistance international search report Continuation of front page (51) Int, C1,5 identification symbol Internal office serial number // (C12P 211 02 C12R1:19) I
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| PCT/US1991/009441 WO1992011360A1 (en) | 1990-12-18 | 1991-12-17 | Analogs of acidic fibroblast growth factor having enhanced stability and biological activity |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4503720A Pending JPH06503962A (en) | 1990-12-18 | 1991-12-17 | Analogs of acidic fibroblast growth factor with enhanced stability and biological activity |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0575339A4 (en) |
| JP (1) | JPH06503962A (en) |
| KR (1) | KR930703439A (en) |
| AU (1) | AU663067B2 (en) |
| CA (1) | CA2098484A1 (en) |
| IE (1) | IE914393A1 (en) |
| IL (1) | IL100418A0 (en) |
| NZ (1) | NZ241021A (en) |
| WO (1) | WO1992011360A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08310966A (en) * | 1995-05-18 | 1996-11-26 | Agency Of Ind Science & Technol | Pharmaceutical composition containing fibroblast growth factor chimera protein |
| JP2012143234A (en) * | 2007-10-12 | 2012-08-02 | National Institute Of Advanced Industrial Science & Technology | Medium for promoting cell proliferation, containing highly functionalized chimeric protein |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2717495B1 (en) * | 1994-03-18 | 1996-04-12 | Rhone Poulenc Rorer Sa | Recombinant viruses, preparation and use in gene therapy. |
| DK0785949T3 (en) * | 1994-10-13 | 2003-08-18 | Amgen Inc | Process for Purification of Keratinocyte Growth Factors |
| AU3828195A (en) * | 1994-10-13 | 1996-08-07 | Amgen, Inc. | Analogs of acidic fibroblast growth factor having enhanced stability and biological activity |
| US6743422B1 (en) | 1996-10-15 | 2004-06-01 | Amgen, Inc. | Keratinocyte growth factor-2 products |
| DE60021360T2 (en) * | 1999-04-15 | 2006-05-24 | Caritas St. Elizabeth's Medical Center of Boston, Inc., Boston | VEGF ANGIOGENIC GROWTH FACTORS FOR THE TREATMENT OF PERIPHERAL NEUROPATHY |
| US7125856B1 (en) | 1999-04-15 | 2006-10-24 | St. Elizabeth's Medical Center Of Boston, Inc. | Angiogenic growth factors for treatment of peripheral neuropathy |
| US20070134204A1 (en) * | 2005-12-09 | 2007-06-14 | Henrich Cheng | Method for treating nerve injury and vector construct for the same |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989000198A1 (en) * | 1987-07-07 | 1989-01-12 | Biotechnology Research Associates, J.V. | Recombinant fibroblast growth factors |
| EP0241136A3 (en) * | 1986-03-07 | 1989-12-27 | The President And Fellows Of Harvard College | Human class 1 heparin-binding growth factor |
| NZ226543A (en) * | 1987-10-22 | 1992-02-25 | Merck & Co Inc | Recombinant mutant acidic fibroblast growth factor |
| DE3909009A1 (en) * | 1989-03-18 | 1990-09-20 | Schloemann Siemag Ag | CONTINUOUS CASTING SYSTEM FOR CASTING PROFILES |
-
1991
- 1991-12-17 NZ NZ241021A patent/NZ241021A/en unknown
- 1991-12-17 JP JP4503720A patent/JPH06503962A/en active Pending
- 1991-12-17 IE IE439391A patent/IE914393A1/en not_active Application Discontinuation
- 1991-12-17 CA CA002098484A patent/CA2098484A1/en not_active Abandoned
- 1991-12-17 WO PCT/US1991/009441 patent/WO1992011360A1/en not_active Application Discontinuation
- 1991-12-17 AU AU91524/91A patent/AU663067B2/en not_active Ceased
- 1991-12-18 IL IL100418A patent/IL100418A0/en unknown
-
1992
- 1992-07-17 EP EP92902895A patent/EP0575339A4/en not_active Withdrawn
-
1993
- 1993-06-17 KR KR1019930701853A patent/KR930703439A/en not_active Application Discontinuation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08310966A (en) * | 1995-05-18 | 1996-11-26 | Agency Of Ind Science & Technol | Pharmaceutical composition containing fibroblast growth factor chimera protein |
| JP2012143234A (en) * | 2007-10-12 | 2012-08-02 | National Institute Of Advanced Industrial Science & Technology | Medium for promoting cell proliferation, containing highly functionalized chimeric protein |
Also Published As
| Publication number | Publication date |
|---|---|
| KR930703439A (en) | 1993-11-30 |
| AU9152491A (en) | 1992-07-22 |
| IE914393A1 (en) | 1992-07-01 |
| NZ241021A (en) | 1993-12-23 |
| AU663067B2 (en) | 1995-09-28 |
| EP0575339A1 (en) | 1993-12-29 |
| CA2098484A1 (en) | 1992-06-19 |
| IL100418A0 (en) | 1992-09-06 |
| WO1992011360A1 (en) | 1992-07-09 |
| EP0575339A4 (en) | 1995-04-05 |
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