JP3012931B1 - SPARC fusion protein and method for producing the same - Google Patents
SPARC fusion protein and method for producing the sameInfo
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- JP3012931B1 JP3012931B1 JP11049826A JP4982699A JP3012931B1 JP 3012931 B1 JP3012931 B1 JP 3012931B1 JP 11049826 A JP11049826 A JP 11049826A JP 4982699 A JP4982699 A JP 4982699A JP 3012931 B1 JP3012931 B1 JP 3012931B1
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Abstract
【要約】
【解決手段】 SPARCとチオレドキシンとを融合させて
なるSPARC融合タンパク質、該タンパク質をコードする
DNA、ならびに該DNAを含有する組換えベクターを
宿主細胞に導入して得られる形質転換体を培地に培養
し、培養物中に該DNAによってコードされるタンパク
質を生成蓄積させ、該培養物から該タンパク質を採取す
ることを特徴とする、SPARC融合タンパク質の製造方
法。
【効果】 本発明によれば、血管新生、抗細胞接着活
性、ECM再構成、骨形成を初めとする種々の生理学的
活性を有することから研究試薬や医薬品としての利用が
期待されるSPARCタンパク質を、その生理学的活性を失
わず、かつ医薬として調製しやすい可溶性の融合タンパ
ク質として、効率良く安価にかつ大量に調製できる。The present invention relates to a SPARC fusion protein obtained by fusing SPARC and thioredoxin, a DNA encoding the protein, and a transformant obtained by introducing a recombinant vector containing the DNA into a host cell. And producing a protein encoded by the DNA in the culture, accumulating the protein in the culture, and collecting the protein from the culture. According to the present invention, a SPARC protein, which has various physiological activities such as angiogenesis, anti-cell adhesion activity, ECM reconstitution, and bone formation, is expected to be used as a research reagent or pharmaceutical because of its various physiological activities. As a soluble fusion protein that does not lose its physiological activity and is easily prepared as a medicament, it can be efficiently and inexpensively prepared in large quantities.
Description
【0001】[0001]
【発明の属する技術分野】本発明はSPARC融合タンパク
質およびその製造方法に関する。The present invention relates to a SPARC fusion protein and a method for producing the same.
【0002】[0002]
【従来技術】SPARC (secreted protein acidic and ric
h in cysteine ;「酸性およびシステインに富む分泌タ
ンパク質」) は、骨中に存在する主要な非コラーゲン性
タンパク質であるオステオネクチン(osteonectin)とし
て最初に報告され(Termine,J.D. et al., Cell, 26 :
99-105, 1981)、コラーゲンやハイドロキシアパタイト
への強い結合性を有し、Ca2+結合能を持つことから機能
性骨特異タンパク質として注目された。このSPARCは、
接着性培養細胞の増殖に伴い細胞外分泌される分子量43
kD蛋白として報告されていたものより遺伝子配列が決定
されたが(Sage.H. et al., J.Biol.Chem., 259, 3993-4
007, 1984) 、後に別のグループによって基底膜から単
離されたBM-40 (Mann, K. et al., FEBS Lett., 218 :
162-172,1987)とも一致することがわかった。SPARCはそ
の後、種々の培養細胞からの分泌が観察され、特に正常
線維芽細胞や血管内皮細胞からの分泌が顕著である(Lan
e.T.F.& Sage, E.H., FASEB J., 8 : 163-173, 1994)。[Prior art] SPARC (secreted protein acidic and ric
h in cysteine; “acidic and cysteine-rich secreted protein”) was first reported as osteonectin, the major non-collagenous protein present in bone (Termine, JD et al., Cell, 26). :
99-105, 1981), which has attracted attention as a functional bone-specific protein because it has strong binding properties to collagen and hydroxyapatite and has Ca 2+ binding ability. This SPARC is
Molecular weight 43 extracellularly secreted with the growth of adherent cultured cells
The gene sequence was determined from that reported as the kD protein (Sage.H. et al., J. Biol. Chem., 259, 3993-4
007, 1984) and BM-40 (Mann, K. et al., FEBS Lett., 218:
162-172, 1987). SPARC was subsequently secreted from various cultured cells, and in particular, remarkable secretion from normal fibroblasts and vascular endothelial cells (Lan
eTF & Sage, EH, FASEB J., 8: 163-173, 1994).
【0003】SPARCの機能としては、SPARCの細胞培養系
への添加によって細胞外マトリックス分子への細胞接着
が濃度依存的に阻害されることが知られており、トロン
ボスポンジンやテネイシンとともに"anti-adhesive pro
tein" と呼ばれる(Sage, E.H. & Bornstein, P., J.Bio
l.Chem., 266 : 14831-14834, 1991) 。この作用はイン
テグリンと細胞接着リガンドとの結合を競合的に阻害す
るのではなく、SPARC細胞表面受容体との結合により、
細胞内シグナル伝達を介してビンキュリン等の細胞骨格
系に作用し、focal adhesion plaque を分解することに
よると報告されている(Murphy-Ullrich, J.E.et al.,
J.Cell.Biochem., 57 : 341-350, 1995)。また、SPARC
の最も着目すべき機能としては、ECM (extracellular m
atrix ;細胞外マトリックス) 産生を低下させ、ECM 分
解性プロテアーゼ産生を亢進するとともに(Lane.T.F.&
Sage, E.H., FASEB J., 8 : 163-173, 1994)、血管新生
部位において高発現し、内皮細胞のフィブロネクチンや
トロンボスポンジンの分泌量を減少させることである。
従って、SPARCはこれらの作用の連動によりECM 再構成
を行い、特に血管内皮での細胞増殖制御や細胞間透過性
上昇を促し、細胞増殖作用や血管新生活性の発現に必須
であると考えられている。上記のような種々の生理活性
を持つSPARCは治療薬としての有用性が注目され、臨床
応用が期待される。[0003] As for the function of SPARC, it is known that addition of SPARC to a cell culture system inhibits cell adhesion to extracellular matrix molecules in a concentration-dependent manner. adhesive pro
tein "(Sage, EH & Bornstein, P., J. Bio
l. Chem., 266: 14831-14834, 1991). This action does not competitively inhibit the binding of integrin to cell adhesion ligands, but by binding to SPARC cell surface receptors,
It is reported to act on the cytoskeletal system such as vinculin via intracellular signal transduction to degrade the focal adhesion plaque (Murphy-Ullrich, JE et al.,
J. Cell. Biochem., 57: 341-350, 1995). Also, SPARC
The most notable features of ECM (extracellular m
atrix; extracellular matrix) production, and increase ECM-degrading protease production (Lane.TF &
Sage, EH, FASEB J., 8: 163-173, 1994), which is highly expressed in angiogenesis sites and reduces the secretion of fibronectin and thrombospondin in endothelial cells.
Therefore, it is thought that SPARC performs ECM reconstitution by linking these actions, and in particular, promotes cell growth control and increased intercellular permeability in vascular endothelium, and is essential for cell growth and angiogenic activity. ing. SPARC having various physiological activities as described above is noted for its usefulness as a therapeutic agent, and clinical application is expected.
【0004】これまで、SPARCを遺伝子工学的手法によ
り生産させる試みとして、宿主細胞に動物細胞を用いた
例があるが、収率が非常に低く量的に満足ができもるの
ではない上、精製に数段階を要し、非常に労力やコスト
がかかるという問題があった。また、宿主細胞に大腸菌
を用いた例もあるが、SPARCが可溶性画分に出てこな
い、若しくは可溶性画分に出てきても極少量であるこ
と、不溶性画分である封入体に蓄積したSPARCは生理活
性を保持していない不活性型であること、不溶性画分か
ら単離精製した不活性型SPARCのS-S 結合を適切な形に
フォールディングさせて可溶性の活性型SPARCを得る方
法が確立されていないこと、またグルタチオン−S−ト
ランスフェラーゼ(GST)などの融合タンパク質として発
現させても封入体に蓄積してしまうこと等の問題があ
り、いずれも大量生産には至っていない。Until now, attempts have been made to produce SPARC by genetic engineering techniques using animal cells as host cells. However, the yield is very low and the yield cannot be satisfactory. There was a problem that several steps were required for purification, and very labor and cost were required. In some cases, Escherichia coli was used as the host cell, but SPARC did not appear in the soluble fraction, or it appeared in the soluble fraction in an extremely small amount, and SPARC accumulated in the inclusion body that was the insoluble fraction. Is an inactive form that does not retain physiological activity, and no method has been established to obtain a soluble, active form of SPARC by folding the SS bond of inactive SPARC isolated and purified from the insoluble fraction into an appropriate form In addition, even when expressed as a fusion protein such as glutathione-S-transferase (GST), the protein accumulates in inclusion bodies.
【0005】[0005]
【発明が解決しようとする課題】本発明は、種々の生理
学的活性を持つことから治療薬としての有用性が高いSP
ARCを効率良く安価にかつ大量に生産することを目的と
する。DISCLOSURE OF THE INVENTION The present invention relates to an SP which has various physiological activities and is therefore highly useful as a therapeutic agent.
The aim is to produce ARC efficiently and inexpensively and in large quantities.
【0006】[0006]
【課題を解決するための手段】本発明者らは、上記課題
を解決すべく鋭意研究を重ねた結果、チオレドキシンを
コードする遺伝子の下流にSPARCをコードする遺伝子を
組込み、これを大腸菌で発現させると、SPARCの有する
生理学的活性を失わず、かつ製剤化しやすい可溶性のタ
ンパク質として大量に生産することができることを見い
だし、本発明を完成するに到った。Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, incorporated a SPARC encoding gene downstream of a thioredoxin encoding gene, and expressed the gene in Escherichia coli. As a result, the present inventors have found that the protein can be produced in a large amount as a soluble protein which is easy to formulate without losing the physiological activity of SPARC, and have completed the present invention.
【0007】すなわち、本発明の第1の発明は、以下の
(a)または(b)に示すタンパク質。 (a) 配列表の配列番号1に示すアミノ酸配列を有する
タンパク質 (b) 配列表の配列番号1に示すアミノ酸配列において
1若しくは数個のアミノ酸が欠失、置換若しくは付加さ
れたアミノ酸配列を有し、かつSPARCの生理学的活性を
有するタンパク質である。 上記のアミノ酸の欠失、置換若しくは付加は、出願前周
知技術である部位特異的変異誘発法により実施すること
ができ、また、1若しくは数個のアミノ酸とは、部位特
異的変異誘発法により欠失、置換若しくは付加できる程
度の数のアミノ酸を意味する。That is, the first invention of the present invention is as follows:
The protein shown in (a) or (b). (a) a protein having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing; And a protein having the physiological activity of SPARC. The deletion, substitution or addition of the above amino acids can be carried out by a site-directed mutagenesis method which is a well-known technique before filing, and one or several amino acids are deleted by the site-directed mutagenesis method. It means the number of amino acids that can be lost, substituted or added.
【0008】また、上記のSPARCの生理学的活性とは、
血管新生、抗細胞接着活性(細胞伸展阻害活性および細
胞球形化活性)、細胞外マトリックス再構成(細胞外マ
トリックス産生の低下と細胞外マトリックス分解性プロ
テアーゼ産生の亢進)、および骨形成(鉱化作用)等を
いう。本発明の第2の発明は、上記タンパク質をコード
するDNA、または配列表の配列番号2に記載の塩基配
列の5209番目から6609番目までの塩基配列からなるDN
Aに関する。[0008] The physiological activity of the SPARC is
Angiogenesis, anti-cell adhesion activity (cell spreading inhibitory activity and cell sphering activity), extracellular matrix reconstitution (decreased extracellular matrix production and increased extracellular matrix degrading protease production), and bone formation (mineralization) ). The second invention of the present invention relates to a DNA encoding the protein or a DN consisting of the nucleotide sequence from the 5209th to the 6609th nucleotide of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.
About A.
【0009】本発明の第3の発明は、上記DNAを含有
する組換えベクターを宿主細胞に導入して得られる形質
転換体を培地に培養し、培養物中に該DNAによってコ
ードされるタンパク質を生成蓄積させ、該培養物から該
タンパク質を採取することを特徴とする、SPARC融合タ
ンパク質の製造方法である。以下、本発明を詳細に説明
する。[0009] A third invention of the present invention is to transform a transformant obtained by introducing a recombinant vector containing the above-mentioned DNA into a host cell in a medium, and to prepare a protein encoded by the DNA in a culture. A method for producing a SPARC fusion protein, comprising producing and accumulating the protein, and collecting the protein from the culture. Hereinafter, the present invention will be described in detail.
【0010】[0010]
【発明の実施の形態】本発明におけるSPARC融合タンパ
ク質とはSPARCとチオレドキシンとの融合タンパク質で
ある。このSPARC融合タンパク質は、例えば、チオレド
キシン遺伝子を含有する市販の発現ベクターを利用し、
チオレドキシン遺伝子の下流にあるマルチクローニング
サイトにSPARCをコードするDNA(以下、SPARC遺伝子
という)を挿入し、これを大腸菌等において大量に発現
させることによって調製することができる。以下、この
調製について具体的に説明する。BEST MODE FOR CARRYING OUT THE INVENTION The SPARC fusion protein in the present invention is a fusion protein of SPARC and thioredoxin. This SPARC fusion protein uses, for example, a commercially available expression vector containing a thioredoxin gene,
It can be prepared by inserting a SPARC-encoding DNA (hereinafter referred to as a SPARC gene) into a multicloning site downstream of the thioredoxin gene and expressing it in E. coli or the like in a large amount. Hereinafter, this preparation is specifically described.
【0011】[1] SPARC遺伝子のクローニング (1) SPARC cDNA 断片の増幅 本発明において、SPARC遺伝子のクローニングは、RT-PC
R法により行う。まず、SPARCが発現している組織または
細胞、例えばマウス大脳皮質から全RNAを調製する。全R
NAを調製する方法としては、酸性チオシアン酸グアニジ
ン・フェノール・クロロホルム(AGPC)法 (P.Chomczyn
ski and N.Sacchi, Analytical Biochemistry, 162 : 1
56, 1987) 、チオシアン酸グアニジン−トリフルオロ酢
酸セシウム法 (H.Okayama et al., Methods in Enzymol
ogy, 154 : 3, 1987) 等が挙げられる。この全RNAを用
いてJ.Sambrookらの方法(J.Sambrook et al., Molecula
rCloning, A Laboratory Manual, Second Edition, Col
d Spring Harbor Laboratory Press, 1989) 等に記載の
方法で逆転写酵素の反応を行い、RNA中のmRNAをcDNAに
変換する。次に、このcDNAを鋳型にし、既に報告されて
いるSPARCのcDNAの塩基配列(J.H. McVey et al., Jour
nal of Biochemistry, 263 : 11111-11116,1988)に基
づいたプライマーを用いてPCRを行い、SPARCのcDNA
断片を増幅する。[1] Cloning of SPARC Gene (1) Amplification of SPARC cDNA Fragment In the present invention, the cloning of SPARC gene is performed by RT-PC
Perform by the R method. First, total RNA is prepared from a tissue or cell expressing SPARC, for example, mouse cerebral cortex. All R
As a method for preparing NA, guanidine acid thiocyanate-phenol-chloroform (AGPC) method (P. Chomczyn)
ski and N. Sacchi, Analytical Biochemistry, 162: 1
56, 1987), guanidine thiocyanate-cesium trifluoroacetate method (H. Okayama et al., Methods in Enzymol
ogy, 154: 3, 1987). Using this total RNA, the method of J. Sambrook et al. (J. Sambrook et al., Molecula
rCloning, A Laboratory Manual, Second Edition, Col
d Spring Harbor Laboratory Press, 1989) and the like, and the mRNA in the RNA is converted to cDNA by performing a reverse transcriptase reaction. Next, using this cDNA as a template, the base sequence of the previously reported SPARC cDNA (JH McVey et al., Journey
nal of Biochemistry, 263: 11111-11116, 1988), and PCR was performed using SPARC cDNA.
Amplify the fragment.
【0012】(2) 増幅断片の塩基配列決定 増幅した断片は、サイクルシークエンス法によりその塩
基配列を決定する。まず、(1) で増幅した断片を、pBlu
escript T-ベクター、pBluescriptII T-ベクター、pT7B
lue T-ベクター、pT7Blue-2 T-ベクター等のTAクロー
ニング用のベクターにサブクローニングし、T7プライマ
ー、T3プライマー、M13(-20)プライマー、リバースプラ
イマー、SKプライマー、KSプライマー、R-20merプライ
マー、U-19merプライマー等のプライマーの存在下、鋳
型DNAを熱変性し、プライマーをアニーリングし、蛍
光標識ddNTP によるシークエンス反応を1サイクルと
し、これを30サイクル程度繰り返す。シークエンス反応
は、Taq ポリメラーゼ等の耐熱性ポリメラーゼを使用
し、市販のPCR用のサーマルサイクラーを用いて行う
ことができる。(2) Determination of Base Sequence of Amplified Fragment The base sequence of the amplified fragment is determined by a cycle sequence method. First, the fragment amplified in (1) is
escript T-vector, pBluescriptII T-vector, pT7B
lue T-vector, pT7Blue-2 Subcloned into a vector for TA cloning such as T-vector, T7 primer, T3 primer, M13 (-20) primer, reverse primer, SK primer, KS primer, R-20mer primer, U In the presence of a primer such as a -19-mer primer, the template DNA is heat-denatured, the primer is annealed, and the sequencing reaction with fluorescently labeled ddNTP is made one cycle, and this cycle is repeated about 30 cycles. The sequencing reaction can be performed using a thermostable polymerase such as Taq polymerase and a commercially available thermal cycler for PCR.
【0013】[2] SPARC融合タンパク質発現用プラスミ
ドベクターの作製 本発明においてSPARC融合タンパク質発現用に用いるこ
とのできるプラスミドベクターとしては、宿主細胞内で
複製可能であること、適当な形質転換マーカー遺伝子、
例えばアンピシリン抵抗性遺伝子、カナマイシン抵抗性
遺伝子、テトランサイクリン抵抗性遺伝子等をもつこ
と、導入した宿主細胞で機能するプロモーターの制御
下、チオレドキシン遺伝子の下流にSPARC遺伝子を接続
した時にチオレドキシンと融合した状態でSPARCを発現
させることのできるものであること、望ましくは誘導物
質が存在しない場合の発現レベルが低く、発現誘導時の
レベルが高い誘導ベクターであること、という条件を具
備するものであればいかなるものでもよい。また、チオ
レドキシン-SPARC cDNA 挿入のため、適当な制限酵素サ
イトがあることが望ましい。このようなベクターとして
は、宿主細胞が大腸菌である場合は、例えばpETベクタ
ー(Novagen社製) 、pTrxFUSベクター(Invitrogen社製)
、pCYBベクター(NEW ENGLAMD Bio Labs社製) 等が、宿
主細胞が酵母である場合は、例えばpESP-1発現ベクター
(STRATAGENE社製) 、pAUR123ベクター(宝酒造社製)、
pPICベクター(Invitrogen社製) 等が、また宿主細胞が
動物細胞である場合は、例えばpMAM-neo発現ベクター
(CLONTECH社製) 、pCDNA3.1ベクター(Invitrogen社製)
、pBK-CMVベクター (STRATAGENE社製) 等が、宿主細胞
が昆虫細胞である場合は、例えばpBacPAKベクター (CLO
NTECH社製) 、pAcUW31ベクター(CLONTECH社製) 、pAcP
(+)IE1ベクター(Novagen社製) 等がそれぞれ挙げられ
る。[2] Preparation of Plasmid Vector for Expression of SPARC Fusion Protein In the present invention, plasmid vectors that can be used for expression of a SPARC fusion protein include those capable of replicating in a host cell, an appropriate transformation marker gene,
For example, having the ampicillin resistance gene, kanamycin resistance gene, tetranecycline resistance gene, etc., under the control of a promoter that functions in the introduced host cell, in the state fused with thioredoxin when the SPARC gene is connected downstream of the thioredoxin gene Any substance that satisfies the condition that it can express SPARC, and desirably an induction vector that has a low expression level in the absence of an inducer and a high expression induction level. May be. In addition, it is desirable to have an appropriate restriction enzyme site for inserting thioredoxin-SPARC cDNA. As such a vector, when the host cell is Escherichia coli, for example, pET vector (Novagen), pTrxFUS vector (Invitrogen)
, PCYB vector (manufactured by NEW ENGLAMD Bio Labs), etc., when the host cell is yeast, for example, pESP-1 expression vector
(STRATAGENE), pAUR123 vector (Takara Shuzo),
pPIC vector (manufactured by Invitrogen) or the like, or when the host cell is an animal cell, for example, a pMAM-neo expression vector
(Manufactured by CLONTECH), pCDNA3.1 vector (manufactured by Invitrogen)
, PBK-CMV vector (manufactured by STRATAGENE) or the like, when the host cell is an insect cell, for example, pBacPAK vector (CLO
NTECH), pAcUW31 vector (CLONTECH), pAcP
(+) IE1 vector (Novagen) and the like.
【0014】プロモーターは、上記ベクターを導入する
各々の宿主細胞で機能しうるものであれば特に限定され
ないが、大腸菌を宿主細胞として用いる場合は、例え
ば、T7プロモーター、T5プロモーター、λPLプロモータ
ー、trpプロモーター、tacプロモーター、lacプロモー
ター等が、酵母を宿主細胞として用いる場合は、例え
ば、PGKプロモーター、ADH1プロモーター、Gal1-Gal10
プロモーター、PHO5プロモーター等が、動物細胞を宿主
細胞として用いる場合は、例えば、SV40プロモーター、
CMVプロモーター、CAGプロモーター、SRαプロモータ
ー、EF1αプロモーター、AMLプロモーター、MMTVプロモ
ーター、MTIIプロモーター等が、昆虫細胞を宿主細胞と
して用いる場合は、例えば、P10プロモーター、ポリヘ
ドロンプロモーター等がそれぞれ挙げられる。[0014] The promoter is not particularly limited as long as it can function in each of the host cell for introducing the vector, when Escherichia coli is used as a host cell, for example, T7 promoter, T5 promoter, .lambda.P L promoter, trp When yeast is used as a host cell, for example, PGK promoter, ADH1 promoter, Gal1-Gal10
Promoter, PHO5 promoter and the like, when using animal cells as host cells, for example, SV40 promoter,
When CMV promoter, CAG promoter, SRα promoter, EF1α promoter, AML promoter, MMTV promoter, MTII promoter, etc. are used as insect cells as host cells, for example, P10 promoter, polyhedron promoter, etc. are exemplified.
【0015】また、適当なプロモーターの制御下に、チ
オレドキシン遺伝子が既に挿入されている市販の発現ベ
クターを利用してもよく、例えば、T7プロモーターを有
するpET32a(+) ベクター(Novagen社製) が好適に使用さ
れる。上記の発現用プラスミドベクターに、SPARC cDNA
断片をチオレドキシン遺伝子のC末側の制限酵素サイト
にタンパク質のフレームが合うように挿入し、組換えD
NAを得ることができる。A commercially available expression vector into which a thioredoxin gene has been inserted under the control of an appropriate promoter may be used. For example, a pET32a (+) vector (manufactured by Novagen) having a T7 promoter is preferable. Used for SPARC cDNA is added to the above expression plasmid vector.
The fragment was inserted into the restriction enzyme site at the C-terminus of the thioredoxin gene so that the protein frame matched it.
NA can be obtained.
【0016】得られた組換えDNAを用いて、宿主細胞
を形質転換することにより、形質転換体を作製する。本
発明においてSPARC融合タンパク質を生産するための宿
主細胞としては、大腸菌、枯草菌、酵母、動物、昆虫細
胞等を用いることができるが、大腸菌が好ましい。具体
的には、Escherichia coli JM109、HB101 、BL21、Nova
Blue、AD494 、B834、HMS174、BLR 等を用いることがで
きる。また、上記株でもlon遺伝子欠損株等のプロテア
ーゼ欠損株を用いることが好ましい。形質転換は、例え
ば塩化カルシウム法、エレクトロポレーション法、塩化
ルビジウム法、リポフェクション法、DEAE−デキストラ
ン法、リチウム法、スフェロプラスト法、ウイルス等に
より行う。A transformant is prepared by transforming a host cell with the obtained recombinant DNA. In the present invention, Escherichia coli, Bacillus subtilis, yeast, animals, insect cells and the like can be used as host cells for producing a SPARC fusion protein, but Escherichia coli is preferred. Specifically, Escherichia coli JM109, HB101, BL21, Nova
Blue, AD494, B834, HMS174, BLR and the like can be used. It is also preferable to use a protease-deficient strain such as a lon gene-deficient strain among the above strains. Transformation is performed by, for example, the calcium chloride method, electroporation method, rubidium chloride method, lipofection method, DEAE-dextran method, lithium method, spheroplast method, virus and the like.
【0017】[3] SPARC融合タンパク質の生産 以上のようにして得られた形質転換体を培地に培養し、
培養物中にタンパク質を生成蓄積させ、これを採取する
ことにより、本発明のSPARC融合タンパク質を得ること
ができる。本発明の形質転換体を培地に培養する方法
は、その宿主細胞の培養に用いられる通常の方法に従っ
て行うことができる。[3] Production of SPARC fusion protein The transformant obtained as described above is cultured in a medium,
The SPARC fusion protein of the present invention can be obtained by producing and accumulating the protein in the culture and collecting the protein. The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing the host cell.
【0018】大腸菌等の原核生物あるいは酵母等の真核
生物を宿主として得られた形質転換体を培養する培地と
しては、該生物が資化し得る炭素源、窒素源、無機塩類
等を含有し、形質転換体の培養を効率的に行える培地で
あれば天然培地、合成培地のいずれを用いてもよい。炭
素源としては、該生物が資化し得るものであればよく、
グルコース、フラクトース、スクロース、これらを含有
する糖蜜、デンプンあるいはデンプン加水分解物等の炭
水化物、酢酸、プロピオン酸等の有機酸、エタノール、
プロパノール等のアルコール類等を用いることができ
る。A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant. The carbon source may be any as long as the organism can assimilate,
Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, organic acids such as propionic acid, ethanol,
Alcohols such as propanol can be used.
【0019】窒素源としては、アンモニア、塩化アンモ
ニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸
アンモニウム等の無機酸もしくは有機酸のアンモニウム
塩、その他の含窒素化合物、並びに、ペプトン、肉エキ
ス、酵母エキス、コーンスチープリカー、カゼイン加水
分解物、大豆粕および大豆粕加水分解物、各種発酵菌
体、およびその消化物等を用いることができる。無機物
としては、リン酸第一カリウム、リン酸第二カリウム、
リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウ
ム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウ
ム等を用いることができる。Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn steep. Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used. As inorganic substances, potassium monophosphate, potassium diphosphate,
Magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
【0020】具体的には、LB培地、H培地、SOB 培地、
SOC 培地、NZYM培地、TB培地、YT培地、トリプトン培
地、TYGPN 培地、λ培地、スーパーブロス培地、M9培
地、M63培地、A培地等で行うことができる。培地のp
Hは、6〜8に調節することが適当である。pHの調整
は、無機または有機の酸、アルカリ溶液、尿素、炭酸カ
ルシウム、アンモニア等を用いて行う。培養は、4〜40
℃、好ましくは36〜38℃で、1〜24時間、好ましく2〜
3時間行い、必要により通気や攪拌を加えてもよい。Specifically, LB medium, H medium, SOB medium,
SOC medium, NZYM medium, TB medium, YT medium, tryptone medium, TYGPN medium, λ medium, super broth medium, M9 medium, M63 medium, A medium and the like can be used. Medium p
It is appropriate that H is adjusted to 6 to 8. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. Culture is 4-40
° C, preferably at 36 to 38 ° C, for 1 to 24 hours, preferably 2 to 24 hours.
The reaction may be performed for 3 hours, and if necessary, ventilation or stirring may be added.
【0021】また、培養中必要に応じて、アンピシリン
やテトラサイクリン等の抗生物質を培地に添加してもよ
い。プロモーターとして誘導性のプロモーターを用いた
発現ベクターで形質転換した微生物を培養するときに
は、必要に応じてインデューサーを培地に添加してもよ
い。例えば、lacプロモーターを用いた発現ベクターで
形質転換した微生物を培養するときにはイソプロピル−
β−D−チオガラクトピラノシド等を、trpプロモータ
ーを用いた発現ベクターで形質転換した微生物を培養す
るときにはインドールアクリル酸等を培地に添加しても
よい。[0021] If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-
When culturing a microorganism transformed with β-D-thiogalactopyranoside or the like using an expression vector using a trp promoter, indoleacrylic acid or the like may be added to the medium.
【0022】また、動物細胞を宿主として得られた形質
転換体を培養する培地としては、一般に使用されている
RPMI1640培地、EagleのMEM培地、DMEM培地等、またはこ
れら培地に牛胎児血清等を添加した培地等を用いること
ができる。上記形質転換体の培養液から、発現させたタ
ンパク質を単離精製するためには、通常のタンパク質の
単離、精製法を用いればよい。As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used medium is used.
RPMI 1640 medium, Eagle's MEM medium, DMEM medium, or the like, or a medium obtained by adding fetal bovine serum or the like to these mediums can be used. In order to isolate and purify the expressed protein from the culture of the above-mentioned transformant, a usual protein isolation and purification method may be used.
【0023】本発明のSPARC融合タンパク質は、細胞質
内に溶解状態で発現しているので、培養終了後、細胞を
遠心分離により回収し水系緩衝液にけん濁後、超音波破
砕機、フレンチプレス、マントンガウリンホモゲナイザ
ー、ダイノミル等により細胞を破砕し、無細胞抽出液を
得る。該無細胞抽出液を遠心分離することにより得られ
た上清から、通常の酵素の単離精製法、即ち、溶媒抽出
法、硫安等による塩析法、脱塩法、有機溶媒による沈殿
法、ジエチルアミノエチル(DEAE)−セファロース、等
レジンを用いた陰イオン交換クロマトグラフィー法、S-
Sepharose FF(ファルマシア社製)等のレジンを用いた
陽イオン交換クロマトグラフィー法、ブチルセファロー
ス、フェニルセファロース等のレジンを用いた疎水性ク
ロマトグラフィー法、分子篩を用いたゲルろ過法、His
Bindレジン(Novagen社製) などを用いたアフィニティー
クロマトグラフィー法、クロマトフォーカシング法、等
電点電気泳動等の電気泳動法等の手法を単独あるいは組
み合わせて用い、精製標品を得ることができる。また、
得られたSPARC融合タンパク質は、エンテロキナーゼ(En
terokinase)、トロンビン(Thrombin)により、SPARCのみ
を切り出すこともできる。Since the SPARC fusion protein of the present invention is expressed in a dissolved state in the cytoplasm, the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, and the like. The cells are crushed with a Manton-Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Diethylaminoethyl (DEAE)-Sepharose, anion exchange chromatography using isoresin, S-
Cation exchange chromatography using a resin such as Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose, phenyl sepharose, gel filtration using a molecular sieve, His
A purified sample can be obtained by using an affinity chromatography method using Bind resin (manufactured by Novagen), a chromatofocusing method, or an electrophoresis method such as isoelectric focusing alone or in combination. Also,
The resulting SPARC fusion protein was identified as enterokinase (En
Only SPARC can be cut out with terokinase) or thrombin (Thrombin).
【0024】[0024]
【実施例】次に実施例を挙げて本発明を詳細に説明する
が、本発明はこれに限定されるものではない。 〔実施例1〕 (SPARC融合タンパク質発現用プラスミド
ベクターの作製) (1) Tベクターの作製 D. Marchukらの方法(Nucleic Acids Research, 19 : 1
154, 1990)に準じた。プラスミド Bluescript SK(-) (1
0μg/μl) (STARATAGENE社製) 1.0μl、10×緩衝液H
(宝酒造社製)2.0μl、EcoRV (10 U/μl) 2.0μl、滅菌
水15.0μlを混合して総量を20.0μlに調製し、37℃で2
時間反応させてプラスミドを完全に切断した。エタノー
ル沈殿で生成した沈殿に、10×PCR緩衝液 (PEアプライ
ドシステムズ社製) 4.0μl、10mM dTTP 8.0μl、Ampli
Taq DNA polymerase (5U/μl)(PEアプライドシス
テムズ社製) 2.0μl、滅菌水24.0μlを加えて総量
を40.0μlに調製し、70℃で2時間反応させた。フェノ
ールクロロホルム抽出およびエタノール沈殿による精製
濃縮後、生成した沈殿に滅菌水20μlに溶解させてTベ
クター溶液を得た。尚、Tベクターは4℃下で保存し
た。Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. [Example 1] (Preparation of plasmid vector for expression of SPARC fusion protein) (1) Preparation of T vector The method of D. Marchuk et al. (Nucleic Acids Research, 19: 1)
154, 1990). Plasmid Bluescript SK (-) (1
0 μg / μl) (STARATAGENE) 1.0 μl, 10 × buffer H
(Takara Shuzo Co., Ltd.) 2.0 μl, EcoRV (10 U / μl) 2.0 μl, and sterile water 15.0 μl were mixed to adjust the total volume to 20.0 μl.
The reaction was allowed to proceed for a period of time to completely cut the plasmid. To the precipitate formed by ethanol precipitation, 4.0 μl of 10 × PCR buffer (manufactured by PE Applied Systems), 8.0 μl of 10 mM dTTP, Ampli
2.0 μl of Taq DNA polymerase (5 U / μl) (manufactured by PE Applied Systems) and 24.0 μl of sterilized water were added to adjust the total volume to 40.0 μl, and reacted at 70 ° C. for 2 hours. After phenol chloroform extraction and purification and concentration by ethanol precipitation, the resulting precipitate was dissolved in 20 μl of sterilized water to obtain a T vector solution. The T vector was stored at 4 ° C.
【0025】(2) マウス大脳皮質からの全RNAの抽
出P.Chomczynskiらの方法(Analytical Biochemistry,
162 : 156-159, 1987)に準じて全 RNAを抽出した。マ
ウス大脳皮質を摘出後に液体窒素で凍結し、ハンマーで
破砕して粉末状にした。この粉末75mgをグアニジン溶液
(4Mグアニジンチオシアネート、25mMクエン酸ナトリウ
ム pH7.0、0.5%サルコシルナトリウム、0.1Mメルカプト
エタノール) 500μlに溶解させ、これに2M酢酸ナトリウ
ム溶液 (pH4.0) 50μl、DEPC水飽和フェノール(pH4.0)
50μl 、クロロホルム 100μl を加えて撹拌した後、4
℃で15分間放置した。10,000 gで20分間の遠心分離後に
水層を回収し、これに同容量の冷却したイソプロパノー
ル600μlとグリコーゲン(20μg/ml) 2μl を加えて-20
℃で1時間放置した。16,000 gで10分間の遠心分離によ
り生成した沈殿は、グアニジン溶液 500μlに完全に溶
解させて上記の過程を再度繰り返した。イソプロパノー
ル沈殿で生成した沈殿にDEPC水50μl を加えて溶解させ
全 RNA溶液を得た。最終的にマウス大脳皮質 0.3gから2
54μgの全RNAが得られた。(2) Extraction of total RNA from mouse cerebral cortex The method of P. Chomczynski et al. (Analytical Biochemistry,
162: 156-159, 1987). After the mouse cerebral cortex was removed, it was frozen with liquid nitrogen and crushed with a hammer to form a powder. 75 mg of this powder in guanidine solution
(4 M guanidine thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarcosyl sodium, 0.1 M mercaptoethanol) dissolved in 500 μl, and 50 μl of 2 M sodium acetate solution (pH 4.0), DEPC water-saturated phenol (pH 4.0 )
After adding 50 μl and 100 μl of chloroform and stirring,
Left at ℃ for 15 minutes. After centrifugation at 10,000 g for 20 minutes, the aqueous layer was recovered, and 600 μl of the same volume of cooled isopropanol and 2 μl of glycogen (20 μg / ml) were added thereto.
It was left at 0 ° C. for 1 hour. The precipitate formed by centrifugation at 16,000 g for 10 minutes was completely dissolved in 500 μl of a guanidine solution, and the above process was repeated again. To the precipitate generated by isopropanol precipitation, 50 μl of DEPC water was added and dissolved to obtain a total RNA solution. Finally from mouse cerebral cortex 0.3g to 2
54 μg of total RNA was obtained.
【0026】(3) RT−PCR法によるマウスSPARC遺
伝子のクローニング(3-1) 一本鎖cDNA合成(2) で得た全
RNA(2.5 μg/μl) 1.2μl にランダムプライマー(100ng
/μl) 1.0μl、DEPC処理滅菌水2.8μlを加えて総量を5.
0μlに調製し、70℃で10分間の熱変性処理をした。この
溶液に5×緩衝液 (GIBCO BRL社製)2.0μl、0.1Mジチオ
スレイトール 2.0μl、1.25mM dNTP 8.0μl、SUPERSCRI
PTTM II Reverse Transcriptase (200U/μl) 1.0μlを
加えて総量を20.0μlに調製し、42℃で2時間反応させ
た。さらに、70℃で15分間の加熱処理により逆転写酵素
を完全に失活させて一本鎖cDNA溶液を得た。(3) Cloning of mouse SPARC gene by RT-PCR (3-1) Synthesis of single-stranded cDNA (2)
RNA (2.5 μg / μl) 1.2 μl with random primer (100 ng)
/ μl) 1.0 μl, 2.8 μl of DEPC-treated sterilized water to add a total volume of
It was adjusted to 0 μl and subjected to a heat denaturation treatment at 70 ° C. for 10 minutes. 2.0 μl of 5 × buffer (GIBCO BRL), 2.0 μl of 0.1 M dithiothreitol, 8.0 μl of 1.25 mM dNTP, SUPERSCRI
The total volume was adjusted to 20.0 μl by adding 1.0 μl of PT ™ II Reverse Transcriptase (200 U / μl), and reacted at 42 ° C. for 2 hours. The reverse transcriptase was completely inactivated by heating at 70 ° C. for 15 minutes to obtain a single-stranded cDNA solution.
【0027】(3-2) PCR反応 (3-1) で得た一本鎖cDNA溶液 5.0μlに 10×PCR緩衝液
(PEアプライドシステムズ社製) 2.5μl、Ampli Taq DNA
polymerase (5U/μl ) (PEアプライドシステムズ社
製)0.25μl、1.25mM dNTP 8.0μl、60ng/μl プライマ
ー1 (5'-CCGAGAGTTCCCAGCATCAT-3' ; 20mer/配列表の
配列番号3)およびプライマー2 (5'-TCAAACCAATTCACC
AGTCT-3' ; 20mer/配列表の配列番号4)を各々2.5μ
l、滅菌水4.25μlを加えて総量を25.0μlに調製して反
応液とした。次に、DNA Thermal cycler PJ2000 Model
480 (PEアプライドシステムズ社)を用いてPCR(変性
反応;94℃、30秒、アニーリング反応;50℃、45秒、合
成反応 ; 72℃、90秒;35サイクル)、続いて72℃で10
分間伸長反応を行った。約1750bpのPCR産物を1%ア
ガロースゲル電気泳動により分離精製し、ウルトラフリ
ー MC フィルター(ミリポア社製)を用いてゲルより回
収した。回収したPCR産物は DNA Ligation Kit Ver.
2(宝酒造社製)を用いて上記Tベクターにサブクロー
ニングした。尚、プライマーはマウスSPARC cDNA塩基配
列(J.H. McVey et. al. Journal of Biochemistry 26
3 , 11111-11116, 1988)の一部に対応するオリゴヌク
レオチド配列を化学合成することにより作製した。プラ
イマー1および 2は各々SPARC cDNA塩基配列の72-91な
らびに1801-1820に対応するオリゴヌクレオチド配列で
ある。(3-2) PCR reaction 10 × PCR buffer was added to 5.0 μl of the single-stranded cDNA solution obtained in (3-1).
(Manufactured by PE Applied Systems) 2.5 μl, Ampli Taq DNA
polymerase (5U / μl) (manufactured by PE Applied Systems) 0.25 μl, 1.25 mM dNTP 8.0 μl, 60 ng / μl Primer 1 (5′-CCGAGAGTTCCCAGCATCAT-3 ′; 20mer / SEQ ID NO: 3 in Sequence Listing) and Primer 2 (5 '-TCAAACCAATTCACC
AGTCT-3 '; 20mer / SEQ ID NO: 4)
l, 4.25 μl of sterilized water was added to adjust the total volume to 25.0 μl, and the mixture was used as a reaction solution. Next, DNA Thermal cycler PJ2000 Model
PCR using 480 (PE Applied Systems) (denaturation reaction; 94 ° C., 30 seconds, annealing reaction; 50 ° C., 45 seconds, synthesis reaction; 72 ° C., 90 seconds; 35 cycles), followed by 10 minutes at 72 ° C.
The extension reaction was performed for minutes. A PCR product of about 1750 bp was separated and purified by 1% agarose gel electrophoresis, and recovered from the gel using an ultra-free MC filter (Millipore). The recovered PCR product is DNA Ligation Kit Ver.
2 (manufactured by Takara Shuzo) and subcloned into the T vector. The primer was a mouse SPARC cDNA base sequence (JH McVey et. Al. Journal of Biochemistry 26).
3, 11111-11116, 1988). Primers 1 and 2 are oligonucleotide sequences corresponding to the SPARC cDNA nucleotide sequences 72-91 and 1801-1820, respectively.
【0028】(3-3) PCR産物のcDNA塩基配列決定 上記PCR産物をサブクローニングしたTベクター(pB
SK-SPARCベクター)はサイクルシークエンス法によりcD
NA塩基配列を決定した。QIAGEN plasmid Minikit (フナ
コシ社製)により精製した上記Tベクター1.5μg を鋳
型として使用し、PRISM Ready Reaction Terminator Cy
cle Sequencing Kit (PEアプライドシステムズ社製)を
用いてシークエンス試料を作製した。尚、PCRはDNA
Thermalcycler PJ2000 Model 480 (PEアプライドシステ
ムズ社)を用い、変性反応;96℃、30秒、アニーリング
反応;50℃、15秒、合成反応 ; 60℃、240秒;25サイク
ルの条件下で行った。PCR産物は、エタノール沈殿に
よって未反応物を除去後、変性溶液(5mg/ml ブルーデ
キストラン、8.3mM EDTA pH 8.0、83.3% ホルムアミ
ド)4.0μlに溶解させて90℃で2分間の熱変性処理を行
った。その後直ちにABI 373Sシーケンサー(PEアプライ
ドシステムズ社製)を用いてcDNA塩基配列の解析を行
い、PCR産物がマウスSPARC cDNA塩基配列 (72-1820)
を有することを確認した。(3-3) Determination of cDNA base sequence of PCR product T vector (pB
SK-SPARC vector)
The NA base sequence was determined. Using 1.5 μg of the above T vector purified by QIAGEN plasmid Minikit (Funakoshi) as a template, PRISM Ready Reaction Terminator Cy
Sequence samples were prepared using the cle Sequencing Kit (manufactured by PE Applied Systems). PCR is DNA
Using Thermalcycler PJ2000 Model 480 (PE Applied Systems), denaturation reaction: 96 ° C, 30 seconds, annealing reaction: 50 ° C, 15 seconds, synthesis reaction; 60 ° C, 240 seconds; 25 cycles were performed. After removing unreacted products by ethanol precipitation, the PCR product is dissolved in 4.0 μl of a denaturing solution (5 mg / ml blue dextran, 8.3 mM EDTA pH 8.0, 83.3% formamide) and subjected to heat denaturation treatment at 90 ° C. for 2 minutes. Was. Immediately thereafter, the cDNA base sequence was analyzed using the ABI 373S sequencer (manufactured by PE Applied Systems), and the PCR product was used as the mouse SPARC cDNA base sequence (72-1820).
Was confirmed.
【0029】(4) pGEX 4T-1ベクターへのサブクローニ
ング (3) で作製した pBSK-SPARC ベクター(6.25μg/μl) 10
μl に10×緩衝液4(NEB社製) 10μl、BamHI 2μl、Xho
I (12 U/μl) 2μl、滅菌水 76μl を加えて総量を100
μlにし、37℃で2時間反応させた。約1800bpのBamHI-X
hoI挿入断片は、1%アガロースゲル電気泳動により分
離精製後にGENE CLEAN II kit(フナコシ社製) を用い
てゲルから回収してTE溶液 (Tris-HCl pH 8.0、0.5mM E
DTA pH 8.0) 48μl に溶解させた。次に、このBamHI-Xh
oI挿入断片溶液 42μlに10×緩衝液4(NEB社製) 10μ
l、BspHI (10 U/μl) 2μl、SmaI (10 U/ μl) 2μl 、
滅菌水44μl を加えて総量を100μlにし、37℃で2時間
反応させた。約1500bpのBspHI-SmaI挿入断片は、1%ア
ガロースゲル電気泳動により分離精製後にGENE CLEAN I
I kit(フナコシ社製)を用いてゲルから回収しTE溶液
48μl に溶解させた。このBspHI-SmaI挿入断片(960ng)
はDNA Blunting Kit(宝酒造社製)を用いて平滑末端化
し、TE溶液10μlに溶解させて平滑末端化挿入断片溶液
を得た。(4) Subcloning into pGEX 4T-1 vector pBSK-SPARC vector (6.25 μg / μl) prepared in (3)
Add 10 × buffer 4 (NEB) 10 μl, BamHI 2 μl, Xho
Add 2 μl of I (12 U / μl) and 76 μl of sterile water to make a total volume of 100
μl and reacted at 37 ° C. for 2 hours. BamHI-X of about 1800bp
The hoI inserted fragment was separated and purified by 1% agarose gel electrophoresis, recovered from the gel using GENE CLEAN II kit (manufactured by Funakoshi), and TE solution (Tris-HCl pH 8.0, 0.5 mM E
DTA pH 8.0). Next, this BamHI-Xh
oI Insert Fragment Solution 10 × Buffer 4 (NEB) 10 μl in 42 μl
l, BspHI (10 U / μl) 2 μl, SmaI (10 U / μl) 2 μl,
The total volume was adjusted to 100 μl by adding 44 μl of sterilized water, and the reaction was carried out at 37 ° C. for 2 hours. The BspHI-SmaI insert of about 1500 bp was separated and purified by 1% agarose gel electrophoresis, followed by GENE CLEAN I
Collect from the gel using I kit (Funakoshi) and extract TE solution
Dissolved in 48 μl. This BspHI-SmaI insert (960 ng)
Was blunt-ended using DNA Blunting Kit (Takara Shuzo) and dissolved in 10 μl of TE solution to obtain a blunt-ended insert fragment solution.
【0030】一方、pGEX-4T1ベクター (500ng/μl) (Ph
armacia Biotech社製) 2μl に10×緩衝液4(NEB社製)
2μl、SmaI (10 U/μl) 1μl 、滅菌水 15μl を加えて
総量を20μlにし、37℃で2時間反応させた。フェノー
ルクロロホルム抽出およびエタノール沈殿による精製濃
縮後、生成した沈殿に10×脱リン酸化緩衝液 (宝酒造社
製)5μl 、子牛小腸由来アルカリフォスファターゼ (10
U/μl) 1μl 、滅菌水 44μl を加えて総量を50μlに
し、37℃で15分間反応させて脱リン酸化処理を施した。
52℃で15分間の熱処理を行って脱リン酸化酵素を失活さ
せた後、ェノールクロロホルム抽出およびエタノール沈
殿による精製濃縮を行った。生成した沈殿はTE溶液 20
μl に溶解させて脱リン酸化処理pGEX-4T1ベクター溶液
を得た。On the other hand, the pGEX-4T1 vector (500 ng / μl) (Ph
armacia Biotech) 10 μl buffer 4 (NEB) in 2 μl
2 μl, 1 μl of SmaI (10 U / μl) and 15 μl of sterilized water were added to make a total volume of 20 μl, and reacted at 37 ° C. for 2 hours. After phenol chloroform extraction and purification and concentration by ethanol precipitation, 5 μl of 10 × dephosphorylation buffer (Takara Shuzo Co., Ltd.) and calf small intestine-derived alkaline phosphatase (10
(U / μl) 1 μl and 44 μl of sterile water were added to make a total volume of 50 μl, and the mixture was reacted at 37 ° C. for 15 minutes for dephosphorylation.
After a heat treatment at 52 ° C. for 15 minutes to deactivate the phosphatase, purification and concentration by enol chloroform extraction and ethanol precipitation were performed. The resulting precipitate is a TE solution 20
The resultant was dissolved in μl to obtain a dephosphorylated pGEX-4T1 vector solution.
【0031】最後に、脱リン酸化処理pGEX-4T1ベクター
溶液 20μlと平滑末端化挿入断片溶液10μlを混合後に
エタノール沈殿による濃縮を行い、DNA Ligation Kit V
er.2(宝酒造社製)によりpGEX-4T1ベクターのSmaI部位
にBspHI-SmaI平滑末端化挿入断片を連結させてpGEX-4T-
SPARC ベクターを得た。尚、BspHI-SmaI平滑末端化挿入
断片がpGEX-4T1ベクターに予定どうり組み込まれている
ことは、pGEX-4T-SPARC ベクターのBamHI-XhoI挿入断片
をpBluescript SK(-) ベクター(STRATAGENE社製) のBam
HI-XhoI部位にサブクローニングしてcDNA塩基配列解析
を行うことにより確認した。Finally, after mixing 20 μl of the dephosphorylated pGEX-4T1 vector solution and 10 μl of the blunt-ended insert fragment solution, the mixture was concentrated by ethanol precipitation, and the DNA Ligation Kit V
er.2 (manufactured by Takara Shuzo Co., Ltd.) to ligate the BspHI-SmaI blunt-ended insert to the Smal site of the pGEX-4T1 vector
SPARC vector was obtained. In addition, the fact that the BspHI-SmaI blunt-ended insert fragment was incorporated into the pGEX-4T1 vector as planned, means that the BamHI-XhoI insert fragment of the pGEX-4T-SPARC vector was converted into a pBluescript SK (-) vector (manufactured by STRATAGENE). Bam
It was confirmed by subcloning into the HI-XhoI site and performing cDNA base sequence analysis.
【0032】(5) pET32a(+)-SPARCベクターの作製 (4) で作製したpGEX-4T-SPARC ベクター (0.25μg/μl)
40μl に10×緩衝液K(宝酒造社製)10μl、BamHI (1
0 U/μl) 2μl、XhoI (12 U/μl) 2μl、滅菌水46μl
を加えて総量を100μlにし、37℃で2時間反応させた。
BamHI-XhoI挿入断片を1%アガロースゲル電気泳動によ
り分離精製し、GENE CLEAN II kit(フナコシ社製)を
用いてゲルよりBamHI-XhoI挿入断片を回収し、TE溶液 4
8μl に溶解させた。pET32a(+)ベクター(Novagen社
製)のBamHI-XhoI部位へサブクローニングした。最終的
に得られたpET32a(+)-SPARCベクターはcDNA塩基配列解
析を行って遺伝子読み枠が合っていることを確認した。
尚、pET32a(+)-SPARCベクターの全cDNA塩基配列は配列
番号2に、このベクターにより生産されるSPARC融合タ
ンパク質の全アミノ酸配列は配列番号1および図1に示
す。また、以上のpET32a(+)-SPARCベクターの構築の概
略を図2に示す。(5) Preparation of pET32a (+)-SPARC vector pGEX-4T-SPARC vector (0.25 μg / μl) prepared in (4)
10 μl of 10 × buffer K (Takara Shuzo) in 40 μl, BamHI (1
0 U / μl) 2 μl, XhoI (12 U / μl) 2 μl, sterile water 46 μl
Was added to make the total volume 100 μl, and the mixture was reacted at 37 ° C. for 2 hours.
The BamHI-XhoI inserted fragment was separated and purified by 1% agarose gel electrophoresis, and the BamHI-XhoI inserted fragment was recovered from the gel using a GENE CLEAN II kit (Funakoshi), and the TE solution 4
Dissolved in 8 μl. It was subcloned into the BamHI-XhoI site of the pET32a (+) vector (Novagen). The finally obtained pET32a (+)-SPARC vector was analyzed for cDNA base sequence to confirm that the gene reading frame was in agreement.
The entire cDNA base sequence of the pET32a (+)-SPARC vector is shown in SEQ ID NO: 2, and the entire amino acid sequence of the SPARC fusion protein produced by this vector is shown in SEQ ID NO: 1 and FIG. FIG. 2 shows the outline of the construction of the pET32a (+)-SPARC vector.
【0033】〔実施例2〕(SPARC融合タンパク質の生
産) 実施例1にて調製したpET32a(+)-SPARCベクターにて大
腸菌AD494(DE3)を形質転換した。この大腸菌
をOD560=0.6 まで37℃で3時間、30μg/mlカナマイシン
を含むLB溶液にて培養後、1mM IPTG存在下、20℃で6
時間振盪培養を行った。菌体は、遠心分離(6,000×g, 3
0 分) により回収し、緩衝液A(5mM Imidazole, 500mM
NaCl, 20mM Tris-HCl, pH7.9)20mlに懸濁させて超音波
処理を行い、遠心分離(30,000×g, 30 分) により可溶
性画分を回収した。この可溶性画分をHis Bind Resinカ
ラム(Novagnen 社) に添加後、緩衝液B(1M Imidazol
e,500mM NaCl, 20mM Tris-HCl, pH7.9) にて吸着画分を
溶出させた。溶出液は約1ml に濃縮後、緩衝液C(10mM
Tris-HCl, pH7.4, 100mM NaCl, 0.1% NP-40) で一晩透
析し、ゲル濾過カラム[Superdex 75 (Pharmacia 社製)]
に添加した。緩衝液D(10mM Tris-HCl pH7.4, 100mM N
aCl)を流速1ml/min で展開して5mlごとに分画を行い、
保持時間40分の単一ピーク(画分8-10) を回収し、緩衝
液Bで一晩透析した。この試料を10%SDS-pAGE で分離後
にCBB 染色を行い、精製試料が25kDaの単一バンドで98%
以上の純度を有することを確認した(図3)。尚、収
率はLB培養液1L当たり約1mgであた。[Example 2] (Production of SPARC fusion protein) Escherichia coli AD494 (DE3) was transformed with the pET32a (+)-SPARC vector prepared in Example 1. The Escherichia coli was cultured in an LB solution containing 30 μg / ml kanamycin at 37 ° C. for 3 hours until OD 560 = 0.6, and then incubated at 20 ° C. in the presence of 1 mM IPTG for 6 hours.
Shaking culture was performed for hours. Cells are centrifuged (6,000 × g, 3
0 minutes) and buffer A (5 mM Imidazole, 500 mM
The suspension was suspended in 20 ml of NaCl, 20 mM Tris-HCl, pH 7.9), sonicated, and the soluble fraction was collected by centrifugation (30,000 × g, 30 minutes). After adding this soluble fraction to a His Bind Resin column (Novagnen), buffer B (1M Imidazol) was added.
e, The adsorbed fraction was eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). The eluate was concentrated to about 1 ml, and buffer C (10 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% NP-40) dialyzed overnight, and gel filtration column [Superdex 75 (Pharmacia)]
Was added. Buffer D (10 mM Tris-HCl pH 7.4, 100 mM N
aCl) at a flow rate of 1 ml / min to fractionate every 5 ml,
A single peak (fractions 8-10) with a retention time of 40 minutes was collected and dialyzed against buffer B overnight. After separating this sample with 10% SDS-pAGE, CBB staining was performed.
It was confirmed that it had the above purity (FIG. 3). The yield was about 1 mg / L of the LB culture solution.
【0034】[0034]
【配列表】 SEQUENCE LISTING <110> Director-General of Agency of Industrial Science and Technology <120> SPARC fusion protein and a process for producing the same <130> 11900252 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 467 <212> PRT <213> Mus musculus <400> 1 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro 115 120 125 Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln 130 135 140 His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met 145 150 155 160 Ala Asp Ile Gly Ser Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu 165 170 175 Ala Gly Arg Ala Leu Ala Ala Pro Gln Gln Thr Glu Val Ala Glu Glu 180 185 190 Ile Val Glu Glu Glu Thr Val Val Glu Glu Thr Gly Val Pro Val Gly 195 200 205 Ala Asn Pro Val Gln Val Glu Met Gly Glu Phe Glu Asp Gly Ala Glu 210 215 220 Glu Thr Val Glu Glu Val Val Ala Asp Asn Pro Cys Gln Asn His His 225 230 235 240 Cys Lys His Gly Lys Val Cys Glu Leu Asp Glu Ser Asn Thr Pro Met 245 250 255 Cys Val Cys Gln Asp Pro Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe 260 265 270 Glu Lys Val Cys Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His 275 280 285 Phe Phe Ala Thr Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys 290 295 300 Leu His Leu Asp Tyr Ile Gly Pro Cys Lys Tyr Ile Ala Pro Cys Leu 305 310 315 320 Asp Ser Glu Leu Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys 325 330 335 Asn Val Leu Val Thr Leu Tyr Glu Arg Asp Glu Gly Asn Asn Leu Leu 340 345 350 Thr Glu Lys Gln Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys 355 360 365 Arg Leu Glu Ala Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe 370 375 380 Glu Lys Asn Tyr Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly 385 390 395 400 Gln Leu Asp Gln His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu 405 410 415 Ala Pro Leu Arg Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg 420 425 430 Phe Phe Glu Thr Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Glu 435 440 445 Glu Trp Ala Gly Cys Phe Gly Ile Lys Glu Gln Asp Ile Asn Lys Asp 450 455 460 Leu Val Ile 465 <210> 2 <211> 7375 <212> DNA <213> Mus musculus <220> <221> CDS <222> (5209)..(6609) <400> 2 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacat atg agc gat 5217 Met Ser Asp 1 aaa att att cac ctg act gac gac agt ttt gac acg gat gta ctc aaa 5265 Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys 5 10 15 gcg gac ggg gcg atc ctc gtc gat ttc tgg gca gag tgg tgc ggt ccg 5313 Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro 20 25 30 35 tgc aaa atg atc gcc ccg att ctg gat gaa atc gct gac gaa tat cag 5361 Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln 40 45 50 ggc aaa ctg acc gtt gca aaa ctg aac atc gat caa aac cct ggc act 5409 Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr 55 60 65 gcg ccg aaa tat ggc atc cgt ggt atc ccg act ctg ctg ctg ttc aaa 5457 Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys 70 75 80 aac ggt gaa gtg gcg gca acc aaa gtg ggt gca ctg tct aaa ggt cag 5505 Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln 85 90 95 ttg aaa gag ttc ctc gac gct aac ctg gcc ggt tct ggt tct ggc cat 5553 Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly His 100 105 110 115 atg cac cat cat cat cat cat tct tct ggt ctg gtg cca cgc ggt tct 5601 Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser 120 125 130 ggt atg aaa gaa acc gct gct gct aaa ttc gaa cgc cag cac atg gac 5649 Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp 135 140 145 agc cca gat ctg ggt acc gac gac gac gac aag gcc atg gct gat atc 5697 Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile 150 155 160 gga tcc atg agg gcc tgg atc ttc ttt ctc ctt tgc ctg gcc ggg agg 5745 Gly Ser Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg 165 170 175 gcc ctg gca gcc cct cag cag act gaa gtt gct gag gag ata gtg gag 5793 Ala Leu Ala Ala Pro Gln Gln Thr Glu Val Ala Glu Glu Ile Val Glu 180 185 190 195 gag gaa acc gtg gtg gag gag aca ggg gta cct gtg ggt gcc aac cca 5841 Glu Glu Thr Val Val Glu Glu Thr Gly Val Pro Val Gly Ala Asn Pro 200 205 210 gtc cag gtg gaa atg gga gaa ttt gag gac ggt gca gag gaa acg gtc 5889 Val Gln Val Glu Met Gly Glu Phe Glu Asp Gly Ala Glu Glu Thr Val 215 220 225 gag gag gtg gtg gct gac aac ccc tgc cag aac cat cat tgc aaa cat 5937 Glu Glu Val Val Ala Asp Asn Pro Cys Gln Asn His His Cys Lys His 230 235 240 ggc aag gtg tgt gag ctg gac gag agc aac acc ccc atg tgt gtg tgc 5985 Gly Lys Val Cys Glu Leu Asp Glu Ser Asn Thr Pro Met Cys Val Cys 245 250 255 cag gac ccc acc agc tgc cct gct ccc att ggc gag ttt gag aag gta 6033 Gln Asp Pro Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val 260 265 270 275 tgc agc aat gac aac aag acc ttc gac tct tcc tgc cac ttc ttt gcc 6081 Cys Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala 280 285 290 acc aag tgc acc ctg gag ggc acc aag aag ggc cac aag ctc cac ctg 6129 Thr Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His Leu 295 300 305 gac tac atc gga cca tgc aaa tac atc gcc ccc tgc ctg gat tcc gag 6177 Asp Tyr Ile Gly Pro Cys Lys Tyr Ile Ala Pro Cys Leu Asp Ser Glu 310 315 320 ctg acc gaa ttc cct ctg cgc atg cgt gac tgg ctc aaa aat gtc ctg 6225 Leu Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys Asn Val Leu 325 330 335 gtc acc ttg tac gag aga gat gag ggc aac aac ctc ctc act gag aag 6273 Val Thr Leu Tyr Glu Arg Asp Glu Gly Asn Asn Leu Leu Thr Glu Lys 340 345 350 355 cag aag ctg cgt gtg aag aag atc cat gag aac gag aag cgc ctg gag 6321 Gln Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys Arg Leu Glu 360 365 370 gct gga gac cac ccc gtg gag ctg ttg gcc cga gac ttt gag aag aac 6369 Ala Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn 375 380 385 tac aat atg tac atc ttc cct gtc cac tgg cag ttt ggc cag ctg gat 6417 Tyr Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp 390 395 400 cag cac cct att gat ggg tac ctg tcc cac act gag ctg gcc cca ctg 6465 Gln His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu Ala Pro Leu 405 410 415 cgt gct ccc ctc atc ccc atg gaa cat tgc acc aca cgt ttc ttt gag 6513 Arg Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg Phe Phe Glu 420 425 430 435 acc tgt gac cta gac aac gac aag tac att gcc ctg gag gaa tgg gcc 6561 Thr Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Glu Glu Trp Ala 440 445 450 ggc tgc ttt ggc atc aag gag cag gac atc aac aag gat ctg gtg atc 6609 Gly Cys Phe Gly Ile Lys Glu Gln Asp Ile Asn Lys Asp Leu Val Ile 455 460 465 taagttcacg cctcctgctg cagtcctgaa ctctctccct ctgatgtgtc ccccctccca 6669 ttaccccctt gtttaaaatg tttggatggt tggctgttcc gcctggggat aaggtgctaa 6729 catagattta actgaataca ttaacggtgc taaaaaaaaa aaaaaaacaa ggtaagaaag 6789 aaactagaac ccaagtctca gcattttccc acataactct gaggccatgg cccatccaca 6849 gcctcctggt cccctgcact acccagtgtc tcactggctg tgttggaaac ggacttgtat 6909 aagctcaccg gccacaagca cgagatatct ctagctttca tttctgtttt gcatttgact 6969 cttaacactc acccagactc tgtgcttatt tcattttggg ggatgtgggc tttttcccct 7029 ggtggtttgg agttaggcag agggaagtta cagacacagg tacaaaattt gggtaaagat 7089 actgtgagac ctgaggaccc accagtcaga acccacatgg caagtcttag tagcctaggt 7149 caaggaaaga cagaataatc cagagctgtg gcacacatga cagactccca gcagcccggt 7209 cgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa 7269 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa 7329 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggat 7375 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 1 <400> 3 ccgagagttc ccagcatcat <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 2 <400> 4 tcaaaccaat tcaccagtct [Sequence List] SEQUENCE LISTING <110> Director-General of Agency of Industrial Science and Technology <120> SPARC fusion protein and a process for producing the same <130> 11900252 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 467 <212> PRT <213> Mus musculus <400> 1 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Gly His Met His His His His His Ser Ser Gly Leu Val Pro 115 120 125 Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln 130 135 140 His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met 145 150 155 160 Ala Asp Ile Gly Ser Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu 165 170 175 Ala Gly Arg Ala Leu Ala Ala Pro Gln Gln Thr Glu Val Ala Glu Glu 180 185 190 Ile Val Glu Glu Glu Thr Val Val Glu Glu Thr Gly Val Pro Val Gly 195 200 205 Ala Asn Pro Val Gln Val Glu Met Gly Glu Phe Glu Asp Gly Ala Glu 210 215 220 Glu Thr Val Glu Glu Val Val Ala Asp Asn Pro Cys Gln Asn His His 225 230 235 240 Cys Lys His Gly Lys Val Cys Glu Leu Asp Glu Ser Asn Thr Pro Met 245 250 255 Cys Val Cys Gln Asp Pro Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe 260 265 270 Glu Lys Val Cys Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His 275 280 285 Phe Phe Ala Thr Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys 290 295 300 Leu His Leu Asp Tyr Ile Gly Pro Cys Lys Tyr Ile Ala Pro Cys Leu 305 310 315 320 Asp Ser Glu Leu Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys 325 330 335 Asn Val Leu Val Thr Leu Tyr Glu Arg Asp Glu Gly Asn Asn Leu Leu 340 345 350 Thr Glu Lys Gln Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys 355 360 365 Arg Leu Glu Ala Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe 370 375 380 Glu Lys Asn Tyr Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly 385 390 395 400 400 Gln Leu Asp Gln His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu 405 410 415 Ala Pro Leu Arg Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg 420 425 430 Phe Phe Glu Thr Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Glu 435 440 445 Glu Trp Ala Gly Cys Phe Gly Ile Lys Glu Gln Asp Ile Asn Lys Asp 450 455 460 Leu Val Ile 465 <210> 2 <211> 7375 <212> DNA <213> Mus musculus <220> <221> CDS <222> (5209) .. (6609) <400> 2 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctatt c 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaa aggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720 gcggtttgcg tattgggcgc cagg gtggtt tttcttttca ccagtgagac gggcaacagc 3780 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560 gccagttgtt gtgccacgcg gttgggaat g taattcagct ccgccatcgc cgcttccact 4620 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacat atg agc gat 5217 Met Ser Asp 1 aaa att att cac ctg act gac gac agt ttt gac acg gat gta ctc aaa 5265 Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys 5 10 15 gcg gac ggg gcg atc ctc gtc gat ttc tgg gca gag tgg tgc ggt ccg 5313 Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro 20 25 30 35 tgc aaa atg atc gcc ccg att ctg gat gaa atc gct gac gaa tat cag 5361 Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln 40 45 50 ggc aaa ctg acc gtt gca aaa ctg aac atc gat caa aac cct ggc act 5409 Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr 55 60 65 gcg ccg aaa tat ggc atc cgt ggt atc ccg act ctg ctg ctg aaa 5457 Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys 70 75 80 aac ggt gaa gtg gcg gca acc aaa gtg ggt gca ctg tct aaa ggt cag 5505 Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln 85 90 95 ttg aaa gag ttc ctc gac gct aac ctg gcc ggt tct ggt tct ggc cat 5553 Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly His 100 105 110 115 atg cac cat cat cat cat cat cat tct tct ggt ctg gtg cca cgc ggt tct 5601 Met His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser 120 125 130 ggt atg aaa gaa acc gct gct gct aaa ttc gaa cgc cag cac atg gac 5649 Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp 135 140 145 agc cca gat ctg ggt acc gac gac gac gac aag gcc atg gct gat atc 5697 Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile 150 155 160 gga tcc atg agg gcc tgg atc ttc ttt ctc ctt tgc ctg gcc ggg agg 5745 Gly Ser Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg 165 170 175 gcc ctg gca gcc cct cag cag act gaa gtt gct gtg gag 5793 Ala Leu Ala Ala Pro Gln Gln Thr Glu Val Ala Glu Glu Ile Val Glu 180 185 190 195 gag gaa acc gtg gtg gag gag aca ggg gta cct gtg ggt gcc aac cca 5841 Glu Glu Thr Val Val Glu Glu Thr Gly Val Pro Val Gly Ala Asn Pro 200 205 210 gtc cag gtg gaa atg gga gaa ttt gag gac ggt gca gag gaa acg gtc 5889 Val Gln Val Glu Met Gly Glu Phe Glu Asp Gly Ala Glu Glu Thr Val 215 220 225 gag gag gtg gtg gct gac aac ccc tgc cag aac cat cat tgc aaa cat 5937 Glu Glu Val Val Ala Asp Asn Pro Cys Gln Asn His His Cys Lys His 230 235 240 ggc aag gtg tgt gag ctg gac gag agc aac acc ccc atg tgt gtg tgc 5985 Gly Lys Val Cys Glu Leu Asp Glu Ser Asn Thr Pro Met Cys Val Cys 245 250 255 cag gac ccc acc agc tgc cct gct ccc att ggc gag ttt gag aag gta 6033 Gln Asp Pro Thrr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val 260 265 270 275 tgc agc aat gac aac aag acc ttc gac tct tcc tgc cac ttc ttt gcc 6081 Cys Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala 280 285 290 acc aag tgc acc ctg gag ggc acc aag aag ggc cac aag ctc cac ctg 6129 Thr Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His Leu 295 300 305 gac tac atc gga cca tgc aaa tac atc gcc ccc tgc ctg gat tcc gag 6177 Asp Tyr Ile Gly Cys Lys Tyr Ile Ala Pro Cys Leu Asp Ser Glu 310 315 320 ctg acc gaa ttc cct ctg cgc atg cgt gac tgg ctc aaa aat gtc ctg 6225 Leu Thr Glu Plu Pro Leu Arg Met Arg Asp Trp Lep Lyu Asn Val Leu 325 330 gtc acc ttg tac gag aga gat gag ggc aac aac ctc ctc act gag aag 6273 Val Thr Leu Tyr Glu Arg Asp Glu Gly Asn Asn Leu Leu Thr Glu Lys 340 345 350 355 cag aag ctg cgt gtg aag aag atc cat gag aac ag aag cgc ctg gag 6321 Gln Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys Arg Leu Glu 360 365 370 gct gga gac cac ccc gtg gag ctg ttg gcc cga gac ttt gag aag aac 6369 Ala Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn 375 380 385 tac aat atg tac atc ttc cct gtc cac tgg cag ttt ggc cag ctg gat 6417 Tyr Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp 390 c395 400 cag ac att gat ggg tac ctg tcc cac act gag ctg gcc cca ctg 6465 Gln His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu Ala Pro Leu 405 410 415 cgt gct ccc ctc atc ccc atg gaa cat tgc acc aca cgt ttc ttt gag 6513 Arg Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg Phe Phe Glu 420 425 430 435 acc tgt gac cta gac aac gac aag tac att gcc ctg gag gaa tgg gcc 6561 Thr Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Glu Glu Trp Ala 440 445 450 ggc tgc ttt ggc atc aag gag cag gac atc aac aag gat ctg gtg atc 6609 Gly Cys Phe Gly Ile Lys Glu Gln Asp Ile Asn Lys Asp Leu Val Ile 455 460 465 taagttcacg cgtg cctg cctgaa ctctctccct ctgatgtgtc ccccctccca 6669 ttaccccctt gtttaaaatg tttggatggt tggctgttcc gcctggggat aaggtgctaa 6729 catagattta actgaataca ttaacggtgc taaaaaaaaa aaaaaaacaa ggtaagaaag 6789 aaactagaac ccaagtctca gcattttccc acataactct gaggccatgg cccatccaca 6849 gcctcctggt cccctgcact acccagtgtc tcactggctg tgttggaaac ggacttgtat 6909 aagctcaccg gccacaagca cgagatatct ctagctttca tttctgtttt gcatttgact 6969 cttaacactc acccagactc tgtgcttatt tcattttggg ggatgtgggc tttttcccct 7029 ggtggtttgg agttaggcag agggaagtta cagacacagg tacaaaattt gggtaaagat 7089 actgtgagac ctgaggaccc accagtcaga acccacatgg caagtcttag tagcctaggt 7149 caaggaaaga cagaataatc cagagctgtg gcacacatga cagactccca gcagcccggt 7209 cgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa 7269 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa 7329 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggat 7375 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 1 <400> 3 ccgagagttc ccagcatcat <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 2 <400> 4 tcaaaccaat tcaccagtct
【0035】[0035]
【配列表フリーテキスト】配列番号3は、プライマー1
のヌクレオチド配列(SPARC cDNA塩基配列の72-91に対
応)を示す。配列番号4は、プライマー2のヌクレオチ
ド配列(SPARC cDNA塩基配列の1801-1820に対応)を示
す。[Sequence List Free Text] SEQ ID NO: 3 is primer 1
(Corresponding to the SPARC cDNA nucleotide sequence 72-91). SEQ ID NO: 4 shows the nucleotide sequence of primer 2 (corresponding to SPARC cDNA nucleotide sequence 1801-1820).
【0036】[0036]
【発明の効果】本発明によれば、血管新生、抗細胞接着
活性、ECM再構成、骨形成を初めとする種々の生理学
的活性を有することから研究試薬や医薬品としての利用
が期待されるSPARCを、その生理学的活性を失わず、か
つ医薬として調製しやすい可溶性の融合タンパク質とし
て、効率良く安価にかつ大量に調製できる。Industrial Applicability According to the present invention, SPARC is expected to be used as a research reagent or pharmaceutical because it has various physiological activities such as angiogenesis, anti-cell adhesion activity, ECM reconstitution, and bone formation. Can be efficiently and inexpensively prepared in large quantities as a soluble fusion protein that does not lose its physiological activity and is easily prepared as a medicament.
【図1】 SPARC融合タンパク質の全アミノ酸配列を示
す。FIG. 1 shows the entire amino acid sequence of the SPARC fusion protein.
A :アラニン(Ala) R :アルギニン (Arg) N :アスパラギン (Asn) D :アスパラギン酸 (Asp) C :システイン(Cys) Q :グルタミン (Gln) E :グルタミン酸 (Glu) G :グリシン (Gly) H :ヒスチジン(His) I :イソロイシン(Ile) L :ロイシン (Leu) K :リジン (Lys) M :メチオニン (Met) F :フェニルアラニン (Phe) P :プロリン (Pro) S :セリン (Ser) T :スレオニン (Thr) W :トリプトファン (Trp) Y :チロシン (Tyr) V :バリン (Val) A: Alanine (Ala) R: Arginine (Arg) N: Asparagine (Asn) D: Aspartic acid (Asp) C: Cysteine (Cys) Q: Glutamine (Gln) E: Glutamic acid (Glu) G: Glycine (Gly) H : Histidine (His) I: Isoleucine (Ile) L: Leucine (Leu) K: Lysine (Lys) M: Methionine (Met) F: Phenylalanine (Phe) P: Proline (Pro) S: Serine (Ser) T: Threonine (Thr) W: Tryptophan (Trp) Y: Tyrosine (Tyr) V: Valine (Val)
【図2】 pET32a(+)-SPARCベクターの構築の概略を示
す。FIG. 2 shows an outline of construction of a pET32a (+)-SPARC vector.
【図3】 精製試料のゲル電気泳動(SDS-PAGE) の結果
を示す。FIG. 3 shows the results of gel electrophoresis (SDS-PAGE) of the purified sample.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI C12P 21/02 C12P 21/02 C // A61K 38/00 A61K 37/02 (58)調査した分野(Int.Cl.7,DB名) C12N 15/09 C07K 14/195 C07K 14/435 C07K 14/47 C07K 19/00 C12P 21/02 A61K 38/17 CAPLUS(STN) REGISTRY(STN)──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 identification code FI C12P 21/02 C12P 21/02 C // A61K 38/00 A61K 37/02 (58) Fields surveyed (Int.Cl. 7 , DB name) C12N 15/09 C07K 14/195 C07K 14/435 C07K 14/47 C07K 19/00 C12P 21/02 A61K 38/17 CAPLUS (STN) REGISTRY (STN)
Claims (4)
タンパク質 (b) 配列表の配列番号1に示すアミノ酸配列において
1若しくは数個のアミノ酸が欠失、置換若しくは付加さ
れたアミノ酸配列を有し、かつSPARCの生理学的活性を
有するタンパク質1. A protein represented by the following (a) or (b): (a) a protein having the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing; (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing , And a protein having the physiological activity of SPARC
DNA。2. A DNA encoding the protein according to claim 1.
5209番目から6609番目までの塩基配列からなるDNA。3. The nucleotide sequence of SEQ ID NO: 2 in the sequence listing
DNA consisting of the nucleotide sequence from the 5209th position to the 6609th position.
した組換えベクターを宿主細胞に導入して得られる形質
転換体を培地に培養し、培養物中に該DNAによってコ
ードされるタンパク質を生成蓄積させ、該培養物から該
タンパク質を採取することを特徴とする、SPARC融合タ
ンパク質の製造方法。4. A transformant obtained by introducing a recombinant vector into which a DNA according to claim 2 or 3 is inserted into a host cell, is cultured in a medium, and a protein encoded by the DNA is cultured in the culture. A method for producing a SPARC fusion protein, comprising producing and accumulating the protein, and collecting the protein from the culture.
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JP11049826A JP3012931B1 (en) | 1999-02-26 | 1999-02-26 | SPARC fusion protein and method for producing the same |
US09/513,442 US6387664B1 (en) | 1999-02-26 | 2000-02-25 | Sparc fusion protein and method for producing the same |
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JP11049826A JP3012931B1 (en) | 1999-02-26 | 1999-02-26 | SPARC fusion protein and method for producing the same |
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JP3012931B1 true JP3012931B1 (en) | 2000-02-28 |
JP2000245467A JP2000245467A (en) | 2000-09-12 |
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EP1856255A4 (en) | 2005-02-11 | 2010-01-27 | Univ Southern California | Method of expressing proteins with disulfide bridges |
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