TW206236B - - Google Patents

Description

經濟部屮央橾準局B3C工消费合作杜印製 Λ 6 --- 五、發明説明（'） 本發明係活化重組蛋白質之方法，尤其適用於原核細 胞（prokaryotes)産生之重組蛋白質之活化。 當重組蛋白質以原核細胞來呈現時，此蛋白質通常係 在宿主細胞中産生，其型態為徹溶性之凝聚物〔折射體 (refractile bodies),包涵體（inclusion bodies IB)〕 ，至少有一部分為無活性態，甚至可能被來自宿主細胞 蛋白質之污染。這些蛋白質在被使用之前，如供治療或 診斷之用途，需轉換成其活性型，方可使用。 使重組蛋白質復性（renaturation)之方法很多，如EP-A 0 1 1 4 5 0 6, W0 86/00610,W0 84/03711,US 4 5 3 0 7 8 7 及EP-A 0 241 022中所描述。但是以這些己知之方法來 活化天然的蛋白質，其産率相當低。本發明之目標即改 善重組蛋白質之活化法，以提高其産率。基本上，改善 産率可藉復性條件之選擇來達成。但是，這並非本發明 所訴求之標的。 本發明標的之逹成，係根據在蛋白質之復性過程中， 加入一輔助序列於其N端或/及其C端後，其産率大增 之驚人發現。 本發明因此提供一種方法來辱化重組蛋白霣，尤其是 來自原核細胞之重組蛋白質，因含有部分無活性型，特 別適用本法。本法仍以已知之肋溶法（solubilization) 或/及復性技術來活化蛋白質‘，但此蛋白質之N端或/ 及C端具有2-50傾胺基酸之輔助序列，而此序列之相對 本紙Λ尺度逍用中囲國家猱準(CNS)甲4規格(2)0X297公*) 81. 2. 20,000 Λ 6 η 6 ^,06236五、發明説明（>) 厭水样（relative hydrophobicity)(由表1之各按基酸 2相對厭水性值之和）具有負值。 本發明所稱之"相對厭水性"這艏名詞傜摘自T . E . Creighton(1983),Proteins,Structure and Molecular Principles,W.H.Freeman and Coipany.New York,P.142 .Table 4.4;G.von Heijne and C.Blomberg(1979)Eur. J · B i 〇 c h e in . 9 5 , 1 7 5 - 1 8 1 and Y.Nozaki and C.Tanford (1971) ,J.Biol.Chem.246,2211-2217.各胺基酸之相對 厭水性，偽依據Nozaki/Tranford以水及非極性溶媒〔 如乙醇/二噁烷〕中，胺基酸在此兩種溶液之分配偽數 來/決定。此相對厭水性傜能量之定量，故其單位為仟卡 /摩爾。此相對厭水性如為正值，表示其較傾向非極性 ，亦即為一非極性之胺基酸。相反的，如果此值小於零 (負值）。表示此胺基酸為極性胺基酸，較易溶於水，而 不是非極性溶媒。當此胺基酸由乙醇轉移到水中時，其 結果會使能量釋出。 各胺基酸之相對厭水性之值，编排於下列之表1之中 表1 經濟部中央楯準局員工消t合作杜印製 睡 \ 卡 仟 /( 袢 ) \f/ V17 酸酸胺酸 胺胺白胺 甘白異錁 /i //\ /V ft\ 本紙Λ尺度逍用中β國家《準(CNS)肀4規格(210x297公釐） 81. 2. 20,000 2(θθ^--^ 五、發明説明（3) 丙苯半甲酥絲色酪醯離醛麩組天精晡 /CV /|\ #fv /i λί\ /Κ /V /V /TV /fv /v /V /IV /IV /1* /1 } 酸酸 酸胺胺 胺丙胱 5 5 8 • ·' 0 2 2 ) 酸 ) 酸冬 } } 酸_}丨_胺_門_ 酸酸 胺酸酸酸酸想酸天酸胺冬 硫胺胺胺胺胺胺胺胺鏃門 4 ο 3 4 ο 3 3 3 2 ο 9 5 9 ο (請先閲請背而之注意事項祚填寫本頁) 裝* 線 經濟部中央標準局只工消费合作杜印製 麩負 是之 其性 尤水 ，厭 酸對 胺相 腩其 酸酸 胺胺 胱離 半和 出酸 看胺 的精 顯， 明酸 可冬 中門 表天 。 上，大 由酸較 胺值 * 值善 ，^,負改 時el為之 化(h和箸 活列總顯 之序之有 質助性化 白輔水活 蛋之厭之 组度對質 重長相白 在酸之蛋 基列組 此 在 若 白 C 令肋 蛋en可輔 質 人序 胺卩重 2 序 艏Μ現以 5lg發度 2itl的長 有 i 奇之 且 加驚列 本紙張尺度边用中a國家楳毕(CNS)T4規格(210X297公》) 81. 2. 20,000 經濟部中央標準局员工消费合作社印製Manufactured by the B3C Industrial and Consumer Cooperation Co., Ltd. of the Ministry of Economic Affairs, B3C. Λ 6 --- V. Description of the invention (') The present invention is a method of activating recombinant proteins, especially suitable for the activation of recombinant proteins produced by prokaryotes. When the recombinant protein is presented in prokaryotic cells, the protein is usually produced in the host cell, and its type is completely soluble aggregates [refractile bodies, inclusion bodies (IB)], at least part of which is Inactive, it may even be contaminated by host cell proteins. Before these proteins are used, they need to be converted to their active form before being used for therapeutic or diagnostic purposes. There are many methods for renaturation of recombinant proteins, such as EP-A 0 1 1 4 5 0 6, W0 86/00610, W0 84/03711, US 4 5 3 0 7 8 7 and EP-A 0 241 022 Described. But using these known methods to activate natural proteins, the yield is quite low. The objective of the present invention is to improve the activation method of recombinant protein to increase its yield. Basically, the improvement of productivity can be achieved by the choice of repetitive conditions. However, this is not the subject of the present invention. The success of the subject of the present invention is based on the surprising discovery that after adding an auxiliary sequence to its N-terminus or / and its C-terminus during the renaturation of the protein, the yield is greatly increased. The present invention therefore provides a method to insult the recombinant protein, especially the recombinant protein from prokaryotic cells, because it contains a partially inactive form, and this method is particularly applicable. This method still uses known solubilization (or solubilization) or / and renaturation techniques to activate the protein ', but the N-terminus or / and C-terminus of this protein has an auxiliary sequence of 2-50 amino acids, and this sequence of Relative to the original paper Λ scale, the Chinese National Standard (CNS) Grade 4 (2) 0X297 (*) 81. 2. 20,000 Λ 6 η 6 ^, 06236 V. Description of the invention (>) Relative hydrophobicity (relative hydrophobicity) ) (From the sum of the relative water repellency value of each base acid 2 in Table 1) has a negative value. The term "relatively water-repellent" referred to in the present invention is taken from T.E. Creighton (1983), Proteins, Structure and Molecular Principles, WHFreeman and Coipany. New York, P.142.Table 4.4; G. von Heijne and C. Blomberg (1979) Eur. J. Bioche in. 9 5, 1 7 5-1 8 1 and Y. Nozaki and C. Tanford (1971), J. Biol. Chem. 246, 2211 -2217. The relative water repellency of each amino acid is based on Nozaki / Tranford's pseudo-numbers for the distribution of amino acids in these two solutions in water and non-polar solvents such as ethanol / dioxane. The relative water-repellent energy is quantitative, so the unit is thousands calories / mole. If the relative water repellency is positive, it indicates that it is more non-polar, that is, a non-polar amino acid. Conversely, if this value is less than zero (negative value). It indicates that this amino acid is a polar amino acid and is more soluble in water than a non-polar solvent. When this amino acid is transferred from ethanol to water, the energy is released as a result. The values of the relative water repellency of each amino acid are arranged in the following Table 1 Table 1 Table 1 of the Ministry of Economic Affairs of the Central Bureau of Economic Cooperation of the People ’s Republic of China to cooperate to print and print \ \ Ka Qian / (袢) \ f / V17 acid acid amino acid Amine leucine Ammonia glycyrrhizine / i // \ / V ft \ This paper is used in the Λ scale of the beta country in the national standard (CNS) 4 (210x297 mm) 81. 2. 20,000 2 (θθ ^-^ Fifth, the invention description (3) Propyl benzene half nail crispy color tyrosin aldehyde bran group Tian Jinghan / CV / | \ #fv / i λί \ / Κ / V / V / TV / fv / v / V / IV / IV / 1 * / 1} Acid Acid Amino Acid Amine Alanine 5 5 8 • '0 2 2) Acid) Acid Winter}} Acid_} 丨 _Amine_Gate_ Acid Acid Amino Acid Acid Acid Acid Aspartame, thiamine, amine, amine, amine, amine, amine, door 4 ο 3 4 ο 3 3 3 2 ο 9 5 9 ο (please read the precautions please fill in this page) install * line Ministry of Economic Affairs The Bureau of Standards only consumes co-products to produce bran, which is particularly characteristic of water. The anaerobic acid is very important for the amine phase. On the top, the big acid is better than the amine value * value, ^, when the negative change is el (the h and 箸 活 are listed in the order of the qualitatively assisted Baishui water live egg, the composition of the weight is relatively obvious The sour egg base group is present in Ruobai C so that the rib egg en can be supplemented with a human sequence amine. The order 2 is now at 5lg and has a length of 2itl. It is strange and surprising. National Twelve (CNS) T4 specification (210X297) 81. 2. 20,000 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

^- 五、發明説明（4) 20個胺基酸為宜，尤其以5-20個胺基酸最適合。 此外，此輔肋序列之相對厭水性水性除以胺基酸數目 ，所得之商，宜為- 2.0仟卡/摩爾或更小，尤其宜為-2.5 仟卡/摩爾或更小且以- 2.8仟卡/摩爾或更小，最為適 合。 將輔肋序列加入重組蛋白質，可藉分子生物學領域中 的常用技術來執行。此宜藉搞有上逑負的相對厭水性之 輔助序列之寡核甘酸，加在所欲表現之重組蛋白質之DNA 排序密碼之一端或兩端來完成〇這些DNAH段中，含有一 < 區域，其密碼中含有與所欲表逹之基因，相同之起端或 末端。合成之寡核甘酸則含有一區域，其密碼為輔肋序 列，藉其他之限制斷裂位置，來***這些DNA之片段之 中》S —種方法為以寡核甘酸序列，完全地取代天然之 DNA片段之基因。藉著加入含有輔助序列訊息之方法， 可使重組蛋白質；έ訊息之D N A排列順序得以改變。 當DNA序列欲接合胺基端之輔助序列時，可方便的利 用已適應此表現橄生物之DNA序列之慣用密碼（E.L. Winnacker.Gene and Klone, Verlay Chemie,1985, 224-241)〇 當以大腸捍菌為表現撇生物時，下列之密碼即較適合 下列之胺基酸： 穌胺酸 ’A C A 脯肢酸 CCA 本紙ft疋度逍用中B B家«準(CNS)甲4規格(210x297公:¢) 81. 2. 20,000 206^36 Λ 6 η 6 五、發明説明（r 白胺酸 離胺酸 丙胺酸 麩胺酸^-V. Description of the invention (4) 20 amino acids are suitable, especially 5-20 amino acids are most suitable. In addition, the relative water repellency of this auxiliary rib sequence is divided by the number of amino acids, and the quotient obtained is preferably -2.0 thousand calories / mole or less, especially preferably -2.5 thousand calories / mole or less and -2.8 Thousand calories / mole or less is most suitable. The addition of the auxiliary rib sequence to the recombinant protein can be performed by common techniques in the field of molecular biology. This should be done by adding oligonucleotides with relatively negative auxiliary sequences that are relatively hydrophobic and adding them to one or both ends of the DNA sequencing code of the recombinant protein to be expressed. These DNAH segments contain a < region , The code contains the same beginning or end as the desired gene. Synthetic oligonucleotides contain a region whose code is the auxiliary rib sequence, which is inserted into the fragments of these DNAs by restricting the position of the break. "S-The method is to completely replace the natural DNA with oligonucleotide sequences. Fragment of genes. By adding a message containing an auxiliary sequence, the recombinant protein; the DN A sequence of the message can be changed. When the DNA sequence is to be joined to the auxiliary sequence at the amine end, it is convenient to use the conventional code (EL Winnacker. Gene and Klone, Verlay Chemie, 1985, 224-241) that has been adapted to the DNA sequence of this expression organism. When defending bacteria to show off the organism, the following codes are more suitable for the following amino acids: sulfanilic acid 'ACA probiotic acid CCA this paper ft stubbornness is used in the BB home «quasi (CNS) A 4 specifications (210x297: ¢) 81. 2. 20,000 206 ^ 36 Λ 6 η 6 V. Description of the invention (r Leucine, Alanine, Alanine, glutamic acid

C T A AAA G C C G AA 經濟部中央櫺準局员工消费合作社印製 DMA之修飾可藉於宿主細胞内之轉形（Iransformation) ，使重組蛋白質加上輔助序列之密碼；此宿主細胞宜為 原核細胞，尤以大腸桿菌為宜。其後，此轉形細胞在適 當之培養基中培養，在這些細胞分解後，此重組蛋白質 即以至少有一部分為無活性之狀態下，以包涵體之型態 呈現，且被分離出來。再將此蛋白質溶解及復性，此宜 在能使此蛋白質恢復其天然構造之酸鹸度下進行。此程 序之步驟，可依描述中之介紹所舉之例子來引證。此蛋 白質之活化宜用例子中之EP-A 0 241 022所掲示之脲衝 式復性法（pulse renaturation)來執行β當在重組蛋白 質之Ν-或/及C-端，存有一輔助序列時，上述活化反應 可藉本發明而有顯#之改善。在上述方法中，輔助序列 宜加在所欲活化之蛋白質之胺基端上。然而，將輔肋序 列加在C -端上，亦有正面之結果。 較佳之輔助序列至少含有二個胺基酸，此胺基酸則由 麩胺酸，天門冬酸，離胺酸，精胺酸和脯胺酸中選出， 然而以離胺酸和麩胺酸殘基為宜，又以麩胺酸殘基最為 適合。此外，此輔助序列宜含有上列所提之帶霜荷之胺 基酸（例如麩胺酸，天門冬酸，離胺酸和精胺酸）且以連 (請先閲讀背而之注意事項典填寫本頁) 裝· 私紙張尺度逍用中Β國家搮毕(CNS)甲4規格(210x297公釐） 81. 2. 20,000CTA AAA GCCG AA The DMA modification printed by the Employee Consumer Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs can be used to transform the protein in the host cell (Iransformation), so that the recombinant protein plus the auxiliary sequence code; this host cell is preferably a prokaryotic cell, especially Escherichia coli is appropriate. Afterwards, the transformed cells are cultured in an appropriate medium. After these cells are decomposed, the recombinant protein appears in the form of inclusion bodies with at least a portion of them inactive, and is isolated. After dissolving and renaturing the protein, this should be done at an acidic degree that allows the protein to restore its natural structure. The steps of this procedure can be cited according to the examples given in the introduction in the description. The activation of this protein is preferably performed by the urea-pulse renaturation method shown in EP-A 0 241 022 in the example. When there is an auxiliary sequence at the N- or / and C-terminus of the recombinant protein The above activation reaction can be significantly improved by the present invention. In the above method, the auxiliary sequence is preferably added to the amine end of the protein to be activated. However, adding the auxiliary rib sequence to the C-terminal has a positive result. The preferred auxiliary sequence contains at least two amino acids. The amino acid is selected from glutamic acid, aspartic acid, lysine, arginine and proline. However, amine and glutamate residues The base is suitable, and glutamic acid residues are the most suitable. In addition, this auxiliary sequence should contain the amino acids mentioned above with frost (such as glutamic acid, aspartic acid, lysine, and arginine) and should be connected (please read the precautionary code (Fill in this page) Packed in the private paper standard for use in the National Beta (CNS) A 4 specifications (210x297 mm) 81. 2. 20,000

-- 五、發明説明（fe 續兩個相同電荷之胺基酸為宜如以兩値連籲之離胺酸 或麩胺酸殘基較宜，其中又以兩個連缠之麩胺酸最適合 〇 如果重組蛋白質依本發明之方法來産生，且將作為治 療用途，其好离是斷裂位置將在輔助序列和所欲用之蛋 白質之接合處。此即意味著此蛋白質可以其天然之胺基 酸序列被獲得。此斷裂處可以是蛋白酶（protease)所認 知之序列，或是可被一專供蛋白質斷裂用之化學製阐〔 如氰化澳（B r C H )〕，其中以蛋白_斷裂處.為宜；又以— 種I g A蛋白酶斷裂處為宜，如w 〇 9 i / u 5 2 〇中所描述。 斷裂時之條件，.如W〇 91/11520中所規定。此外，被Xa 因子所斷裂之序列亦可使用。 當此重組蛋白質傜供分析之用時，則在蛋白質之序列 上，並不需要一斷裂處之序列存在。 適合用來改菩蛋白質活化之輔助序列，如下列之實例 所示，逭些序列可加在蛋白質之胺基端。-5. Description of the invention (fe It is advisable to continue two amino acids of the same charge. For example, it is more appropriate to use two consecutive amino acid residues or glutamic acid residues. Among them, two consecutive glutamic acids are the most suitable. Suitable 〇 If the recombinant protein is produced according to the method of the present invention, and will be used for therapeutic purposes, its cleavage position will be at the junction of the auxiliary sequence and the desired protein. This means that this protein can be its natural amine The base acid sequence is obtained. This break can be a sequence recognized by a protease, or it can be chemically elaborated for protein cleavage [e.g. Cyanide O (B r CH)], where protein_ It is appropriate to break. Another type of I g A protease is suitable for breakage, as described in w 〇9 i / u 5 2 〇. The conditions at break, as specified in W〇91 / 11520. In addition The sequence broken by factor Xa can also be used. When this recombinant protein is used for analysis, the sequence of the protein does not require a sequence at the break. It is suitable for modifying the auxiliary sequence of protein activation , As shown in the following example, some preface It can be added at the amino terminus of the protein.

Met-Glu (SEQ ID NO: 1) 經濟部屮央標準局工消费合作社印製Met-Glu (SEQ ID NO: 1) Printed by the Industrial and Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

Met-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ id no： 2) Met-Thr-Pro-Leu-His-His-Pro-Arg-Pro-Pro (SEQ ID NO: 3)Met-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ id no: 2) Met-Thr-Pro-Leu-His-His-Pro-Arg-Pro-Pro (SEQ ID NO: 3)

Met-Thr-Pro-Leu-Lys-Lys-Pro-Arg-Pro-Pro (SEQ ID NO: 4)Met-Thr-Pro-Leu-Lys-Lys-Pro-Arg-Pro-Pro (SEQ ID NO: 4)

Met—Thr—Pro—Leu—Glu**Glu—Gly—Pro—Arg~Piro—Pro (SEQ ID NO: 5) , 本《張尺度边用中B B家搮準(CNS)甲4規格(210x297公釐> 81. 2. 20,000 Λ 6 Π 6 發明説明（7) 請 先 閲 背 之 '注 意 事 項 再 填 寫 本 頁Met—Thr—Pro—Leu—Glu ** Glu—Gly—Pro—Arg ~ Piro—Pro (SEQ ID NO: 5), this "Zhang Scale Side Use Chinese BB Family Support Standard (CNS) Grade 4 Specification (210x297 %> 81. 2. 20,000 Λ 6 Π 6 Description of the invention (7) Please read the "Notes" on the back before filling this page

Het-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 6)Het-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 6)

Het-Thr-Pro-Leu-Glu-Glu-Gln-Pro-Pro (SEQ ID NO: 7) Het-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro~Arg-Pro-Pro (SEQ ID NO： 8)Het-Thr-Pro-Leu-Glu-Glu-Gln-Pro-Pro (SEQ ID NO: 7) Het-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro ~ Arg-Pro-Pro ( SEQ ID NO: 8)

Met-Thr-Pro-Leu-Glu-Glu-Gly-Ile-Glu-Gly-Arg (SEQ ID NO: 9)Met-Thr-Pro-Leu-Glu-Glu-Gly-Ile-Glu-Gly-Arg (SEQ ID NO: 9)

Met-Thr-Pro-Leu-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro-Arg-Pro-Pro (SEQ ID NO: 10) 這些輔肋序列中，编號SEQ ID H0:5,6,7及9,均含有 二個連纗之麩肢酸殘基，用於復性（Anaturation)時， 其産率較高。 依本發明之方法，尤其適合用來活化重组之人類蛋白 質及其由原核細胞産生之衍生物，如胞漿素原活化劑< P 1 a s m i η 〇 g e n a c t i v a t 〇 r s )，干擾素（i π t e r f e r ο n s )，介 白素（interleukins)和顆粒球種株剌激因子（granulocyte colony stimulating factors，G-CSF)e 如所欲活化之 蛋白質傜G-CSF,而又帶有ACACCA序列之起始DNA，則最 宜以本法來活化。在£?4 0 456 200中所掲示之G-CSF 之衍生物，亦宜用本法來活化。 經濟部中央標準局员工消费合作社印製 而此載體（vector)pKK 177-3-G-CSF,僳以编號 DSM 5867貯存於 Deutsche Same lung fur Mikro organisnen,Met-Thr-Pro-Leu-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro-Arg-Pro-Pro (SEQ ID NO: 10) In these auxiliary rib sequences, the number SEQ ID H0: 5,6,7 and 9, all contain two consecutive bran acid residues, and when used in anaturation, the yield is higher. According to the method of the present invention, it is particularly suitable for activating recombinant human protein and its derivatives produced by prokaryotic cells, such as cytoplasmin activator < P 1 asmi η 〇genactivat 〇rs), interferon (i π terfer ο ns), interleukins and granulocyte colony stimulating factors (G-CSF) e such as the desired activation protein G-CSF and the starting DNA with ACACCA sequence , It is best to use this method to activate. The derivatives of G-CSF shown in £? 4 0 456 200 should also be activated by this method. Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. The vector pKK 177-3-G-CSF, numbered DSM 5867, is stored in Deutsche Same lung fur Mikro organisnen,

Grisebachstr.8,D- 3 4 0fl Gottingen. 以下之例子和圖，目的在進一步鬭明本發明》 圈1顯示此愎性（renaturation)之産率和濃度間之依 存醑傺（精胺酸濃度0.2摩爾/公升），圃中所含之序列 81. 2. 20,000 本紙張尺度逍用中國B家榫準(CNS)甲4規格(210X2974*)Grisebachstr. 8, D- 3 4 0fl Gottingen. The following examples and figures are intended to further clarify the invention. Circle 1 shows the dependence of the yield and concentration of this renaturation (arginine concentration 0.2 Molar / L), the sequence contained in the garden 81. 2. 20,000 paper-scale free use Chinese B home tenon (CNS) A 4 specifications (210X2974 *)

CC

An 2 經濟部屮央標準局员工消费合作杜印製 -- 五、發明説明（2 ) 和表2所列之序列相同，序列〇為曲線1，s E 〇 1 D N 0 : 3 為曲線2, SEQ ID N0:5為曲線3，及SEQ ID N0:8為曲線 4〇 圖2顯示後性之産率和濃度之依存關精胺酸濃度 為0.8摩爾/升），圈中所含之序列，和表2相同，分別 為序列 〇(曲線 1)，SEQ ID N0:3(曲線 2) ，SEQ ID N0: 5 (曲線 3)及 SEQ ID N0:8(曲線 4>。 匾3顯示復性之産率和精胺酸濃度之依存鬭傲（曲線 所代表之序列與圈1，豳2中相同。 圖4顯示此再活化之産率與培餐時間之關係（精胺酸 濃度為0.2摩爾/公升，曲線名與鼷1及圈2中相同）β 例 1 : 載體之結構 此載賭 pKK 177-3 G-CSF Bg(DSM 5 8 6 7 )傜浸漬於 EcoRI (部分地）及Apal及此寡核甘酸 EcoRI Apal AATTCGGAGGAAAAATTA I ATG......... |ACACCACTGGGCC I Met.......... JG-CSF sequence • without ATG 被嵌入到此線狀之媒介者片段之中（c a , 3 4 5 0 b p )而形成 AATTCGGAGGAAAAATTA： SEQ ID NO： 11 ACACCACTGGGCC: SEQ ID NO： 12 每一 DN A序列之基因密碼。僳用來埔補表2中所列之胺 基酸密碼之缺口，如某寡核甘酸具有之基因碼為Met- -10 本紙張尺度遑用中βΒ家《準(CNS)甲4規格(210父297公釐) 81. 2. 20,000 (請先閲讀背而之注意事項#'蜞裒本^ 裝- 線- 2( C C < 66 ΛΠ 五、發明説明（9) 桃充 經濟部屮央標準局员工消t合作社印製 1'111'-?1'〇-1^11-?1'〇-/^轻-?1>〇-?1'〇(3£9 10«0:2)係用來構 成例（2)。由寡核甘酸與斷裂之載體，結合所形成之質 體（plasmids),被轉形（transformed)到大腸桿菌 HB101 之内。為了確保tac促進子能有更佳之調節作用，此細 胞株再加入可與質體相容之PBP01Q(依歐洲專利號碼 9111155.7製成），其含有laciq基因。此13(；1(1基因之 蓮用，偽基因工程中很早就已知悉之一項技術，並且容 易獲得。 可與PBP010相容之質體，包括已嵌有l.aciq基因之 PACYC 177(DSM 3693 P)或由下面例子衍生之質體〔如 基因 85(1989)， 109-114和 EP-A 0373365〕。由此獲得 之種株（clones)經由康纳徽素（kanamycin)(50微克/毫 升）/安比西林（Anpicillin)(50微克/毫升）篩選，並 藉約束分析（restriction analysis)來鑑定。當以EcoRI 和EcoRV來分割時，會産生長度分別為ca.3.15kb，ca. O’.3kb(視結構而定）和4.85 kb之片段。 例 2 : a)酷酵（Fermentation) 依例1方法鑑定為陽性之種株，在5毫升之LB培餐基 中生長，此培餐基含有康纳徽素和安比西林（濃度同例1) 直到其0D 500之值到達0.5 ,以濃度5摩爾/公升之IPTG 誘生，且在3 7 °C下培養3小時。如此可獲得1 〇 〇 D之誘 生培餐液，並由此萃取得全部細胞萃取液，此萃取液經 11 本紙張尺度逍用中國《家楳準(CNS) 規怙(210X297公龙) 81. 2. 20,000 2 經濟部中央櫺準局只工消合作社印製 五、發明説明（丨〇) A 6 Π 6 由SDS膠膜來分析β 當所痛要之蛋白質己明確的産生時，此培餐液再以1:1 之比例繼鑛培餐，此種株即已獲得，再以ΙΒ配方來完成。 b ) I Β 製備（I B p r e p a r a t i ο η ) 此細胞株偽以離心法收成，以100毫升之Tris鎂，缓 衝液（10¾摩爾/公升Tris, pH 8.0，1毫摩爾/公升 之)處理，並以溶菌酵素（IjYS0Z0ME，0.3毫克/ ¾ 升）來溶解。 它們在37 °C下培餐15分鐘，且處於一法式壓榨機（ F r e n c h p r e s s，1 2 0 0 p s i)之一個通道之下。 其後以DNA酶（2毫克之DNAse I)在37°C下消化30分鏡。 加入20毫升之氣化納（〇.5摩爾/公升，HaCl), 20毫 摩爾之 EDTA, pH 8.0和 3 毫升之 20%Trition X100,在 室溫下培養10分鐘。 此懸液在15000rp·, 4°C下離心10分鐘。其離心物加 30毫升溶液（其中含Tris 50毫摩爾/公升，pH 8.0, 5 0毫摩爾/公升之EDTA和0.5%之Triton X100)且以超 音波處理。再離心一次後，再以上迷溶液用超音波處理 。此種步驟再重覆2次。由此方式獲得之離心産物，即 用在例3中之IBs(包涵體>。 例 3 : 肋溶作用（solubilization)/ 愎性作用（renaturation) a)肋溶作用 -12- 本紙張尺度边用中a國家樣準(CNS)甲4規格(210x297公：it) 81. 2. 20,000 (請先IW讀背而之注意事項孙璜¾本页) Λ 6 Π6 五、發明説明（q 經濟部中央標準局s：工消费合作社印製 助溶作用缓衝液： 6 摩爾/ 升鹽酸胍（guanidine hydrochloride) 0.1摩爾/升 Tris缓衝液，pH 8.0 1毫摩爾/升 EDTA 100毫摩爾/升DTE (二硫代赤蘚酵） 透析缓衝液 / (Dialysis buffer 1): 6摩爾/升鹽酸胍 3毫摩爾/升 EDTA於pH 3 . 0 1公'克之包涵.醱（inclusion bodies，I,Bs)加入30牽升 之助溶緩衝液中，以超音波將其均質化5分鐘，再於室 溫下培養1小時。加入鹽酸調其PH值至3.0後，其不溶 物以離心法移除。 將其以透析缓衝液1進行透析，直到DTE完全移除（忘 1毫摩爾/升之DTE) b)_ 脈衝式再活化（pulse reactivation): 復性缓衝液（renaturation buffer): 0.8摩爾/ 升鹽酸精胺酸（arginine hydrochloride) 0.1摩爾/升 Tris緩衝液，pH 8.0 0.5毫摩爾/升GSH(運原麩胺基硫） 0.5毫摩爾/升 GSSG(氣化麩胺基硫） 1 毫摩爾/升 EDTA 透析緩衝液2 : 10牽摩爾/升 Tris緩衝液，pH 8.0 13- 本紙張尺度逍用中曲Η家«準(CNS)甲4規格(210X297公; 81, 2. 20,000 (請先閱讀濟而之注意事項孙塡寫本頁)An 2 Du-printed by the employee consumption cooperation of the Central Standards Bureau of the Ministry of Economic Affairs-V. Invention description (2) The same sequence as listed in Table 2, the sequence 〇 is curve 1, s E 〇1 DN 0: 3 is curve 2, SEQ ID N0: 5 is curve 3, and SEQ ID N0: 8 is curve 4. Figure 2 shows the dependence of the subsequent yield and concentration on the concentration of arginine (0.8 mol / L), the sequence contained in the circle, The same as Table 2, the sequence 〇 (curve 1), SEQ ID N0: 3 (curve 2), SEQ ID N0: 5 (curve 3) and SEQ ID N0: 8 (curve 4>. Plaque 3 shows the refolding Dependence of yield and arginine concentration (the sequence represented by the curve is the same as in circle 1 and bin 2. Figure 4 shows the relationship between the yield of this reactivation and the time of meal preparation (arginine concentration is 0.2 mol / Liters, the curve name is the same as that in Nai 1 and Circle 2) β Example 1: The structure of the carrier is pKK 177-3 G-CSF Bg (DSM 5 8 6 7), which is impregnated with EcoRI (partially) and Apal and this Oligonucleotide EcoRI Apal AATTCGGAGGAAAAATTA I ATG ......... | ACACCACTGGGCC I Met .... JG-CSF sequence • without ATG is embedded in this linear mediator segment (Ca, 3 4 5 0 bp) to form AATTCGGAGGAAAAATTA: SEQ ID NO: 11 ACACCACTGGGCC: SEQ ID NO: 12 the genetic code of each DNA sequence. It is used to fill the gap in the amino acid code listed in Table 2, such as an oligonucleotide The gene code is Met- -10. The standard of this paper is the beta β standard (CNS) A 4 specifications (210 father 297 mm) 81. 2. 20,000 (please read the back notes beforehand # '蜞 裒This ^ Pack-Line-2 (CC &66; ΛΠ V. Description of the invention (9) Printed by the Employee Consumer Cooperative of the Piaoyang Standards Bureau of the Ministry of Economy, Taochong 1'111 '-? 1'〇-1 ^ 11-? 1 '〇-/ ^ 轻-? 1> 〇-? 1'〇 (3 £ 9 10 «0: 2) is used to constitute the example (2). The oligonucleotide formed by the combination of oligonucleotides and the broken carrier, formed (Plasmids), transformed into E. coli HB101. In order to ensure better regulation of tac promoter, this cell line was added with PBP01Q (made according to European Patent No. 9111155.7) which is compatible with plastids ), Which contains the laciq gene. This 13 (; 1 (1 gene is used for lotus, a technology known from the early pseudo-gene engineering, and is easy to obtain. Plastids that are compatible with PBP010 include PACYC 177 (DSM 3693 P) with the l.aciq gene embedded or plastids derived from the following examples [eg Gene 85 (1989), 109-114 and EP-A 0373365] . The clones thus obtained were screened by kanamycin (50 μg / ml) / Anpicillin (50 μg / ml) and identified by restriction analysis. When divided by EcoRI and EcoRV, fragments of ca.3.15kb, ca. O’.3kb (depending on the structure) and 4.85 kb will be generated. Example 2: a) Fermentation The strain identified as positive according to the method of Example 1 is grown in a 5 ml LB culture base, which contains Connexin and Ampicillin (same concentration as Example 1) Until the value of 0D 500 reached 0.5, it was induced with IPTG with a concentration of 5 mol / liter and incubated at 37 ° C for 3 hours. In this way, the inducible meal solution of 100D can be obtained, and all the cell extracts are extracted therefrom. This extract is used in 11 paper scales and is used in China's "China Family Standard (CNS) Regulations (210X297 Gonglong) 81 2. 20,000 2 Printed by the Central Bureau of Trade and Industry of the Ministry of Economic Affairs, only the Consumers ’Co-operatives Co., Ltd. 5. Description of Invention (丨 〇) A 6 Π 6 Analysis of β by SDS film The meal solution is then followed by a 1: 1 ratio of ore cultivation, this strain has been obtained, and then completed with ΙΒ formula. b) I Β preparation (IB preparati ο η) This cell line was pseudo-harvested by centrifugation, treated with 100 ml of Tris magnesium, buffer (10¾ mol / L Tris, pH 8.0, 1 mmol / L) and treated with Lysozyme (IjYS0Z0ME, 0.3 mg / ¾ liter) to dissolve. They are cooked for 15 minutes at 37 ° C, and are under a channel of a French press (F r e n c h p r e s s, 1 2 0 0 p s i). Thereafter, digest with DNase (2 mg of DNAse I) at 37 ° C for 30 minutes. Add 20 ml of vaporized sodium (0.5 mol / L, HaCl), 20 mmol of EDTA, pH 8.0 and 3 ml of 20% Trition X100, and incubate at room temperature for 10 minutes. This suspension was centrifuged at 15000rp ·, 4 ° C for 10 minutes. The centrifuge was added with 30 ml of solution (containing Tris 50 mmol / L, pH 8.0, 50 mmol / L EDTA and 0.5% Triton X100) and treated with ultrasound. After centrifuging again, the above solution was treated with ultrasound. Repeat this step 2 more times. The centrifuged product obtained in this way is the IBs (inclusions) used in Example 3. Example 3: Solubilization / renaturation a) Rib dissolution -12- Edge of this paper scale Use China National Standards (CNS) Grade 4 specifications (210x297 g: it) 81. 2. 20,000 (please read the notes before IW Sun Huang ¾ this page) Λ 6 Π6 V. Description of invention (q Ministry of Economic Affairs Central Bureau of Standards: Industrial and Consumer Cooperatives printing solubilization buffer: 6 mol / L guanidine hydrochloride 0.1 mol / L Tris buffer, pH 8.0 1 mmol / L EDTA 100 mmol / L DTE (2 Thioerythromycin) Dialysis buffer 1 / (Dialysis buffer 1): 6 mol / L guanidine hydrochloride 3 mmol / L EDTA at pH 3.0. 1 g of the inclusion of grams. Inclusion bodies (I, Bs) added 30 In the lifted solubilization buffer, homogenize it with ultrasound for 5 minutes, and then incubate at room temperature for 1 hour. After adding hydrochloric acid to adjust its pH to 3.0, the insoluble matter was removed by centrifugation. Dialysis buffer 1 is used for dialysis until the DTE is completely removed (forget the DTE of 1 mmol / L) b) _ Pulse rea (pulse rea) ctivation): Renaturation buffer: 0.8 mol / L arginine hydrochloride 0.1 mol / L Tris buffer, pH 8.0 0.5 mmol / L GSH (Yunyuan glutamine sulfide) 0.5 mmol Molar / liter GSSG (gasified glutamine sulfide) 1 millimolar / liter EDTA dialysis buffer 2: 10 mol / liter Tris buffer, pH 8.0 13- This paper scale is easy to use Zhongqu A 4 specifications (210X297; 81, 2. 20,000 (please read the precautions of Ji Er first to write this page)

2|υό ⑽ G Λ 6 Π 6 五、發明説明（ι>) 1毫摩爾/升 EDTA 此脲衝式再活化係依EP-A 0 241 022中所敘述之方法 實施，如圖5所示之設備偽EP-A 0 241 022中所使用。 將此蛋白質加入此再反應容積（10 0毫升復性緩衝液） 之中，以30分鐘為間隔，每次脈衝時，容積中之蛋白質 環度增加50微克/毫升。總共脈衝次數為2Q(再反應容 積之最後濃度約為1毫克/毫升。 當此脈衝式再活化之混濁物，以離心法將其由再反應 容積中移除後；全部再反應容積以透析緩衝液2進行透 析，直至精胺酸被移除為止（含50毫摩爾/升）。（此可 利用導電度測量而得知，當透析緩衝液和再反應容積之 導電度完全相同時，即可停止透析）。各锢蛋白質再活 化之産率，僳藉活性測試來決定，示於表 2» 先 閲 ih 背 之 注 意 事 項 再 寫 木 頁 經濟部中央標準局貝工消费合作社印製 本紙張尺度逍用中B B家樣準(CNS)T4規格(210X297公*) 81. 2· 20,000 五、發明説明（丨夕） Λ 6 Π 62 | υό ⑽ G Λ 6 Π 6 V. Description of the invention (ι >) 1 mmol / l EDTA This urea punch reactivation is implemented according to the method described in EP-A 0 241 022, as shown in FIG. 5 Used in equipment pseudo-EP-A 0 241 022. Add this protein to the re-reaction volume (100 ml of renaturation buffer), and at 30-minute intervals, the protein ring in the volume will increase by 50 μg / ml per pulse. The total number of pulses is 2Q (the final concentration of the re-reaction volume is about 1 mg / ml. When the pulse-reactivated turbidity is removed from the re-reaction volume by centrifugation; the entire re-reaction volume is buffered by dialysis Solution 2 is dialyzed until arginine is removed (contains 50 mmol / L). (This can be determined by conductivity measurement, when the conductivity of the dialysis buffer and the re-reaction volume are exactly the same, it can be Stop dialysis). The yield of reactivation of each protein is determined by the activity test, shown in Table 2 »Read the notes on the back of ih first, and then write the wooden page. Printed paper size BB family sample standard (CNS) T4 specification (210X297 g *) for free use 81. 2 · 20,000 V. Description of invention (丨 eve) Λ 6 Π 6

(0) Met-G-CSF- (1) Met-Glu-G-CSF (2) Met-dlr 丨 pro-Leu—pro-ATg-pro-pro-G-CSF (3) MetlThr-pro-Leu—liisllIis_pro-Arglpro-pro-G-csr,3 ^ w —ΜνΜ?Μ§ΙΜ^ΟΙΜ^Μ!^ΗΟΙ>5Ι^ΗΟΙ^ΟΙΟΛΜ^ (5) Metlulr-pro-Leu-Glu-Glu-Glylpro-ATg-prTpro-G-CSF (6) Met-Ihripro-Leu-Glu-Glu-Giylnlr-pro-Leu-pro-Arg丨I (8} MetlLys-Ala-Lys-Arg-phe-Lys-Lys-JIie-pro-AT^-pro-pro-G-CSF (9) Met-lhr-pr?Leu-Giu-Glu-Giy—Ile-Glu-GlylArg-G-CSF (1°) Μβί.—Ί11Γ—ΡΓΟ-Ι£υι—ΙΥω-Λ1£1,—Ιγω—ΑΙ:9-ρι1β-ΙΥ5ΙΙΙ;7ΜΙΙΙ13—ΡΓ0-ΛΓ'£3— pr?pro-G-CSF >2 (請先閲讀背而之注意事項#填寫本頁} 裝- 線_ 10s 2P 20 s 80 丨 90 000 00 s 80 50 s- § 經濟部屮央標準局员工消t合作杜印製 — 45,4 丨 2'co 丨8 -20 -19 -29 — s 丨41 丨26 丨44 — 38 — 2 — 2 丨2 • 3 丨2 丨2 — 3 I 3 本紙張尺度边用中國國家«準(CNS)甲4規格(210x297公ft) {kcal/nor 81. 2. 20,000 2〇ρ^β- 五、發明説明 Λ 6 η 6 1 .此例並非依本發明 2 .依例3之活性試驗而測定 (1)至（10): SEQ ID N0:1至10與G-CSF序列接合，並無 胺基端之甲硫胺酸之殘基。 例 4 : G - C S F活性之測定 6-08?活性之測定係依據81〇(：116!11.(1.253(1988) 213- 218,Exp.HeBatol.17(1989)116-119,Proc.Natl.Acad. Sci.USA 83(1986)5010,所描述之方法測試。使用完金 依賴G-CSF之白血病鼠株NFS 60,此培養.基（RPMI培養 基，Boehringer Ma,nnheim GmbH,訂貨號碼 2099445含有 10%之仔牛血清）所含之G-CSF保持在1〇〇〇 U / b 1 ,以保存 對此因子有依賴性之細胞株。 此測試直接測量以6-CSF刺激之NFS 60細胞之增植， 藉測量氚一胸腺核甘（3 H-thymidine)之结合量而測定(0) Met-G-CSF- (1) Met-Glu-G-CSF (2) Met-dlr pro-Leu—pro-ATg-pro-pro-G-CSF (3) MetlThr-pro-Leu— liisllIis_pro-Arglpro-pro-G-csr, 3 ^ w —ΜνΜ? Μ§ΙΜ ^ ΟΙΜ ^ Μ! ^ ΗΟΙ > 5Ι ^ ΗΟΙ ^ ΟΙΟΛΜ ^ (5) Metlulr-pro-Leu-Glu-Glu-Glylpro-ATg- prTpro-G-CSF (6) Met-Ihripro-Leu-Glu-Glu-Giylnlr-pro-Leu-pro-Arg 丨 I (8) MetlLys-Ala-Lys-Arg-phe-Lys-Lys-JIie-pro- AT ^ -pro-pro-G-CSF (9) Met-lhr-pr? Leu-Giu-Glu-Giy—Ile-Glu-GlylArg-G-CSF (1 °) Μβί.—Ί11Γ—ΡΓΟ-Ι £ υι —ΙΥω-Λ1 £ 1, —Ιγω—ΑΙ: 9-ρι1β-ΙΥ5ΙΙΙ; 7ΜΙΙΙ13—ΡΓ0-ΛΓ '£ 3— pr? Pro-G-CSF > 2 (please read the back-end notes before filling in #fill this page } Installation-line _ 10s 2P 20 s 80 丨 90 000 00 s 80 50 s- § Printing by the employees of the Ministry of Economic Affairs Bureau of Standards-45,4 丨 2'co 丨 8 -20 -19 -29 — s 丨 41 丨 26 丨 44 — 38 — 2 — 2 丨 2 • 3 丨 2 丨 2 — 3 I 3 The size of the paper is used in the Chinese National Standards (CNS) A 4 specifications (210x297 ft) (kcal / nor 81 . 2. 20,000 2〇ρ ^ β- V. Description of the invention Λ 6 η 6 1. This example is not in accordance with the invention 2 . Determined according to the activity test of Example 3 (1) to (10): SEQ ID N0: 1 to 10 are joined to the G-CSF sequence, and there is no residue of methionine at the amine end. Example 4: G- Determination of CSF activity 6-08? Activity determination is based on 81〇 (: 116! 11. (1.253 (1988) 213-218, Exp. He Batol. 17 (1989) 116-119, Proc. Natl. Acad. Sci. USA 83 (1986) 5010, tested by the method described. Using the gold-dependent G-CSF leukemia mouse strain NFS 60, this medium. (RPMI medium, Boehringer Ma, nnheim GmbH, order number 2099445 contains 10% calf serum ) The contained G-CSF is maintained at 1000 U / b 1 to preserve cell lines that are dependent on this factor. This test directly measures the proliferation of NFS 60 cells stimulated with 6-CSF, and is determined by measuring the binding amount of tritium-thymidine (3 H-thymidine)

此測試方法依下列之描述而實施。 以指數生長期之NFS 60細胞（細胞密度為IX 1〇5個細 胞/毫升）移至撤量皿（ιχίο4韬細胞/槽），並以G_cS 經 濟 部 屮 央 標 準 局 员 工 消 货 合 作 社 印 製 遞減之濃度培餐。第1槽其G-CSF濃度最高，與維持0 餐液相同（1000單位/毫升）（專一活性lx 108單位/ $ 克蛋白質）。稀釋步隳之因子為1〇。 培餐24小時後，加入3 H -胸腺甘酸（〇·1 "ci〆檐）° 再培養16小時。 -1 5-Λ 本紙張尺度逍用中B B家«準(CHS)甲4規格(210X297公») 81. 2. 2〇,〇〇0 ㊃發明説明⑻ 經濟部中央標準局员工消费合作杜印製 為了評估此測試，此細胞在微量皿上冷凍，以將其溶 解《其細胞溶解物以玻璃纖維過戶，沖洗，乾燥，再以 閃爍計數器測量。3 H-胸腺甘酸結合量與C-CSF刺撖之 NFS 60細胞之增殖量成正比。 例5 :單次加入之變性蛋白質濃度，與復性産率之依存關係 之測定。 起始物質：具有表2中编號0/3/5和8組成之包涵龌。 肋溶作用和初次透析： 包涵體依例3助溶，並透析以移除邏原劑，其後再調其 濃度成 30毫克 / 毫升〔M.M.Bradford,Anal.Biochem.72 (1976)255] 後性（renataration): 此再活化僳在0.8摩爾/升或0.2摩爾/升之鹽酸精胺酸 ,10毫摩爾/升之EDTA, 0.5毫摩爾/升之GSH和0.5毫 摩爾/升之GSSG,於2Q-C和PH8.0之下執行。 依各自的後性製劑，將蛋白質之濃度調成0.3至3毫克 /毫升，並在所有之製剤中加入濃度為0.55摩爾/升之 鹽酸胍。 在室溫培餐3小時後，此反匾藉酸化（pH 4.5)來停止。 變性蛋白質與復性蛋白質之比值，以HPLC來測定。 移動相緩衝液A: 0.12% (V/V)之三氟乙酸 (trifluoroacetic acid) 16- 本紙張尺度逍用中《國家《準(CNS)甲4規格(210x297公*) 81/2. 20,000 先 閲 讀 背 而 之 意 事 項 再 填 寫 本 裝 訂 線 20 經濟部屮央標準局只工消费合作社印製 Λ 6η 6This test method is implemented as described below. Move the NFS 60 cells (cell density is IX 105 cells / ml) to the measuring dish (ιχίο4 Tao cells / tank) in the exponential growth phase, and print them with the G_cS Ministry of Economic Affairs Bureau of Standards and Merchandise Consumers ’Cooperatives. Concentration of food. The first tank has the highest G-CSF concentration, which is the same as maintaining 0 meals (1000 units / ml) (specific activity lx 108 units / $ gram protein). The factor of the dilution step is 10. After cultivating food for 24 hours, add 3 H-thymic acid (〇 · 1 " ci〆 eaves) ° and incubate for another 16 hours. -1 5-Λ The standard of this paper is BB's quasi (CHS) Jia 4 specification (210X297) »81. 2. 2〇, 〇〇0 ㊃ Description of invention ⑻ Employee consumption cooperation Du Central of the Ministry of Economic Affairs Central Standards Bureau To evaluate this test, the cells were frozen on a micro-dish to dissolve them. The cell lysates were transferred to glass fibers, rinsed, dried, and measured with a scintillation counter. 3 The amount of H-thymic acid binding is proportional to the proliferation of C-CSF-pricked NFS 60 cells. Example 5: Determination of the dependence of the denatured protein concentration in a single addition on the refolding yield. Starting substance: inclusion inclusions with the composition 0/3/5 and 8 in Table 2. Ribolysis and initial dialysis: Inclusion bodies were solubilized according to Example 3, and dialyzed to remove the original agent, and then adjusted to a concentration of 30 mg / ml [MMBradford, Anal.Biochem.72 (1976) 255] (Renataration): This reactivation is at 0.8 mol / L or 0.2 mol / L arginine hydrochloride, 10 mmol / L EDTA, 0.5 mmol / L GSH and 0.5 mmol / L GSSG, at Executed under 2Q-C and PH8.0. Adjust the protein concentration to 0.3 to 3 mg / ml according to the respective subsequent formulations, and add guanidine hydrochloride with a concentration of 0.55 mol / L to all preparations. After cooking at room temperature for 3 hours, this anti-plaque was stopped by acidification (pH 4.5). The ratio of denatured protein to renatured protein was determined by HPLC. Mobile phase buffer A: 0.12% (V / V) of trifluoroacetic acid (trifluoroacetic acid) 16- This paper is used in the "National Standards (CNS) A 4 specifications (210x297 g *) 81/2. 20,000 first Read the back-to-back matters and fill out this binding line. 20 Printed by the Consumer Labor Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Λ 6η 6

/五、發明説明（<i〇 移動相緩衝液B: 90% (V/V)之乙腈 0.1% (V/V)之三氟乙酸 B之升降率（gradient): 40至70%於30分鐘 流速：1毫升/分，於280nffl處檢測。 其結果示於圖1和2。 例子6 : 復性和精胺酸濃度之依存關係 由表2中，编號0, 3, 5和8組成之透析助溶物（蛋白質濃 度為10毫克/毫升），依例 5同法配製，.供為起始物質。 在培養3小時後，其反應以酸化（pH 4.5)使其停止，其 後依例5同法以HPLC來評估。 其結果示於圖3。 例 7 : 在0.2摩爾/升精胺酸缓衝液中，再活化之動力學 起始物質：例6中所用之助溶産物（solubilisatesh 再活化像在下列組成之溶液中進行（室溫，pH 8.0) 0.2摩爾/升鹽酸精胺酸 100 摩爾/升 Tris * 10 摩爾/升 EDTA 0.5摩爾/升 GSH 0.5摩爾/升 GSSG -17- 本紙張尺度边用中a B家搮準(CNS) T4規格(210X297公釐) 81. 2· 20,000 (< 發明説明（“） Λ 6 Η 6 反應配方中之蛋白質濃度，係一次調成1毫克/毫升， 胍之濃度則調成ϋ.55摩爾/升。分別在反應開始後 5, 10, 15, 60及180分鐘取樣，並藉酸化（pH 4.5)使其 反應停止。接著以Η P L C測定此再活化之動力學（如例5 ) 圖4顯示再活化之産率和培養時間長短之依存關傜β ····.............··.·裝...........線 (請先閲讀背而之注意t項再塡寫木頁) 經濟部中央標準局貝工消份合作杜印製 -1 8 - 本紙張尺度逍用中Β Β家樣準(CNS) Τ 4規格(210X297公釐） 81. 2. 20,000/ V. Description of the invention (< i〇 mobile phase buffer B: 90% (V / V) of acetonitrile 0.1% (V / V) of trifluoroacetic acid B (gradient): 40 to 70% in 30 Minute flow rate: 1 ml / min, measured at 280nffl. The results are shown in Figures 1 and 2. Example 6: The dependence of renaturation and arginine concentration is composed of No. 0, 3, 5 and 8 in Table 2. The dialysis solubilizer (protein concentration is 10 mg / ml), prepared in the same way as in Example 5, is used as the starting material. After 3 hours of incubation, the reaction was stopped by acidification (pH 4.5), and then The same method of Example 5 was evaluated by HPLC. The results are shown in Figure 3. Example 7: Kinetic starting material for reactivation in 0.2 mol / L arginine buffer: Solubilisatesh used in Example 6 (solubilisatesh Reactivation is performed in a solution of the following composition (room temperature, pH 8.0) 0.2 mol / L arginine hydrochloride 100 mol / L Tris * 10 mol / L EDTA 0.5 mol / L GSH 0.5 mol / L GSSG -17- This For paper-scale edge use, a B Family Standard (CNS) T4 specification (210X297mm) 81.2 · 20,000 (< Description of the invention (“) Λ 6 Η 6 protein concentration in the reaction formula It was adjusted to 1 mg / ml at a time, and the concentration of guanidine was adjusted to ϋ.55 mol / l. Samples were taken at 5, 10, 15, 60 and 180 minutes after the start of the reaction, and the reaction was stopped by acidification (pH 4.5) Then, the kinetics of this reactivation was measured by HPLC (as in Example 5). Figure 4 shows the dependence of the yield of reactivation and the length of the cultivation time. . ··. · Installation .............. line (please read the note t first and then write the wooden page) Central Bureau of Standards of the Ministry of Economic Affairs Cooperate with Duong Printing -1 8 -The size of this paper is for the use of the standard (CNS) Τ 4 specifications (210X297mm) 81. 2. 20,000

Claims (1)

經濟部中央標準局貝工消贽合作社印製 2〇623δ ^_- U； 7f _ 六、申請專利範園 第8 11 0 1 1 1 4號「重組體蛋白質的改良活化法」專利案 (82年4月修正） 1. 一種重組蛋白質之活化方法，而此蛋白質至少有一部 分以非活性呈現；在其中，蛋白質愾籍已知之肋溶或 /及復性技術來活化，所稱之蛋白質具有長度為2-50 個胺基酸之輔助序列，在其胺基端或/及羧基端相接 ，且其相對相水性，依各胺基酸之相對厭水性之和來 計算，為一負值；其相對厭水性與胺基酸個數之比值 為-2 . 5仟卡/摩爾或更小；其中，輔助序列係加在待 活化之蛋白質之胺基端；其中，輔助序列像加在待活· 化之蛋白質之羧基端；輔助序列至少有兩値胺基酸， 且偽由麩胺酸，天門冬酸，離胺酸，稍肢酸和脯胺酸 中挑選而得；輔助序列至少含2傾麩胺酸殘基； 其中，輔助序列含有2値連缠之胺基酸，且具有相 同之電荷，俗由下列胺基酸中選出：麩胺酸，天門冬 酸，離胺酸和精胺酸；其中，輔助序列含有2個連绩 之麩胺酸殘基。 2. 如’申請專利範圍第1項之方法，其中欲活化之蛋白質 有一斷裂處位於輔肋序列和所需之蛋白質之間。 3..如申請專利範圍第2項之方法，其中斷裂處為一可被 蛋白8毎認知之序列。Printed by the Beigong Xiaozhi Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs, 2〇623δ ^ _- U; 7f _ VI. Patent application No. 8 11 0 1 1 1 4 "Recombinant Protein Improved Activation Method" Patent Case (82 (Amended in April 2014) 1. A method of activating a recombinant protein in which at least a portion of the protein is inactive; in which the protein is activated by known ribolysis or / and renaturation techniques, the protein is said to have a length It is an auxiliary sequence of 2-50 amino acids, which is connected at the amine end or / and carboxyl end, and the relative phase water is calculated as a negative value according to the sum of the relative water repellency of each amino acid; The ratio of its relative water repellency to the number of amino acids is -2.5 thousand calories / mole or less; where the auxiliary sequence is added to the amine end of the protein to be activated; where the auxiliary sequence is added to be activated · The carboxyl terminus of the chemical protein; the auxiliary sequence has at least two amino acids, and is pseudo-selected from glutamic acid, aspartic acid, lysine, limpet acid and proline; the auxiliary sequence contains at least 2 Pour glutamic acid residues; where the auxiliary sequence contains 2 intertwined amine groups And having the same phase of the charge, selected from the following amino acids are popular: glutamic acid, aspartic acid, lysine and arginine; wherein the helper sequences contain two glutamic acid residues connected group of performance. 2. The method as described in item 1 of the 'Patent Scope', in which the protein to be activated has a break between the auxiliary rib sequence and the desired protein. 3. The method as claimed in item 2 of the patent application, wherein the break is a sequence that can be recognized by protein 8. ί請先閱讀背面之注意事項再嗔寫本頁) .¾. .訂. •線. 本紙张尺度遑用中B國家樣準（CNS)甲4規格(210x297公釐）' …., mi--· | ——"·ΠΝ·丨· 206236 A 7 B7 C7 D7 經濟部中央標準局貝工消贽合作社印製 六'申請專利範ffl4. 如申請専利範圓第3項之方法，其中斷裂處為一種可被I g A蛋白酶切斷之序列。5. 如申請專利範颺第4項之方法，其中斷裂處為一因子 Xa断裂處。6. 如申請專利範菌第1項之方法，其中欲活化蛋白霣含 有一胺基端之輔助序列，係由下列序列之一遂出： Met-Glu (SEQ ID N0:1) Met-Thr-Pro—Leu-Pro—Arg-Pro-Pro (SEQ ID NO: 2) Met>Thr-Pro-Leu-His-His-Pro-Arg-Pro-Pro (SEQ ID NO: 3) ^ Het-Thr-Pro-Leu-Lys-Lys-Pro-Arg-Pro-Pro (SEQ ID NO； 4) Met-Thr-Pro-Leu-Glu-Glu-Gly-Pro-Arg-Pro-Pro (SEQ ID NO： 5) / Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 6) Met-Thr-Pro-Leu-Glu-Glu-Gln-Pro-Pro (SEQ ID NO: 7) Het-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro-Arg-Pro'-Pro (SEQ ID NO: 8) Met-Thr-Pro-Leu-Glu-Glu-Gly-Ile-Glu-Gly-Arg (SEQ ID NO: 9)Met: 一 Thr_p：ro 一；Leu_I*ys 一 Ala_]Lys 一 Arg_Phe_：Lys 命 Eys—His^Pro 一 Arg-Pro-Pro (SEQ ID NO: 10)7·如申請専利範圍第i或6項之方法.其中活化之蛋 白質俱指顆粒球種株刺激因子（G-CSF)或其衍生物。 -2- (請先閔請背面<注意事項再填寫本頁) 本紙張尺度適用中a國家樣準(CNS)甲4規格(210x297公釐)ί Please read the precautions on the back before writing this page). ¾.. Order. • Line. This paper uses the Chinese National Standard B (CNS) A 4 specifications (210x297 mm) '…., mi- -· | —— " · ΠΝ · 丨 · 206236 A 7 B7 C7 D7 The Ministry of Economic Affairs Central Standards Bureau Beigong Xiaozhi Cooperative printed six 'application patent model ffl4. Such as applying for the method of item 3 of the Lifan circle, which breaks It is a sequence that can be cleaved by I g A protease. 5. For example, the method of applying for patent Fan Yang item 4, where the fracture is a factor Xa fracture. 6. The method as claimed in Item 1 of the patent application, in which the protein to be activated contains an auxiliary sequence at the end of an amine group, which is derived from one of the following sequences: Met-Glu (SEQ ID N0: 1) Met-Thr- Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 2) Met > Thr-Pro-Leu-His-His-Pro-Arg-Pro-Pro (SEQ ID NO: 3) ^ Het-Thr-Pro -Leu-Lys-Lys-Pro-Arg-Pro-Pro (SEQ ID NO; 4) Met-Thr-Pro-Leu-Glu-Glu-Gly-Pro-Arg-Pro-Pro (SEQ ID NO: 5) / Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 6) Met-Thr-Pro-Leu-Glu-Glu-Gln-Pro- Pro (SEQ ID NO: 7) Het-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro-Arg-Pro'-Pro (SEQ ID NO: 8) Met-Thr-Pro-Leu-Glu -Glu-Gly-Ile-Glu-Gly-Arg (SEQ ID NO: 9) Met: one Thr_p: ro one; Leu_I * ys one Ala_] Lys one Arg_Phe_: Lys life Eys—His ^ Pro one Arg-Pro-Pro (SEQ ID NO: 10) 7. The method as claimed in item i or 6 of the scope of application. The activated protein refers to the granulosa bulb stimulating factor (G-CSF) or its derivative. -2- (Please first please back < Note before filling this page) This paper size is applicable to the National Sample Standard (CNS) Grade 4 (210x297 mm)