TWI382847B - A protein having a protease activity, a nucleic acid sequence encoding the protein, a method for producing the protein, and a method for producing the same - Google Patents
A protein having a protease activity, a nucleic acid sequence encoding the protein, a method for producing the protein, and a method for producing the same Download PDFInfo
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本發明係關於一種具有脯胺酸寡胜肽酶活性之蛋白質及其基因、生產方法與應用，尤關於一種具有碎葉鬼傘之脯胺酸寡胜肽酶活性之蛋白質及其基因、生產方法與應用。The present invention relates to a protein having proline oligopeptide activity and a gene, a production method and application thereof, and more particularly to a protein having the activity of valinoic acid oligopeptide activity of the genus Echinops and its gene, and a production method thereof And application.
脯胺酸寡胜肽酶(prolyl oligopeptidase)(EC 3.4.21.26)；也稱為脯胺酸內切胜肽酶(prolyl endopeptidase)或切脯胺酸後酶(post－proline cleaving enzyme)，能夠水解含脯胺酸之胜肽，其作用位置是在脯胺酸之羧基端(carboxyl side)(Polgr,1994；Polgr,2002)。Prolyl oligopeptidase (EC 3.4.21.26); also known as prolyl endopeptidase or post-proline cleaving enzyme, capable of hydrolyzing A peptide containing a proline that acts at the carboxyl side of the proline (Polg) r, 1994; Polg r, 2002).
脯胺酸寡胜肽酶近來應用性研究很廣泛，一方面是脯胺酸寡胜肽酶能降解許多與記憶及學習有關的胜肽，被認為與記憶喪失症(amnesia)(如帕金森氏症(Parkinsonism))有關，所以有研究尋找其抑制劑以作為治療(Yoshimotoet al. ,1987；Atacket al. ,1991；Marighettoet al. ,2000；Leeet al. ,2004；Sorensenet al ,2004；Atta－ur－Rahmanet al. ,2005；Jarhoet al. ,2005)；另一方面研究脯胺酸寡胜肽酶應用在解決因富含脯胺酸(proline)之麩質(gluten)所引起之腹瀉(celiac disease)(Piperet al. ,2004；Martiet al. ,2005；Matysiak－Budniket al. ,2005；Pyleet al. ,2005；Gasset al. ,2005；)；或用以純化回收異源性表現胜肽酶(Xiuet al ., 2002)；另外也有應用在治療癌症上的研究使藥物先以前體藥物(prodrug)存在，減少對正常細胞的傷害，經脯胺酸寡胜肽酶作用後成為有功效的藥物(Heiniset al .,2004)。Proline oligopeptidase has been widely used in recent applications. On the one hand, proline oligopeptidase can degrade many peptides related to memory and learning, and is considered to be associated with memory loss (amnesia) (such as Parkinson's). (Parkinsonism) is related, so there are studies to find its inhibitors for treatment (Yoshimoto et al. , 1987; Atack et al. , 1991; Marighetto et al. , 2000; Lee et al. , 2004; Sorensen et al , 2004; Atta-ur-Rahman et al. , 2005; Jarho et al. , 2005); on the other hand, the study of proline oligopeptidase is applied to solve gluten due to proline-rich gluten Caused by celiac disease (Piper et al. , 2004; Marti et al. , 2005; Matysiak-Budnik et al. , 2005; Pyle et al. , 2005; Gass et al. , 2005;); Purification of heterologous performance of peptidase (Xiu et al . , 2002); in addition, there are also applications in the treatment of cancer to make the drug prodrug, reduce damage to normal cells, by lysine Oligopeptides act as potent drugs (Heinis et al ., 2004).
脯胺酸寡胜肽酶存在動物、植物及微生物，但活性普遍不高。微生物方面脯胺酸寡胜肽酶曾在腦膜炎敗血黃桿菌(Flavobacterium meningosepticum )(0.30 U/ml；Yashimotoet al .,1980)、乳酸菌(Lactobacillus casei )(0.15 U/g；Habibi-Najafi and Lee,1994)、費氏丙酸桿菌(Propionibacterium freudenreichii )(4.3 mU/ml；Tobiassenet al .,1996)、洋菇(Agaricus bisporus )發酵液(0.15 U/ml；Abdus Sattaret al .,1990)、黃單胞菌(Xanthomonas spp.)(0.15 U/ml；Szwajcer-Deyet al .,1992)中被發現。Proline oligopeptidase is present in animals, plants and microorganisms, but activity is generally not high. In the microbial aspect, the proline oligopeptidase was found in Flavobacterium meningosepticum (0.30 U/ml; Yashimoto et al ., 1980) and Lactobacillus casei (0.15 U/g; Habibi-Najafi and Lee, 1994), Propionibacterium freudenreichii (4.3 mU/ml; Tobiassen et al ., 1996), Agaricus bisporus fermentation broth (0.15 U/ml; Abdus Sattar et al ., 1990) It was found in Xanthomonas spp. (0.15 U/ml; Szwajcer-Dey et al ., 1992).
以基因工程方式將脯胺酸寡胜肽酶基因選殖(cloning)後以大腸桿菌(Eschericia coli ,E .coli )等作為宿主細胞，進行異源性大量表現可提高酶活性，如莢膜鞘單胞菌(Sphingomonas capsulata )在大腸桿菌中表現脯胺酸寡胜肽酶活性(0.2 U/ml)比原始母株高約七倍(Yoshimotoet al .,1998)；腦膜炎敗血黃桿菌之脯胺酸寡胜肽酶基因在大腸桿菌中表現最高可得0.7 U/ml(Diefenthalet al .,1993)，Uchiyama等人將腦膜炎敗血黃桿菌之脯胺酸寡胜肽酶重新構築在大腸桿菌表現之活性最高可得8.1 U/ml，純化後其比活性可高達124 U/mg(Uchiyamaet al .,2000)；嗜水氣單胞菌(Aeromonas hydrophila )脯胺酸寡胜肽酶基因在大腸桿菌中表現為原始母株100倍(1.48 U/ml)，純化後其比活性為8.8 U/mg(Kanataniet al. ,1993)；斑點氣單胞菌(Aeromonas punctata )脯胺酸寡胜肽酶基因在大腸桿菌中表現為原始母株112倍，純化後其比活性為67 U/mg(Liet al. ,2000)。After genetic engineering manner proline oligopeptide gene cloning (Cloning) Escherichia coli (Eschericia coli, E. Coli) as a host cell and the like, for improved performance of a large number of heterologous enzymatic activity, such as a sheath capsular Sphingomonas capsulata exhibits prothrombin activity (0.2 U/ml) in E. coli approximately seven-fold higher than the original parent strain (Yoshimoto et al ., 1998); The proline oligopeptidase gene showed the highest expression in E. coli at 0.7 U/ml (Diefenthal et al ., 1993), and Uchiyama et al . reconstructed the proline oligopeptidase from M. septicum. The activity of E. coli is up to 8.1 U/ml, and its specific activity can be as high as 124 U/mg after purification (Uchiyama et al ., 2000); Aeromonas hydrophila proline oligopeptidase The gene expressed 100 times (1.48 U/ml) of the original parent strain in E. coli, and its specific activity was 8.8 U/mg after purification (Kanatani et al. , 1993); Aeromonas punctata proline The oligopeptidase gene is 112 times as large as the original parent strain in E. coli, and its specific activity is 67 U/mg after purification. (Li et al. , 2000).
另外，極端嗜熱厭氧古菌(Pyrococcus furiosus )脯胺酸寡胜肽酶基因在大腸桿菌中表現後純化之比活性為232 U/mg(但其活性定義與前述各相關研究所使用之定義有相當大的不同，為每分鐘使OD410 吸光值產生0.1的改變即為1U，Harwoodet al. ,1997)及4 U/mg(Harwood and Schreier,2001)。In addition, the specific activity of the hyperthermophilic anaerobic archaea ( Pyrococcus furiosus ) proline oligopeptidase gene after purification in E. coli was 232 U/mg (but its activity definition and the definitions used in the relevant studies mentioned above) There is a considerable difference between a 1 U change in the OD 410 absorbance per minute, Harwood et al. , 1997) and 4 U/mg (Harwood and Schreier, 2001).
以上各例顯示出利用大腸桿菌表現脯胺酸寡胜肽酶基因並加以純化，確可提高其活性。然而各種脯胺酸寡胜肽酶分別適合不同的反應條件。在適合之反應條件下方可充分發揮其活性，而在不適合之反應條件下則僅能發揮部分活性。一般而言，活性高，且其所適用之環境條件貼近實際應用的環境條件之脯胺酸寡胜肽酶，比較具有應用潛力。而在評價其應用潛力時，通常觀察其反應最適溫度及最適pH值，而其適用範圍之寬窄，則視其於非最適環境條件下保有之活性比例高低，並測量其受熱後所餘之活性，以知其熱安定性。The above examples show that the use of Escherichia coli to express the prolyl oligopeptidase gene and purification thereof can indeed improve its activity. However, various proline oligopeptidase enzymes are suitable for different reaction conditions, respectively. The activity can be fully exerted under suitable reaction conditions, and only partial activity can be exerted under unsuitable reaction conditions. In general, prolyl oligopeptidase, which has high activity and suitable environmental conditions close to the actual application environment, has potential application potential. When evaluating its application potential, it is usually observed the optimum temperature and optimum pH value of the reaction, and the wide range of its application depends on the proportion of activity retained under non-optimal environmental conditions, and the activity remaining after heating is measured. To know its thermal stability.
就前揭各脯胺酸寡胜肽而言，嗜水氣單胞菌之脯胺酸寡胜肽酶之最適溫度為30℃，最適pH為8.0，在42℃預熱30分鐘時活性會減少50%；斑點氣單胞菌之脯胺酸寡胜肽酶之最適溫度為34℃，最適pH為8.4；腦膜炎敗血黃桿菌之脯胺酸寡胜肽酶最適pH為7.0，最適反應溫度為40℃，在42℃預熱15分鐘活性會減少50%(Yashimotoet al. ,1980)，而經易錯PCR(error－prone PCR)突變後之脯胺酸寡胜肽酶在60℃預熱1小時後活性(於pH7.0,30℃測定)會減少50%(Uchiyamaet al. ,2000)。目前利用大腸桿菌表現脯胺酸寡胜肽酶基因所得脯胺酸寡胜肽酶之中，以腦膜炎敗血黃桿菌之脯胺酸寡胜肽酶比活性最高，且經易錯PCR突變後其耐熱性也最佳。但腦膜炎敗血黃桿菌之為一株病源菌，在應用上有所疑慮，而其他的脯胺酸寡胜肽酶則有耐熱性不佳的缺點。For the glutamate oligopeptides, the optimum temperature for the valeric acid oligopeptidase of Aeromonas hydrophila is 30 ° C, the optimum pH is 8.0, and the activity is reduced when preheated at 42 ° C for 30 minutes. 50%; the optimum temperature of the valinoic acid oligopeptidase of Aeromonas sphaeroides is 34 ° C, the optimum pH is 8.4; the optimum pH of the proline oligopeptidase of Flavobacterium meningitidis is 7.0, the optimum reaction temperature At 40 ° C, the activity is reduced by 50% by preheating at 42 ° C for 15 minutes (Yashimoto et al. , 1980), while the proline oligopeptidase after mutation by error-prone PCR is pre-treated at 60 ° C. After 1 hour of heat, the activity (measured at pH 7.0, 30 ° C) was reduced by 50% (Uchiyama et al. , 2000). Among the prolyl oligopeptidase enzymes which use the E. coli expression of the prolyl oligopeptidase gene, the specific activity of the prolyl oligopeptidase of M. septicum is the highest, and after the error-prone PCR mutation Its heat resistance is also the best. However, the meningococcal bacillus is a pathogenic bacterium, which has doubts in application, while other lysine oligopeptidase has the disadvantage of poor heat resistance.
然而，由於生物體種類繁多，找出適當之生物體，並從生物體中分離純化出具有所需性質之脯胺酸寡胜肽酶，具有相當之困難，非本發明所屬技術領域中具有通常知識者可輕易得知或思及。例如，以往針對黑麴菌(Aspergillus niger )進行研究而最初篩選到之脯胺酸寡胜肽酶，其後經序列比對發現應屬另一種絲胺酸蛋白酶(serine protease)，而非脯胺酸寡胜肽酶；而到目前為止，在真菌中之絲狀真菌，尚未曾發現具有脯胺酸寡胜肽酶者，僅曾經在擔子菌中發現具有脯胺酸寡胜肽酶之真菌，目前也未見將真菌之脯胺酸寡胜肽酶在大腸桿菌中大量表現的案例。However, due to the wide variety of organisms, it is quite difficult to find a suitable organism and to isolate and purify the proline oligopeptidase having the desired properties from the organism, which is not common in the technical field to which the present invention pertains. Knowledgeable people can easily learn or think about it. For example, the lysine oligopeptidase originally screened for the study of Aspergillus niger was later sequenced and found to be another serine protease instead of guanamine. Acid oligopeptides; and so far, filamentous fungi in fungi have not been found to have glutamate oligopeptidase, and only fungi with valinoic acid oligopeptidase have been found in basidiomycetes. There have been no cases in which a large number of fungal glutamate oligopeptidases are expressed in Escherichia coli.
有鑒於前述脯胺酸寡胜肽酶種類不足，而難以配合不同應用條件之問題，本發明之目的在於提供一種具有優異的脯胺酸寡胜肽酶活性，且耐熱性較佳的蛋白質及其基因、生產方法與應用。In view of the above-mentioned problems of insufficient proline oligopeptidase and difficulty in adapting to different application conditions, the object of the present invention is to provide a protein having excellent proline oligopeptidase activity and better heat resistance. Genes, production methods and applications.
本發明之發明人經研究及努力，終於自碎葉鬼傘(Coprinus clastophyllus )中，分離出可達成本發明目的之脯胺酸寡胜肽酶及其基因與生產方法。Through research and efforts, the inventors of the present invention finally isolated the proline oligopeptidase and its gene and production method which can achieve the purpose of the invention from Coprinus clastophyllus .
為達成上述目的，本發明提供一種經分離的蛋白質，該蛋白質具有脯胺酸寡胜肽酶之活性，其係選自由下列組成之群(a)具有胺基酸序列SEQ ID NO：6或SEQ ID NO：10之蛋白質；(b)由具有序列SEQ ID NO：5或SEQ ID NO：9之核酸序列所編碼之蛋白質；(c)具有與蛋白質(a)或(b)之胺基酸序列序列相似性大於60%的胺基酸序列，且具有同一活性之蛋白質。In order to achieve the above object, the present invention provides an isolated protein having the activity of a proline oligopeptidase which is selected from the group consisting of (a) having an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: a protein of 10; (b) a protein encoded by the nucleic acid sequence having the sequence of SEQ ID NO: 5 or SEQ ID NO: 9; (c) having an amino acid sequence with the protein (a) or (b) Amino acid sequence having a sequence similarity greater than 60% and having the same active protein.
其中由於胺基酸序列之變異並不必然影響其構成蛋白質之活性，因此只要該胺基酸序列之變異不影響該蛋白質之活性，仍屬於本發明之範圍。Since the variation of the amino acid sequence does not necessarily affect the activity of the constituent protein, it is within the scope of the present invention as long as the variation of the amino acid sequence does not affect the activity of the protein.
本發明亦提供一種經分離的核酸，其係選自由下列組成之群：(a)編碼具有前述蛋白質之核酸；(b)具有序列SEQ ID NO：5之核酸；(c)具有與核酸(b)之核酸序列的序列相似性大於60%，且編碼具有同一活性之蛋白質之核酸；(d)具有序列SEQ ID NO：9之核酸；(e)具有與核酸(d)之核酸序列的序列相似性大於60%之核酸序列，且編碼具有同一活性之蛋白質之核酸；(f)與核酸(a)至(e)編碼具有相同胺基酸序列的蛋白質之核酸；(g)核酸(a)至(f)之反義核酸。The present invention also provides an isolated nucleic acid selected from the group consisting of: (a) a nucleic acid having the aforementioned protein; (b) a nucleic acid having the sequence of SEQ ID NO: 5; (c) having a nucleic acid (b) a nucleic acid sequence having a sequence similarity greater than 60% and encoding a nucleic acid having the same active protein; (d) a nucleic acid having the sequence of SEQ ID NO: 9; (e) having a sequence similar to the nucleic acid sequence of nucleic acid (d) a nucleic acid sequence greater than 60%, and a nucleic acid encoding a protein having the same activity; (f) a nucleic acid encoding a protein having the same amino acid sequence as the nucleic acid (a) to (e); (g) a nucleic acid (a) to (f) antisense nucleic acid.
其中由於核酸序列之變異並不必然影響本發明所提供之序列編碼蛋白質之活性，因此只要在核酸序列之變異不影響該蛋白質之活性，仍屬於本發明之範圍。Where the variation of the nucleic acid sequence does not necessarily affect the activity of the sequence-encoded protein provided by the present invention, it is within the scope of the present invention as long as the variation in the nucleic acid sequence does not affect the activity of the protein.
本發明提供一種核酸探針，其係可藉由下列高度嚴格條件與前述之核酸雜合：(a)在FastHyb溶液中於50℃下反應16小時；(b)以適量2 X SSC,0.1 % SDS溶液於室溫下沖洗五分鐘；(c)反覆前述步驟(b)一次；(d)以0.5 X SSC,0.1 % SDS溶液於65℃下充分作用15分鐘；(e)反覆前述步驟(d)一次。The present invention provides a nucleic acid probe which can be heterozygously mixed with the aforementioned nucleic acid by the following highly stringent conditions: (a) reacting in a FastHyb solution at 50 ° C for 16 hours; (b) taking an appropriate amount of 2 X SSC, 0.1 % The SDS solution is rinsed at room temperature for five minutes; (c) the above step (b) is repeated once; (d) is fully effected with 0.5 X SSC, 0.1% SDS solution at 65 ° C for 15 minutes; (e) repeating the foregoing steps (d) )once.
本發明提供一種具有前述核酸之嵌合基因，其係可操作的連結到一驅動子，而該驅動子可將該嵌合基因於一細胞中加以驅動表現。The present invention provides a chimeric gene having the aforementioned nucleic acid, which is operably linked to a driver which can drive the chimeric gene to be expressed in a cell.
一種核酸構築體，其包括前述之核酸，且該核酸係可操作地連接到控制序列，而該控制序列能夠在特定細胞中驅動該核酸所編碼的蛋白。A nucleic acid construct comprising the nucleic acid described above, and the nucleic acid is operably linked to a control sequence which is capable of driving a protein encoded by the nucleic acid in a particular cell.
本發明提供一種載體，其包括有前述之核酸或前述之核酸構築體。The present invention provides a vector comprising the nucleic acid described above or the nucleic acid construct described above.
本發明提供一種轉形株(transformant)，其包括有前述之載體。The present invention provides a transformant comprising the aforementioned vector.
前述之轉形株，其係一BL21(DE3)大腸桿菌菌株。The aforementioned transformant strain is a BL21 (DE3) Escherichia coli strain.
前述之轉形株，其係一DH10B大腸桿菌菌株。The aforementioned transformed strain is a DH10B E. coli strain.
本發明提供一種具有解決因富含脯胺酸之麩質所引起腹瀉之功能的醫藥組成物，其含有前述之蛋白質作為有效成分，並含有醫藥上可接受之賦形劑。The present invention provides a pharmaceutical composition having a function of solving diarrhea caused by glutamic acid-rich gluten, which comprises the aforementioned protein as an active ingredient and contains a pharmaceutically acceptable excipient.
本發明提供一種組成物，其含有一前體藥物、一醫藥上可接受之賦形劑與如申請專利第1項所述之蛋白質，該蛋白質係作為有效成分以處理該前體藥物而使其成為一有功效藥物。The present invention provides a composition comprising a prodrug, a pharmaceutically acceptable excipient, and the protein of claim 1, which is used as an active ingredient to treat the prodrug Become a potent drug.
本發明提供一種使用申請專利第1項所述之蛋白質之應用，其係用於製造純化回收異源性表現胜肽之回收處理劑。The present invention provides an application using the protein of claim 1, which is used for the production of a recovery treatment for purifying and recovering a heterologous performance peptide.
本發明提供一種生產具有脯胺酸寡胜肽酶活性之蛋白質之方法，其包括有以下步驟：(a)提供前述轉形株；(b)在可令具有脯胺酸寡胜肽酶活性之蛋白質被表現之環境下培養該轉形株；及(c)進行純化以獲取該蛋白質。The present invention provides a method for producing a protein having glutamate oligopeptidase activity, which comprises the steps of: (a) providing the aforementioned transformant; (b) allowing for the activity of proline oligopeptidase The protein is cultured in the presence of the transformed strain; and (c) purified to obtain the protein.
由上述可知本發明所提供碎葉鬼傘脯胺酸寡胜肽酶之性質及其基因序列與生產方法之實施步驟，藉由該脯胺酸寡胜肽酶之性質，其所適用之範圍恰可彌補既有脯胺酸寡胜肽酶適用範圍之不足，具有更優秀之應用潛力；另一方面，藉由本發明所提供脯胺酸寡胜肽酶基因，可有效的利用前述之生產方法加以生產以供應用。It can be seen from the above that the nature of the genomic acid oligopeptidase provided by the present invention and the sequence of the gene sequence and the production method thereof are carried out by the nature of the glutamic acid oligopeptidase. It can make up for the deficiency of the application range of the pro- lysine oligopeptidase and has better application potential; on the other hand, the proline oligopeptidase gene provided by the invention can effectively utilize the aforementioned production method. Production for supply.
相同度(Identity)：術語「相同度」於本文定義為二核酸序列之間或二胺基酸序列之間相互不歧異的程度。在本說明書中該相同度係採用基礎區域排比搜尋工具(Basic Local Alignment Search Tool，BLAST)程式測出Identity之讀值。Identity: The term "identity" is defined herein to the extent that the two nucleic acid sequences or the diamino acid sequences are not different from each other. In this specification, the identity is measured using the Basic Local Alignment Search Tool (BLAST) program.
相似度(Similarity)：術語「相似度」於本文定義為二序列之間相關連的程度，其可以序列間相同比率及/或保留比率定之。在本說明書中該相同度係採用基礎區域排比搜尋工具(Basic Local Alignment Search Tool，BLAST)程式測出Similarity之讀值。Similarity: The term "similarity" is defined herein as the degree of association between two sequences, which can be determined by the same ratio and/or retention ratio between sequences. In this specification, the same degree is measured by the Basic Local Alignment Search Tool (BLAST) program to measure the value of Similarity.
嵌合基因(chimeric gene)：術語「嵌合基因」於本文定義為攜帶來自不同來源之核酸之重組核酸形成之基因。例如來自相互無關之不同基因之核酸組成者，或由來自一基因的一部分片段與另一基因的一部分片段以重組技術形成者。Chimeric gene: The term "chimeric gene" is defined herein as a gene that is formed by recombinant nucleic acids carrying nucleic acids from different sources. For example, a nucleic acid component from a different gene that is not related to each other, or a part of a fragment from one gene and a part of another gene are formed by recombinant techniques.
控制序列(controlling sequence)：術語「控制序列」於本文定義為一種能使一基因之核酸序列啟動或關閉，從而影響該基因核酸序列表現之序列；例如該控制序列可為一驅動子或一編碼訊號胜肽(signal peptide)之序列。Controlling sequence: The term "control sequence" is defined herein as a sequence that enables a nucleic acid sequence of a gene to be turned on or off, thereby affecting the expression of the nucleic acid sequence of the gene; for example, the control sequence can be a driver or an encoding The sequence of the signal peptide.
載體(vector)：術語「載體」於本文定義為一種用以轉移一核酸進入一宿主細胞之物，例如質體、噬菌體和其他病毒等。該載體亦可為一表現載體，其可例行地接受諸如具有重組核酸序列之核酸，並在將該核酸送入該宿主細胞之後，引起該核酸之核酸序列表現。其中載體與宿主細胞在配對使用上，係典型地根據載體與宿主細胞之間的相容性加以選擇。又，該載體可為線性或封閉環狀之核酸所構成之質體。Vector: The term "vector" is defined herein as a substance used to transfer a nucleic acid into a host cell, such as plastids, bacteriophages, and other viruses. The vector may also be an expression vector that routinely accepts, for example, a nucleic acid having a recombinant nucleic acid sequence and, after the nucleic acid is introduced into the host cell, causes the nucleic acid sequence of the nucleic acid to behave. Where the vector is used in pair with the host cell, it is typically selected based on the compatibility between the vector and the host cell. Further, the vector may be a plastid composed of a linear or closed circular nucleic acid.
由於脯胺酸寡胜肽酶如前述與記憶學習相關胜肽之降解、以及富含脯胺酸之麩質所引起之腹瀉均有所關聯，且可用以純化回收異源性表現胜肽，或作用於前體藥物使之形成有效藥物，因此具有相當之研究與應用價值。Since the proline oligopeptidase is associated with the degradation of the memory learning related peptide and the diarrhea caused by the proline-rich gluten, and can be used for purification to recover the heterologous peptide, or It acts on prodrugs to form effective drugs, so it has considerable research and application value.
本發明提供具有碎葉鬼傘脯胺酸寡胜肽酶活性之蛋白質及其基因序列與生產方法之相關數據與具體實施步驟。本發明之具有脯胺酸寡胜肽酶活性之蛋白質所適用之範圍恰可彌補既有脯胺酸寡胜肽酶適用範圍之不足，而該脯胺酸寡胜肽酶更具有極高之活性而具有更優秀之應用潛力；另一方面，可有效的利用本發明所提供分離自碎葉鬼傘之脯胺酸寡胜肽酶基因，以本發明所揭露之生產方法並進行製造醫藥組成物等相關應用。The present invention provides data and specific implementation steps for a protein having the activity of the genus Pseudomonas oligopeptide activity and its gene sequence and production method. The protein of the present invention having valeric acid oligopeptidase activity is suitable for the deficiency of the application range of the glutamic acid oligopeptidase, and the proline oligopeptidase is extremely active. In addition, the present invention can effectively utilize the proline oligopeptidase gene isolated from the genus Coprinus comatus, and the manufacturing method disclosed in the present invention and the manufacture of the pharmaceutical composition Related applications.
又，藉由本發明提供之具有碎葉鬼傘脯胺酸寡胜肽酶活性之蛋白質，可用以製造醫藥組成物以解決因富含脯胺酸之麩質所引起腹瀉、或處理前體藥物使之成為有功效藥物；亦可用以製造能純化回收異源性表現胜肽之回收處理劑。Further, the protein having the activity of the genus Echinopin oligopeptidase provided by the present invention can be used to produce a pharmaceutical composition for solving diarrhea caused by proline-rich gluten or treating a prodrug. It can be used as an effective drug; it can also be used to manufacture a recovery treatment agent capable of purifying and recovering heterologous performance peptides.
除此之外，本發明亦揭露了相關之核酸、載體及轉形株以作為生產與進一步研究之用。In addition, the present invention also discloses related nucleic acids, vectors and transformants for use in production and further research.
根據本發明可作之不同修正及變化，以及所生產之脯胺酸寡胜肽酶在濃度數據或活性數據之小幅差距等，對於熟悉該項技術者而言均顯然不會偏離本發明的範圍與精神。雖然本發明已敘述特定的較佳具體事實，必須瞭解的是本發明不應被不當地限制於該等特定具體事實上。在實施本發明之已述模式方面，對於熟習該項技術者而言，諸如採用不同的既有生物學方法或其他所屬技術領域中的習知技術等，均屬於顯而易知之不同修正而同樣的被涵蓋於本文之申請專利範圍之內。Different modifications and variations may be made in accordance with the present invention, as well as small differences in concentration data or activity data produced by the proline oligopeptidase, etc., and it will be apparent to those skilled in the art that they do not depart from the scope of the present invention. With the spirit. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In terms of implementing the modes of the present invention, those skilled in the art, such as using different existing biological methods or other prior art techniques, are well-known and differently modified. It is covered by the scope of the patent application herein.
蛋白質 本發明係相關一種經分離且具有脯胺酸寡胜肽酶活性之蛋白質。為了提高脯胺酸寡胜肽酶之活性，在本發明中由原先所篩選到含有熱安定性較佳的脯胺酸寡胜肽酶(胞外活性0.03 U/ml)的碎葉鬼傘菌株(財團法人食品工業發展研究所生物資源保存及研究中心，編號BCRC 36074)中，選殖出脯胺酸寡胜肽酶之cDNA，並將其利用大腸桿菌作為宿主細胞以大量表現而獲取該蛋白質。 Proteins The present invention relates to a protein which has been isolated and has valyanate oligopeptide activity. In order to increase the activity of the prolyl oligopeptidase, in the present invention, the previously selected hyaluronan oligopeptidase (extracellular activity 0.03 U/ml) having a better thermal stability is screened. (The Center for Bioresource Conservation and Research, Institute of Food Industry Development, No. BCRC 36074), the cDNA of proline oligopeptidase was selected and used to obtain the protein in large quantities using E. coli as a host cell. .
若定義在pH為7.0，反應溫度為45℃之條件下，每分鐘產生1微莫耳(μmole)重氮化對硝基苯胺之脯胺酸寡胜肽酶活性為1 U，則該具有脯胺酸寡胜肽酶活性之蛋白質之活性在大腸桿菌菌株中於胞內所表現之脯胺酸寡胜肽酶活性為7.0 U/ml至8.0 U/ml，更佳的是7.2 U/ml至7.7 U/ml，最佳的是7.2 U/ml與7.7 U/ml；將該蛋白質純化後，其比活性為55.0 U/mg至70.0 U/mg，更佳的是56.1 U/mg至66.8 U/mg，最佳的是56.1 U/mg與66.8 U/mg。If it is defined at a pH of 7.0 and a reaction temperature of 45 ° C, a micromolecular (μmole) diazotized p-nitroaniline valeric acid oligopeptidase activity of 1 U per minute is obtained. The activity of the protein of the amino acid oligopeptide activity in the E. coli strain is in the range of 7.0 U/ml to 8.0 U/ml, more preferably 7.2 U/ml. 7.7 U/ml, optimally 7.2 U/ml and 7.7 U/ml; after purification of the protein, the specific activity is 55.0 U/mg to 70.0 U/mg, more preferably 56.1 U/mg to 66.8 U /mg, the best is 56.1 U/mg and 66.8 U/mg.
又，該蛋白質最適反應pH為pH6至pH8，更佳的是pH6至pH7，最佳的是pH7。其於pH7之最適反應溫度係45℃，而其於pH8之最適反應溫度係37℃，且在受到未達55℃之短時間預熱時，仍保有相當之脯胺酸寡胜肽酶活性。Further, the optimum pH of the protein is pH 6 to pH 8, more preferably pH 6 to pH 7, and most preferably pH 7. The optimum reaction temperature at pH 7 is 45 ° C, and the optimum reaction temperature at pH 8 is 37 ° C, and still retains comparable glutamate luciferase activity when preheated for less than 55 ° C for a short period of time.
該蛋白質，較佳的是一種具有胺基酸序列SEQ ID NO：6或SEQ ID NO：10之蛋白質，或由具有序列SEQ ID NO：5或SEQ ID NO：9之核酸所編碼之蛋白質。又，該蛋白質具有與前述胺基酸序列SEQ ID NO：6、SEQ ID NO：10、或由SEQ ID NO：5、SEQ ID NO：9編碼之胺基酸序列之序列相似性大於60%以上的胺基酸序列，且具有同一活性；較佳的是上述序列相似度較佳是70%以上，較佳的是上述序列相似度較佳是80%以上，更佳的是上述序列相似度較佳是90%以上，最佳的是上述序列相似度較佳是95%以上。即使胺基酸序列之間有所變異，其蛋白質仍可能發揮本發明所提供脯胺酸寡胜肽酶之活性；因此所屬領域中具有通常知識者可以理解：只要序列之變異不影響該蛋白質之功能或活性，仍屬於本發明之範圍。The protein is preferably a protein having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 10, or a protein encoded by the nucleic acid having the sequence of SEQ ID NO: 5 or SEQ ID NO: 9. Further, the protein has a sequence similarity to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 10, or the amino acid sequence encoded by SEQ ID NO: 5, SEQ ID NO: 9 of more than 60%. The amino acid sequence has the same activity; preferably, the sequence similarity is preferably 70% or more, and preferably the sequence similarity is preferably 80% or more, and more preferably the sequence similarity is Preferably, it is 90% or more, and it is preferable that the above sequence similarity is preferably 95% or more. Even if there is a variation between the amino acid sequences, the protein may still exert the activity of the proline oligopeptidase provided by the present invention; therefore, those of ordinary skill in the art can understand that as long as the variation of the sequence does not affect the protein Function or activity is still within the scope of the invention.
再者，該蛋白質係由具有與序列SEQ ID NO：5或SEQ ID NO：9的序列相似性大於60%之核酸所編碼，且與前述蛋白質具有同一功能之蛋白質。Further, the protein is a protein which is encoded by a nucleic acid having a sequence similarity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 9 and which has the same function as the aforementioned protein.
另外，該蛋白質係由一核酸所編碼，該核酸係可藉由：在FastHyb溶液中於50℃下反應16小時；以適量2 X SSC,0.1 % SDS溶液於室溫下沖洗五分鐘；如前一步驟再反覆沖洗一次；以0.5 X SSC,0.1 % SDS溶液於65℃下充分作用15分鐘；如前一步驟再反覆作用一次；之高度嚴格條件而得以與具有序列SEQ ID NO：5或SEQ ID NO：9之核酸雜合之核酸，且該蛋白質與前述本發明之蛋白質具有同一功能。In addition, the protein is encoded by a nucleic acid by: reacting in a FastHyb solution at 50 ° C for 16 hours; washing with an appropriate amount of 2 X SSC, 0.1% SDS solution at room temperature for five minutes; Rinse again in one step; use 0.5 X SSC, 0.1% SDS solution at 65 ° C for 15 minutes; repeat the previous step as in the previous step; with high stringency conditions and with sequence SEQ ID NO: 5 or SEQ ID NO: A nucleic acid heterozygous for a nucleic acid of 9, and the protein has the same function as the aforementioned protein of the present invention.
核酸 本發明亦相關於一種經分離之核酸，其具有編碼前述本發明蛋白質之核酸序列。該核酸係編碼具有前述蛋白質之核酸，較佳的是，其係具有序列SEQ ID NO：5或SEQ ID NO：9之核酸，或是具有與序列SEQ ID NO：5或SEQ ID NO：9之序列相似性大於60%之核酸序列，且編碼具有同一活性之蛋白質之核酸，皆屬於本發明涵蓋之範圍；較佳的是上述序列相似度較佳是70%以上，較佳的是上述序列相似度較佳是80%以上，更佳的是上述序列相似度較佳是90%以上，最佳的是上述序列相似度較佳是95%以上。 Nucleic Acids The invention also relates to an isolated nucleic acid having a nucleic acid sequence encoding a protein of the invention described above. The nucleic acid encoding a nucleic acid having the aforementioned protein, preferably having the nucleic acid of the sequence of SEQ ID NO: 5 or SEQ ID NO: 9, or having the sequence of SEQ ID NO: 5 or SEQ ID NO: A nucleic acid sequence having a sequence similarity greater than 60% and encoding a nucleic acid having the same active protein is within the scope of the present invention; preferably, the sequence similarity is preferably 70% or more, and preferably the above sequence is similar. The degree is preferably 80% or more, and more preferably the above sequence similarity is preferably 90% or more. Most preferably, the above sequence similarity is preferably 95% or more.
其中，序列SEQ ID NO：5與SEQ ID NO：9之間以及上述為本發明所涵蓋之核酸序列之間雖有變異，但其所編碼具有如前述SEQ ID NO：6及SEQ ID NO：10等胺基酸序列或與之具有相當序列相似性之胺基酸序列的蛋白質均能發揮前述脯胺酸寡胜肽酶之功能，因此只要序列之變異不影響其所編碼蛋白質之功能或活性，仍屬於本發明之範圍。Wherein the sequence between SEQ ID NO: 5 and SEQ ID NO: 9 and the nucleic acid sequences encompassed by the present invention are mutated, but are encoded as SEQ ID NO: 6 and SEQ ID NO: 10 above. A protein having an amino acid sequence or an amino acid sequence having comparable sequence similarity can function as the aforementioned proline oligopeptidase, so that as long as the variation of the sequence does not affect the function or activity of the protein encoded thereby, Still falls within the scope of the invention.
又，與前述核酸編碼具有相同胺基酸序列的蛋白質之核酸，以及前述核酸之反義核酸，對所屬領域中具有通常知識者而言並不會偏離本發明之範圍，仍屬於本發明之範圍。Further, the nucleic acid encoding the protein having the same amino acid sequence as the nucleic acid described above, and the antisense nucleic acid of the nucleic acid described above, without departing from the scope of the present invention, are still within the scope of the present invention. .
核酸探針(probe) 本發明亦相關於一種核酸探針，其係可藉由：在FastHyb溶液中於50℃下反應16小時；以適量2 X SSC,0.1 % SDS溶液於室溫下沖洗五分鐘；如前一步驟再反覆沖洗一次；以0.5 X SSC,0.1 % SDS溶液於65℃下充分作用15分鐘；如前一步驟再反覆作用一次；之高度嚴格條件而得以與前述之核酸雜合。 Nucleic acid probes The invention also relates to a nucleic acid probe which can be prepared by reacting in a FastHyb solution at 50 ° C for 16 hours; washing with an appropriate amount of 2 X SSC, 0.1% SDS solution at room temperature. Minute; repeat the previous step as in the previous step; fully act for 15 minutes at 0.5 °C SSC, 0.1% SDS solution at 65 °C; once again in the previous step; highly stringent conditions to be heterozygous with the aforementioned nucleic acid .
嵌合基因 本發明亦相關於一種具有前述核酸之嵌合基因，其係可操作的連結到一驅動子，而該驅動子可將該嵌合基因於一細胞中加以驅動表現。為研究或商業上之應用而將一核酸配合其他之核酸組合成嵌合基因以達其應用之目的，係為所屬技術領域中具有通常知識者可充份理解而能配合需要實施者。 Chimeric Genes The present invention also relates to a chimeric gene having the aforementioned nucleic acid operably linked to a driver which can be driven to display the chimeric gene in a cell. The combination of a nucleic acid and other nucleic acids into a chimeric gene for research or commercial use for the purpose of its application is well understood by those of ordinary skill in the art and can be adapted to the needs of the practitioner.
核酸構築體(construct)及載體 本發明亦相關於一種核酸構築體，其包括前述之核酸，且該核酸係可操作地連接到一控制序列，而該控制序列至能夠在特定細胞中驅動該核酸所編碼的蛋白質。該等核酸構築體，例如重組質體，在研究或商業上均有多種應用，以一核酸構築一核酸構築體係為所屬技術領域中具有通常知識者可充份理解而能配合需要實施者。 Nucleic acid constructs and vectors The present invention is also related to a nucleic acid construct comprising the nucleic acid described above, and the nucleic acid is operably linked to a control sequence which is capable of driving the nucleic acid in a particular cell The encoded protein. Such nucleic acid constructs, such as recombinant plastids, are used in research or commercial applications, and a nucleic acid constructing nucleic acid constructing system can be fully understood by those skilled in the art and can be implemented as needed.
又，本發明亦相關於一種載體，該載體係包括有前述本發明所相關之核酸或前述核酸構築體。Further, the present invention is also related to a vector comprising the nucleic acid or the nucleic acid construct of the present invention as described above.
轉形株 本發明亦相關於一種轉形株(transformant)，其係一接受前述本發明所相關之核酸而被轉形之宿主細胞。該宿主細胞可為一大腸桿菌，該大腸桿菌菌株之例示包括有BL21(DE3)或DH10B等大腸桿菌菌株。 Transformation strain of the present invention is also related to one Transformation strain (transformant), which system accepts a nucleic acid of the present invention is related to the type of host cell to be transfected. The host cell may be an Escherichia coli, and the Escherichia coli strain is exemplified by an Escherichia coli strain such as BL21 (DE3) or DH10B.
組成物 本發明亦相關於一種組成物，例如醫藥組成物，其包括有前述本發明所提供之蛋白質。 Composition The present invention is also related to a composition, such as a pharmaceutical composition, comprising the protein provided by the aforementioned invention.
解決因富含脯胺酸之麩質所引起腹瀉，且本發明之具有脯胺酸寡胜肽酶活性之蛋白質所適用之範圍恰可彌補既有脯胺酸寡胜肽酶適用範圍之不足，因此本發明之組成物更具優秀之應用潛力。其中，該有效成分係指該蛋白質成為該組成物之成分，而能發揮該組成物所欲達成之功效或功能；且本發明之組成物可更進一步包括有一賦形劑，以讓該組成物被調配成例如固體、半固體或液體等適當形態，使之更便於使用。To solve the diarrhea caused by glutamic acid-rich gluten, and the protein of the present invention having valeric acid oligopeptidase activity is suitable for the deficiency of the application range of the glutamic acid oligopeptidase. Therefore, the composition of the present invention has an excellent application potential. Wherein, the active ingredient means that the protein becomes a component of the composition, and can exert the effect or function desired by the composition; and the composition of the present invention may further comprise an excipient to allow the composition to It is formulated into a suitable form such as a solid, semi-solid or liquid to make it easier to use.
又，本發明亦相關於一種可配合具有哺胺酸殘基(residue)之前體藥物使用之組成物，其含有至少一醫藥上可接受之賦形劑與前述之蛋白質，並利用該蛋白質作為有效成分以發揮其脯胺酸寡胜肽酶活性，以處理該前體藥物而使其成為一有功效藥物。更佳的是，其進一步含有一與前述蛋白質結合之抗體。藉由將該抗體與前述蛋白質相結合，當該組成物被送入生物體之循環系統時，該蛋白質在生物體中可透過該抗體與特定組織之結合而定位。再接著將可配合使用之前體藥物送入生物體之循環系統，則由於僅在前述蛋白質定位處，該前體藥物方得以被該蛋白質所發揮之脯胺酸寡胜肽酶活性剪切為有效之藥物，因此可大幅增加該前體藥物之選擇性，而使作用之範圍更為精確，降低對其他組織產生影響。Further, the present invention is also related to a composition which can be used in combination with a drug having a precursor of a glutamic acid residue, which contains at least one pharmaceutically acceptable excipient and the aforementioned protein, and utilizes the protein as an effective The component exerts its prolyl oligopeptidase activity to treat the prodrug to make it an effective drug. More preferably, it further contains an antibody that binds to the aforementioned protein. By binding the antibody to the aforementioned protein, when the composition is delivered to the circulatory system of the organism, the protein can be localized in the organism by binding of the antibody to a particular tissue. Then, the medicinal system can be administered to the circulatory system of the living body, and the prodrug moiety can be effectively cut by the proline oligopeptidase activity exerted by the protein only at the aforementioned protein localization. The drug, therefore, can greatly increase the selectivity of the prodrug, and the scope of action is more precise, reducing the impact on other tissues.
用途 本發明亦相關於一種使用前述本發明之蛋白質之應用，其係利用前述蛋白質作為有效成分，發揮其脯胺酸寡胜肽酶活性，從而處理異源性表現胜肽以供回收，以用於製造純化回收異源性表現胜肽之回收處理劑。 Uses The present invention is also related to the use of the aforementioned protein of the present invention, which utilizes the aforementioned protein as an active ingredient to exert its valeric acid oligopeptidase activity, thereby treating the heterologous expression peptide for recovery, A recovery treatment agent for purifying and recovering a heterologous performance peptide.
生產方法 本發明亦相關於一種生產具有脯胺酸寡胜肽酶活性之蛋白質之方法，其係以提供前述轉形株；在可令具有脯胺酸寡胜肽酶活性之蛋白質被表現之環境下培養該轉形株；以及進行純化以獲取該蛋白質等步驟所組成。其中，該宿主細胞可採用但不限於前述BL21(DE3)或DH10B等大腸桿菌菌株形成之轉形株。 Production method The present invention is also related to a method for producing a protein having glutamate oligopeptidase activity, which is to provide the aforementioned transformant strain; an environment in which a protein having proline oligopeptidase activity can be expressed The transformed strain is cultured; and the step of purifying to obtain the protein is used. Wherein, the host cell can adopt, but is not limited to, the transformed strain formed by the Escherichia coli strain such as BL21 (DE3) or DH10B.
本發明之實際實施態樣及實施方法如下所示，但本發明所屬領域中常用、眾所周知或可得而知的各種已確立之技術手段，則酌減冗贅重覆之說明。又，各實施例係用以說明本發明而非限定本發明於該等實施例上。The actual embodiments and implementation methods of the present invention are as follows, but various established technical means that are commonly used, well-known or available in the field to which the present invention pertains are described as redundant and redundant. Further, the embodiments are intended to illustrate the invention and not to limit the invention to the embodiments.
建立碎葉鬼傘cDNA庫(cDNA library)並選殖脯胺酸寡胜肽酶之cDNA 1.碎葉鬼傘脯胺酸寡胜肽酶基因片段之選殖及探針之製備 本發明先以人類的脯胺酸寡胜肽酶之胺基酸序列在NCBI網站上以blast分析真菌之基因體序列，找到較相關的灰蓋鬼傘岡山7#130菌株(Coprinopsis cinerea okayama7#130)與黃孢原毛平革菌RP－78菌株(Phanerochaete chrysosporium RP－78)之脯胺酸寡胜肽酶，並在本實施例中以灰蓋鬼傘岡山7#130菌株(http：//www.ncbi.nlm.nih.gov/sites/entrez？db＝genomeprj&cmd＝Retrieve&dopt＝Overview&list_uids＝9596)與黃孢原毛平革菌RP－78菌株(http：//www.ncbi.nlm.nih.gov/sites/entrez？db＝genomeprj&cmd＝Retrieve&dopt＝Overview&list_uids＝9525)二者之脯胺酸寡胜肽酶基因相似度高的區域設計引子(primer)，獲取Pro16引子：「5’－tacggcggmttcascatctc－3’(SEQ ID NO：1)」及Pro17引子：「5’－tgccaytcytcwccraactc－3’(SEQ ID NO：2)」，再以碎葉鬼傘DNA作為模板DNA(template DNA)，進行PCR(polymerase chain reaction，聚合酶連鎖反應)。 Establishing a cDNA library and selecting a cDNA of glutamate oligopeptidase 1. Selection of the fragment of the genomic acid oligopeptidase gene and preparation of the probe The invention firstly The amino acid sequence of the human proline oligopeptidase was analyzed by blast on the NCBI website to find the more relevant genus of the genus Ganoderma lucidum Okayama 7#130 ( Coprinopsis cinerea okayama7#130) and Xanthomonas. The lysine oligopeptidase of P. stipitis RP-78 strain ( Phanerochaete chrysosporium RP-78), and in this example, G. sylvestris Okayama 7#130 strain (http://www.ncbi.nlm) .nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=9596) with P. chrysosporium RP-78 strain (http://www.ncbi.nlm.nih.gov/sites/entrez?db =genomeprj&cmd=Retrieve&dopt=Overview&list_uids=9525) Primer for the region with high similarity of the proline oligopeptidase gene, obtain Pro16 primer: "5'-tacggcggmttcascatctc-3' (SEQ ID NO: 1) And Pro17 primer: "5'-tgccaytcytcwccraactc-3' (SEQ ID NO: 2)", and then broken leaves Umbrella DNA as template DNA (template DNA), performed PCR (polymerase chain reaction, polymerase chain reaction).
為進行PCR所調製之PCR混合液包括有：「最終濃度成份為1倍之PCR緩衝液、dNTP 0.2 mM、Pro16引子及Pro17引子各1 μM、5 U pfu DNA複製酶(pfu DNA polymerase)(Roach)以及適量的模板DNA(即前述碎葉鬼傘或cDNA)」並令該PCR混合液總體積為50 μl。The PCR mixture prepared for PCR includes: "1 time PCR buffer, dNTP 0.2 mM, Pro16 primer and Pro17 primer 1 μM, 5 U pfu DNA polymerase (Roach) And an appropriate amount of template DNA (ie, the aforementioned leafhopper or cDNA) and the total volume of the PCR mixture was 50 μl.
PCR所使用儀器為ABI 9700(Applied Bioscience)，所使用條件為：94℃,3分鐘[循環1次]；94℃,30秒,64℃,30秒,72℃,1分鐘[循環5次]；94℃,30秒,60℃,30秒,72℃,1分鐘[循環5次]；94℃,30秒,56℃,30秒,72℃,1分鐘[循環35次]；72℃,7分鐘[循環1次]。The instrument used for PCR was ABI 9700 (Applied Bioscience) under the conditions of 94 ° C, 3 minutes [1 cycle]; 94 ° C, 30 seconds, 64 ° C, 30 seconds, 72 ° C, 1 minute [5 cycles] ; 94 ° C, 30 seconds, 60 ° C, 30 seconds, 72 ° C, 1 minute [5 cycles]; 94 ° C, 30 seconds, 56 ° C, 30 seconds, 72 ° C, 1 minute [cycle 35 times]; 72 ° C, 7 minutes [1 cycle].
藉由上述之PCR，可自前述模板DNA上擴增出一長度為128 bp(base pair，鹽基)之擴增片段，再利用生物學方法將該擴增片段選殖到PCR2.1－TOPO載體(購自Invitrogen)上，以獲取一質體(plasmid)，並稱該質體為p128，且利用可行之定序方法確認包含擴增片段的區域之序列。An amplification fragment of 128 bp (base pair) can be amplified from the template DNA by PCR as described above, and the amplified fragment can be cloned into PCR by biological methods. The 2.1-TOPO vector (purchased from Invitrogen) was used to obtain a plasmid, and the plasmid was designated as p128, and the sequence of the region containing the amplified fragment was confirmed by a feasible sequencing method.
接著利用p128之序列設計Pro20引子「5’－tacggcggattcagcatctc－3’(SEQ ID NO：3)」與Pro21「5’－tgccactcctcaccaaactc－3’(SEQ ID NO：4)」引子，再以PCR DIG標定套組(PCR DIG labeling kit，購自Roche)製作探針。製作探針過程中之PCR條件為：94℃,3分鐘[循環1次]；94℃,30秒,58℃,30秒,72℃,40秒[循環35次]；72℃,七分鐘[循環1次]。Then use the sequence of p128 to design the Pro20 primer "5'-tacggcggattcagcatctc-3' (SEQ ID NO: 3)" and Pro21 "5'-tgccactcctcaccaaactc-3' (SEQ ID NO: 4)" primer, and then use the PCR DIG calibration set. A probe (PCR DIG labeling kit, available from Roche) was prepared. The PCR conditions during the preparation of the probe were: 94 ° C, 3 minutes [1 cycle]; 94 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 40 seconds [cycle 35 times]; 72 ° C, seven minutes [ Cycle once]].
依照該PCR DIG標定套組之操作方法並進行該PCR，可獲得一探針。A probe can be obtained by performing the PCR DIG calibration kit and performing the PCR.
2.培養菌株 將碎葉鬼傘在YMA(購自Difco，編號Difco 0712)之平板(添加瓊膠製成之凝態培養基)上於25℃培養18天後，接菌於培養液N。該培養液N含有：2%葡萄糖(glucose，購自Merck),0.3%黃豆粉(soybean flour),1%胰化蛋白腖(Tryptone),0.3% KH2 PO4 (Merck),0.1% MgSO4 (Merck)，且其pH值為6。接著於25℃、轉速200 rpm下培養七日。 2. Culture strains Coprinus comatus was cultured on a plate of YMA (purchased from Difco, No. Difco 0712) (coagulated medium made of agar) at 25 ° C for 18 days, and then incubated in culture solution N. The culture solution N contained: 2% glucose (purchased from Merck), 0.3% soybean flour, 1% Tryptone, 0.3% KH 2 PO 4 (Merck), 0.1% MgSO 4 ( Merck) and its pH is 6. It was then cultured at 25 ° C for 2 days at 200 rpm.
3.建立碎葉鬼傘互補DNA文庫 將碎葉鬼傘如上述培養至第七天，取1.2 g除水菌絲體，依據TRIZOL反應劑(購自Invitrogen)之操作流程抽取全部RNA(total RNA)。 3. Establish a complementary DNA library of the genus Coprinus comatus. The cultivar was cultured to the seventh day as above, and 1.2 g of water-removing mycelium was taken. All RNA was extracted according to the TRIZOL reagent (purchased from Invitrogen). ).
首先係抽取碎葉鬼傘之全部RNA，在本實施例中取得約325 μg，其OD260 /OD280 為2.05；但全部RNA之抽取量並不嚴格限定，取得多達500 μg之全部RNA亦無不可。First, the total RNA of the genus Coprinus comatus was extracted, and in this example, about 325 μg was obtained, and its OD 260 / OD 280 was 2.05; however, the total RNA extraction amount was not strictly limited, and all RNAs of up to 500 μg were obtained. Nothing.
再者，依據PolyATractmRNA單離系統套組(PolyATractmRNA isolation systems kit，購自Promega)之操作流程，對所取得之全部RNA進行mRNA的萃取，取得6 μg且OD260 /OD280 為2.01之polyA mRNA(具有聚腺苷酸之mRNA)。Furthermore, according to PolyATract mRNA single-off system kit (PolyATract The mRNA isolation systems kit, purchased from Promega, was used to extract mRNA from all of the obtained RNA to obtain 6 μg of polyA mRNA (with poly(A) mRNA) with an OD 260 /OD 280 of 2.01.
接著，取2－7 μg polyA mRNA依據ZAP－cDNAGigapackIII Gold選殖套組(ZAP－cDNAGigapackIII Gold Cloning Kit，購自Stratagen)之操作流程建立碎葉菌互補DNA文庫。在本實施例中，係接著藉由該ZAP－cDNAGigapackIII Gold選殖套組回收0.75－3 kb DNA片段，並篩選8 x 105 顆溶菌斑(plaque)以建立互補DNA文庫。Next, take 2-7 μg of polyA mRNA according to ZAP-cDNA Gigapack III Gold Selection Kit (ZAP-cDNA Gigapack The III Gold Cloning Kit, purchased from Stratagen, was used to create a Fragmenter complementary DNA library. In this example, the ZAP-cDNA is then Gigapack III Gold cloning kit 0.75-3 kb DNA fragment recovered, and screened Ke 8 x 10 5 plaques (plaque) to establish a complementary DNA library.
該碎葉菌互補DNA文庫之中的個別菌株分別帶有不同的質體，且各質體係由至少一載體與至少一cDNA所構成。其中該載體上具有複數之限制酶切位(restriction site)，在本實施例中，該載體至少包括Eco RI與Hind III等限制酶(restriction enzyme)之切位，且其位於cDNA兩端處之序列係分別為對應於T3引子與T7引子之序列。Each of the individual strains of the leaflet complementary DNA library carries a different plastid, and the various systems are composed of at least one vector and at least one cDNA. Wherein the vector has a plurality of restriction sites, and in this embodiment, the vector comprises at least a cleavage site of a restriction enzyme such as Eco RI and Hind III, and is located at both ends of the cDNA. The sequence lines are sequences corresponding to the T3 primer and the T7 primer, respectively.
4.挑選溶菌斑 再依照GigapackIII Gold Cloning Kit(購自Stratagen)之後續操作流程進行溶菌斑雜合(Plaque hybridization)，得到複數由輔助噬菌體(helper phage)所形成之溶菌斑，並製作一溶菌斑採測材料(plaque lift)，其中通常採用硝化纖維素膜(nitrocellulose membrane)作為該溶菌斑採測材料。 4. Pick plaque and follow Gigapack The subsequent operation of the III Gold Cloning Kit (purchased from Stratage) was performed by Plaque hybridization, and a plurality of plaques formed by helper phage were obtained, and a plaque lift was prepared. Among them, a nitrocellulose membrane is usually used as the plaque-measuring material.
該溶菌斑採測材料係先於FastHyb溶液(購自Biochain)在50℃下反應2小時進行預雜合(prehybridization)，再製備一含有探針之FastHyb溶液，並將該溶菌斑採測材料移入含有探針之FastHyb溶液中於50℃下反應16小時。The plaque-preventing material was prehybridized by reacting with FastHyb solution (purchased from Biochain) at 50 ° C for 2 hours, preparing a FastHyb solution containing the probe, and moving the plaque-preserving material into the material. The reaction was carried out in a FastHyb solution containing the probe at 50 ° C for 16 hours.
接著進行沖洗過程：(1)以適量2 X SSC,0.1 % SDS溶液於室溫下沖洗該溶菌斑採測材料五分鐘；(2)反覆前述步驟(1)一次；(3)對該溶菌斑採測材料以0.5 X SSC,0.1 % SDS溶液於65℃下充分作用15分鐘；(4)反覆前述步驟(3)一次。Then, the rinsing process is carried out: (1) rinsing the plaque test material with an appropriate amount of 2 X SSC, 0.1% SDS solution at room temperature for five minutes; (2) repeating the aforementioned step (1) once; (3) the plaque The test material was fully acted at 65 ° C for 15 minutes in a 0.5 X SSC, 0.1% SDS solution; (4) the previous step (3) was repeated.
經上述沖洗該溶菌斑採測材料後，再使用針對探針所附DIG之抗體進行抗體偵測，並以X光底片進行自動放射顯影，可得到不同的訊號，以區分出具有訊號之溶菌斑以及不具訊號之溶菌斑。After the plaque test material is washed as described above, the antibody against the DIG antibody attached to the probe is used for antibody detection, and the X-ray film is used for automatic radiography to obtain different signals to distinguish the plaque with signal. And plaque without signal.
5.取得轉形株 經由上述方法，在本實施例中確定出93個有意義的訊號。依據ZAP－cDNAGigapackIII Gold Cloning Kit操作流程，經過二次篩選得到純淨單一的溶菌斑，以T3引子與T7引子進行PCR，挑選PCR產物分子量最大的11個溶菌斑，並進行生體內剪接作用(in vivo excision)。 5. Obtaining a transformed strain By the above method, 93 meaningful signals are determined in this embodiment. Based on ZAP-cDNA Gigapack III Gold Cloning Kit operation process, after the second screening to obtain a pure single plaque, T3 primer and T7 primer for PCR, select the 11 plaques with the largest molecular weight of PCR products, and in vivo excision.
所得之轉形株質體以Eco RI與Hind III限制酶(restriction enzyme)同時作用進行確認，並用膠體電泳(gel electrophoresis)分析，並挑選出四個長度較長的轉形株，49－1、71－1、76－3與91－1。The obtained transformed plastids were confirmed by the simultaneous action of Eco RI and Hind III restriction enzyme, and analyzed by gel electrophoresis, and four long-length transgenic plants were selected, 49-1. 71-1, 76-3 and 91-1.
6.進行定序6. Perform sequencing
在此定序步驟中，先以前述質體之載體上現有引子進行定序得知cDNA兩端之序列後，再依據所得知的序列更進一步設計引子並定序，以逐步完成全長cDNA之定序。In this sequencing step, the sequence of the two ends of the cDNA is first sequenced by using the existing primers on the carrier of the plastid, and then the primers are further designed and sequenced according to the known sequence to gradually complete the full-length cDNA. sequence.
7.獲取碎葉鬼傘脯胺酸寡胜肽酶之cDNA並確認其性質及相關數據7. Obtain the cDNA of the lysergic acid oligopeptidase and confirm its properties and related data.
藉由上述步驟，可定出碎葉鬼傘脯胺酸寡胜肽酶cDNA之核酸序列(SEQ ID NO：5)，其全長2,508 nt，在第65 nt開始有一個2,217 nt之ORF，編碼739個胺基酸(SEQ ID NO：6)，該739個胺基酸之分子量為83.9 kD並構成一具有碎葉鬼傘脯胺酸寡胜肽酶活性之蛋白質。該碎葉鬼傘脯胺酸寡胜肽酶cDNA之核酸序列(SEQ ID NO：5)係：「 」。又該碎葉鬼傘脯胺酸寡胜肽酶cDNA之核酸序列(SEQ ID NO：5)所編碼739個胺基酸(SEQ ID NO：6)係：「 By the above procedure, the nucleic acid sequence (SEQ ID NO: 5) of the genus Pseudomonas oligopeptidase cDNA, which has a full length of 2,508 nt and a 2,217 nt ORF starting at the 65th nt, encodes 739. Amino acid (SEQ ID NO: 6), the 739 amino acid has a molecular weight of 83.9 kD and constitutes a protein having the activity of the Phytophthora bismuth peptidase. The nucleic acid sequence (SEQ ID NO: 5) of the genus Pseudomonas oligopeptidase cDNA is: " "." Further, the 739 amino acid (SEQ ID NO: 6) encoded by the nucleic acid sequence of the genus falciparum oligopeptidase cDNA (SEQ ID NO: 5) is:
又如表一所示，將碎葉鬼傘脯胺酸寡胜肽酶胺基酸序列以套裝軟體GCG(Accelrys)之GAP程式與其他物種脯胺酸寡胜肽酶胺基酸序列比較，結果以非洲爪蟾(Xenopus tropicalis )相同性最高(45.8%)，其次為玉米黑穗菌(Ustilago maydis )(44.7%)，與人、鼠、豬、藍綠藻、***芥及牛間之相同性(40~43.9%)反較同屬擔子菌屬之新型隱球菌(Cryptococcus neoformans )相同性(31.6~32%) 高。As shown in Table 1, the amino acid sequence of the valeric acid oligopeptide peptidase was compared with the amino acid sequence of the other species of proline oligopeptidase by the GAP program of the packaged software GCG (Accelrys). Xenopus tropicalis is the most identical (45.8%), followed by Ustilago maydis (44.7%), and is identical to human, mouse, pig, blue-green algae, Arabidopsis and cattle. (40~43.9%) is higher than the similarity of Cryptococcus neoformans (31.6~32%).
由上述可知，在不同物種之中具有與SEQ ID NO：5所編碼之胺基酸序列SEQ ID NO：6之相似度在60%以下之各種胺基酸序列，其中由於胺基酸序列之變異並不必然影響其所構成蛋白質之活性，因此只要胺基酸序列之變異不影響該蛋白質之活性，仍屬於本發明之範圍。From the above, it is known that among the different species, various amino acid sequences having a similarity to the amino acid sequence of SEQ ID NO: 5 of SEQ ID NO: 6 of 60% or less, wherein the amino acid sequence is mutated, It does not necessarily affect the activity of the protein it constitutes, so it is within the scope of the invention that the variation of the amino acid sequence does not affect the activity of the protein.
上述相似度較佳是70%以上。The above similarity is preferably 70% or more.
上述相似度更佳是80%以上。The above similarity is more preferably 80% or more.
上述相似度更較佳是90%以上。The above similarity is more preferably 90% or more.
上述相似度最佳是95%以上。The above similarity is preferably 95% or more.
碎葉鬼傘脯胺酸寡胜肽酶cDNA在大腸桿菌之表現Performance of Escherichia coli peptidase cDNA in Escherichia coli
以Pro 31引子「5’-atggtgaccaaaacctgggt-3’(SEQ ID NO：7)」與Pro 32引子「5’-ctagagtgtagctttatctttc-3’(SEQ ID NO：8)」，使用pfu複製酶進行PCR增生一含有終止密碼子(stop codon)之完整脯胺酸寡胜肽酶cDNA。Using Pro 31 primer "5'-atggtgaccaaaacctgggt-3' (SEQ ID NO: 7)" and Pro 32 primer "5'-ctagagtgtagctttatctttc-3' (SEQ ID NO: 8)", PCR amplification was performed using pfu replicase. Stop codon complete glutamate oligopeptidase cDNA.
該PCR所使用條件為：94℃,3分鐘[循環1次]； 94℃,30秒，58℃,30秒，72℃,180秒[循環35次]；72℃,3分鐘[循環1次]。The conditions used for the PCR were: 94 ° C, 3 minutes [1 cycle]; 94 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 180 seconds [35 cycles]; 72 ° C, 3 minutes [1 cycle].
將上述PCR所得到的完整脯胺酸寡胜肽酶cDNA，以及一含有對應T7引子序列及N端His-tag(組胺酸標幟)的pET 151/D-TOPO表現載體(Invitrogen)進行接合反應，以構成一基因重組質體，並將該基因重組質體送入DH10B大腸桿菌菌株(購自Invitrogen)中並於37℃以LB培養液(購自USB)或LB平板(購自USB)培養之，以獲取複數菌落(colony)。從該複數菌落中挑選其中三菌落，並依生物學方法自各菌落之大腸桿菌中取得基因重組質體，再將該等基因重組質體送入BL21(DE3)大腸桿菌菌株(購自Invitrogen)中進行表現。The complete prolyl oligopeptidase cDNA obtained by the above PCR and a pET 151/D-TOPO expression vector (Invitrogen) containing the corresponding T7 primer sequence and the N-terminal His-tag (histidine tag) were ligated. Reaction to form a recombinant plastid, and the recombinant plastid was sent to DH10B E. coli strain (purchased from Invitrogen) and LB medium (purchased from USB) or LB plate (purchased from USB) at 37 °C. Cultivate to obtain a plurality of colonies (colony). Three colonies were selected from the plurality of colonies, and the recombinant plastids were obtained from the E. coli of each colony by biological methods, and the recombinant plastids were then sent to BL21(DE3) Escherichia coli strain (purchased from Invitrogen). Perform performance.
上述三組含基因重組質體之BL21(DE3)大腸桿菌菌株以LB培養液於搖瓶中培養至OD600 大約0.4-0.6時，加入終濃度為0.4 mM的IPTG繼續培養20小時，使各含有基因重組質體之BL21(DE3)大腸桿菌菌株表現其基因重組質體之碎葉鬼傘脯胺酸寡胜肽酶cDNA，其所表現之具有脯胺酸寡胜肽酶活性的蛋白質係參考pET系統(pET system，購自Novagen)使用手冊所揭露之蛋白質純化方法進行純化，以Bio-Rad蛋白質試驗(Bio-Rad Protein Assay)(購自Bio-Rad)測定蛋白質量，並使用牛血清白蛋白(bovine serum albumin，BSA)做為標準，以其作出標準曲線(standard curve)後據以測定蛋白質量。The above three groups of recombinant BL21(DE3) E. coli strains containing the recombinant plasmid were cultured in LB medium to a OD 600 of about 0.4-0.6, and then added to IPTG at a final concentration of 0.4 mM for 20 hours to make each contained. The BL21(DE3) E. coli strain of the recombinant plastid expresses the recombinant plastid of the genus Phaeocystis oligopeptidase cDNA, which shows the protein system with peptidyl luciferase activity reference pET The system (pET system, purchased from Novagen) was purified by the protein purification method disclosed in the manual, and the amount of protein was determined by Bio-Rad Protein Assay (purchased from Bio-Rad), and bovine serum albumin was used. (bovine serum albumin, BSA) was used as a standard to determine the amount of protein by making a standard curve.
此外，亦可配合實際上應用或研究等需求，以上述之 技術手段，將上述脯胺酸寡胜肽酶cDNA置於一可驅動表現該脯胺酸寡胜肽酶cDNA之驅動子或控制序列的控制之下，使該脯胺酸寡胜肽酶cDNA可操作地連結到該驅動子或該控制序列以構築成一嵌合基因或一核酸構築體，並藉由該驅動子或該控制序列驅動表現之能力，令該嵌合基因或該核酸構築體於一細胞中被驅動表現。In addition, it can also be used in conjunction with actual application or research needs. Technically, the glutamic acid oligopeptidase cDNA is placed under the control of a promoter or a control sequence which can drive the cDNA of the proline oligopeptidase to make the proline oligopeptidase cDNA The chimeric gene or the nucleic acid construct is operably linked to the driver or the control sequence to construct a chimeric gene or a nucleic acid construct and to drive expression by the driver or the control sequence The cells are driven to perform.
測定碎葉鬼傘脯胺酸寡胜肽酶活性Determination of valeric acid oligopeptide activity
1.挑選pProHN14及pProHN17並測定其所編碼蛋白質脯胺酸寡胜肽酶之活性1. Select pProHN14 and pProHN17 and determine the activity of the encoded protein proline oligopeptidase
取適當稀釋之脯胺酸寡胜肽酶溶液先與1 N HCl終止液反應，再加入其他試劑以作為控制組並建立OD410 對於重氮化對硝基苯胺(p-nitroaniline，購自Fluka)產生量的標準曲線，以作為測定之根據，並定義前述在pH為7.0，反應溫度為45℃之條件下，每分鐘產生1微莫耳(μmole)重氮化對硝基苯胺之脯胺酸寡胜肽酶活性為1 U。The appropriately diluted prolyl oligopeptidase solution was first reacted with 1 N HCl stop solution, and then other reagents were added as a control group and OD 410 was established for p-nitroaniline (plunitroaniline, purchased from Fluka). A standard curve of the amount is produced as a basis for the determination, and defines the above-mentioned methionine of p-nitroaniline at a pH of 7.0 and a reaction temperature of 45 ° C to produce 1 micromole per minute. The oligopeptidase activity is 1 U.
於微量離心管中加入400 μl之0.1 M鈉-磷酸緩衝液(Na-phosphate buffer)、50μl之10mM Z-甘胺醯基-L-脯胺酸-4-硝基苯胺(Z-glycyl-L-proline-4-nitroanilide，購自Fluka)及50 μl之經適當稀釋具有脯胺酸寡胜肽酶活性蛋白質之溶液，反應5-60分鐘，加入500 μl之1 N HCl終止反應之後，以13,000 rpm離心5分鐘，取上清液測OD410 。Add 400 μl of 0.1 M sodium-phosphate buffer (Na-phosphate buffer), 50 μl of 10 mM Z-glycine-L-proline -4-nitroaniline (Z-glycyl-L) to a microcentrifuge tube. -proline-4-nitroanilide, purchased from Fluka) and 50 μl of a solution diluted with a protein of prothrombin luciferase activity, reacted for 5 to 60 minutes, and after adding 500 μl of 1 N HCl to stop the reaction, 13,000 Centrifuge at rpm for 5 minutes, and take the supernatant to measure OD 410 .
取前述上清液所測得之OD410 ，對應上述標準曲線，分別求得前述三組BL21(DE3)大腸桿菌菌株所表現脯胺酸寡胜肽酶之活性，並從中選出表現活性較高之二組，其分別由基因重組質體pProHN14及pProHN17所編碼，而pProHN14及pProHN17之蛋白質在BL21(DE3)大腸桿菌菌株中於胞內所表現之脯胺酸寡胜肽酶活性分別為7.2 U/ml與7.7 U/ml。Taking the OD 410 measured by the above supernatant, corresponding to the above standard curve, respectively, the activity of the lysine oligopeptidase exhibited by the above three groups of BL21 (DE3) Escherichia coli strains was determined, and the activity activity was selected from the above. The two groups were encoded by the recombinant plastids pProHN14 and pProHN17, respectively, and the proline oligopeptidase activities of the proteins of pProHN14 and pProHN17 in the BL21(DE3) E. coli strain were 7.2 U/, respectively. Ml with 7.7 U/ml.
2.對pProHN14及pProHN17之完整脯胺酸寡胜肽酶cDNA進行定序2. Sequencing the complete proline oligopeptidase cDNA of pProHN14 and pProHN17
將pProHN14及pProHN17以可行之定序方法進行定序。依定序結果可知pProHN17之完整脯胺酸寡胜肽酶cDNA之核酸序列符合原先設計，具有SEQ ID NO：5所記載之核酸序列。pProHN14 and pProHN17 were sequenced in a viable sequencing method. According to the sequencing results, the nucleic acid sequence of the complete proline oligopeptidase cDNA of pProHN17 conforms to the original design and has the nucleic acid sequence of SEQ ID NO: 5.
另一方面，pProHN14則具有SEQ ID NO：9所示之核酸序列。相較於pProHN17，pProHN14由於在C端缺少4個核酸(CTAG)而造成讀框轉移(frame-shift)，使得pProHN14所表現之具有脯胺酸寡胜肽酶活性之蛋白質的胺基酸序列(SEQ ID NO：10)在C端比預期多出24個胺基酸，其係：「RASSDPAANKA RKEAELAAAT AEQ」。該24個胺基酸亦可記載為：「Arg Ala Ser Ser Asp Pro Ala Ala Asn Lys Ala Arg Lys Glu Ala Glu Leu Ala Ala Ala Thr Ala Glu Gln」。其中，前述pProHN14所具有SEQ ID NO：9之核酸序列係：「 」。前述pProHN14所表現之具有脯胺酸寡胜肽酶活性之蛋 白質的胺基酸序列(SEQ ID NO：10)係：「 755 760」。以BCRC 36074為模版，於引子設計時特意使相對應之引子缺漏上述pProHN14在C端所缺少之4個核酸(CTAG)，即可利用PCR方式得到一序列，其C端較完整寡胜肽酶之DNA序列缺少4個核酸；進一步將該序列接入一表現載體，可獲取SEQ ID NO：6之核酸序列和SEQ ID NO：10之胺基酸序列。在一可行之實施態樣中，可採用pET151/D-TOPO作為該表現載體。In another aspect, pProHN14 has the nucleic acid sequence set forth in SEQ ID NO:9. Compared to pProHN17, pProHN14 caused a frame-shift due to the lack of four nucleic acids (CTAG) at the C-terminus, resulting in the amino acid sequence of the protein having prolyl oligopeptidase activity exhibited by pProHN14 ( SEQ ID NO: 10) 24 more amino acids than expected at the C-terminus, which is: "RASSDPAANKA RKEAELAAAT AEQ". The 24 amino acids can also be described as: "Arg Ala Ser Ser Asp Pro Ala Ala Asn Lys Ala Arg Lys Glu Ala Glu Leu Ala Ala Ala Thr Ala Glu Gln". Wherein the aforementioned pProHN14 has the nucleic acid sequence of SEQ ID NO: 9: "." The amino acid sequence (SEQ ID NO: 10) of the protein having prolyl oligopeptidase activity expressed by the aforementioned pProHN14 is: 755 760". BCRC 36074 is used as a template. When the primer is designed, the corresponding primers are deprived of the four nucleic acids (CTAG) lacking at the C-terminus of pProHN14, and a sequence can be obtained by PCR. The C-terminal is more complete than the oligopeptidase. The DNA sequence lacks 4 nucleic acids; further accessing the sequence to a expression vector provides the nucleic acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 10. In a possible embodiment, pET151/D-TOPO can be used as the expression carrier.
3.比較pProHN14與pProHN17所表現之之蛋白質以及既有脯胺酸寡胜肽酶之活性與比活性3. Compare the activity of the protein expressed by pProHN14 and pProHN17 with the activity and specific activity of the glutamic acid oligopeptidase
如上所述，pProHN14及pProHN17之蛋白質在BL21(DE3)大腸桿菌菌株中於胞內所表現之脯胺酸寡胜肽酶活性分別為7.2 U/ml與7.7 U/ml，該活性近於腦膜炎敗血黃桿菌脯胺酸寡胜肽酶在大腸桿菌表現之活性(8.1 U/ml)，而優於其他大部分既有脯胺酸寡胜肽酶所表現之活性。As described above, the proline oligopeptidase activities of pProHN14 and pProHN17 proteins in the BL21(DE3) E. coli strain were 7.2 U/ml and 7.7 U/ml, respectively, which was close to meningitis. The activity of Flavobacterium fuliginea glutamate oligopeptidase in E. coli (8.1 U/ml) is superior to that exhibited by most other proline oligopeptidases.
經由Ni-NTA管柱(管柱是自行裝填(packing)，其樹脂(resin)為購自Invitrogen之Ni-NTA瓊膠)親和性步驟可得pProHN14與pProHN17之蛋白質經純化後，可測得純化蛋白質之脯胺酸寡胜肽酶比活性分別為56.1 U/mg與66.8 U/mg，低於純化後之腦膜炎敗血黃桿菌脯胺酸寡胜肽酶比活性(124 U/mg)，而優於其他大部分既有脯胺酸寡胜肽酶在純化後所表現之比活性。The purified protein of pProHN14 and pProHN17 can be purified by a Ni-NTA column (the column is self-packing and the resin is Ni-NTA agar from Invitrogen). The specific activities of the proline oligopeptidase of the protein were 56.1 U/mg and 66.8 U/mg, respectively, which was lower than the purified activity of the lysine luciferase (124 U/mg). It is superior to most other glutamate oligopeptidase enzymes in the specific activity after purification.
pProHN14與pProHN17之脯胺酸寡胜肽酶基本性質分析Analysis of basic properties of proline oligopeptidase from pProHN14 and pProHN17
1.最適合碎葉鬼傘脯胺酸寡胜肽酶反應之pH值1. The most suitable pH value for the lysine oligopeptide reaction
最適反應pH之測定方法係以pH3-8之檸檬酸-亞磷酸氫鈉緩衝溶液(citric acid-Na2 HPO4 buffer solution)或pH9-12之甘胺酸-氫氧化鈉緩衝溶液(glycine-NaOH buffer solution)取代標準酶活性測定所用之緩衝液，其餘反應條件同上述脯胺酸寡胜肽酶活性之測定方法。The optimum reaction pH is determined by a pH 3-8 citric acid-Na 2 HPO 4 buffer solution or a pH 9-12 glycine-sodium hydroxide buffer solution (glycine-NaOH). Buffer solution) Substitute the buffer used for the standard enzyme activity assay, and the rest of the reaction conditions are the same as the above-described method for determining the activity of the proline oligopeptide activity.
pProHN14與pProHN17之蛋白質皆於pH 7.0反應時表現出最高脯胺酸寡胜肽酵素酶活性。又pProHN14之蛋白質酶可較pProHN17之蛋白質適應更廣的pH範圍。其比較的結果如第一圖所示，在pH 6.0時，pProHN14之蛋白質約有90%活性而pProHN17之蛋白質僅約有50%活性。Both pProHN14 and pProHN17 proteins exhibited the highest proline oligopeptide activity when reacted at pH 7.0. Moreover, the proteinase of pProHN14 can adapt to a wider pH range than the protein of pProHN17. The results of the comparison are shown in the first panel. At pH 6.0, the protein of pProHN14 is approximately 90% active and the protein of pProHN17 is only approximately 50% active.
2.最適合碎葉鬼傘脯胺酸寡胜肽酶之反應溫度2. The most suitable reaction temperature for the lysine oligopeptide
最適反應溫度之測定方法係令酶反應分別於25、30、37、45或55℃之溫度下進行，其餘反應條件同上述脯胺酸寡胜肽酶活性之測定方法。The optimum reaction temperature is determined by subjecting the enzymatic reaction to a temperature of 25, 30, 37, 45 or 55 ° C, respectively, and the rest of the reaction conditions are the same as those of the above lysine oligopeptide activity.
其在pH 7.0時測定的結果如第二圖所示，pH 7.0時pProHN14之蛋白質與pProHN17之蛋白質皆在45℃反應時表現出最高酶活性，可知其最適溫度為45℃，而此最適溫度比現有比活性高的脯胺酸寡胜肽酶的最適溫度都高。The results of the measurement at pH 7.0 are shown in the second figure. At pH 7.0, the protein of pProHN14 and the protein of pProHN17 showed the highest enzyme activity when reacted at 45 ° C, and the optimum temperature was 45 ° C, and the optimum temperature ratio was obtained. The optimum temperature for the high specific activity of the prolyl oligopeptidase is high.
又在pH 8.0時測定的結果如第三圖所示，在pH 8.0時，pProHN14之蛋白質與pProHN17之蛋白質皆以37℃反應的酶活性最高。The results of the measurement at pH 8.0 are shown in the third figure. At pH 8.0, both the protein of pProHN14 and the protein of pProHN17 have the highest enzyme activity at 37 °C.
3.碎葉鬼傘脯胺酸寡胜肽酶之熱安定性3. Thermal stability of acetaminophen oligopeptide
酶之熱安定性之測定方法係將樣品於分別於30℃、37℃或45℃先預熱0、20、40、60或80分鐘，或於55℃下先預熱0、5、10、15、20、25或30分鐘，再以上述脯胺酸寡胜肽酶活性之測定方法進行酶活性測定。The thermal stability of the enzyme is determined by preheating the sample at 30 ° C, 37 ° C or 45 ° C for 0, 20, 40, 60 or 80 minutes, respectively, or preheating 0, 5, 10 at 55 ° C, The enzyme activity was measured by the above-described method for measuring the activity of the proline oligopeptide activity at 15, 20, 25 or 30 minutes.
其測定結果如第四圖所示，pProHN14之蛋白質於30℃與37℃分別預熱80分鐘後，尚保有99%與93%之活性；而pProHN17之蛋白質於同樣條件下預熱後，則保有80%與73%之活性。As shown in the fourth figure, the protein of pProHN14 is maintained at 99 °C and 37 °C for 80 minutes, and still retains 99% and 93% of the activity; while the protein of pProHN17 is preheated under the same conditions, the protein remains. 80% and 73% active.
在45℃預熱60分鐘後，pProHN14之蛋白質保有67%活性，而pProHN17之蛋白質僅保有32%活性，可知pProHN14之蛋白質之熱安定性高於pProHN17之蛋白質之熱安定性。After preheating at 45 ° C for 60 minutes, the protein of pProHN14 retains 67% activity, while the protein of pProHN17 retains only 32% activity. It is known that the thermal stability of the protein of pProHN14 is higher than the thermal stability of the protein of pProHN17.
pProHN14之蛋白質與pProHN17之蛋白質在55℃預熱5分鐘均僅殘存9%活性。The protein of pProHN14 and the protein of pProHN17 were only 9% active after preheating for 5 minutes at 55 °C.
第一圖：係揭露不同pH值對pProHN14與pProHN17之蛋白質之脯胺酸寡胜肽酶活性影響之折線圖。其中「●」符號之折線表示pProHN14之蛋白質；「▲」符號之折線表 示pProHN17之蛋白質。First panel: A line graph showing the effect of different pH values on the activity of proline oligopeptidase of pProHN14 and pProHN17 proteins. The polyline of the "●" symbol indicates the protein of pProHN14; the line of the "▲" symbol The protein of pProHN17.
第二圖：係揭露pH7.0時溫度對pProHN14與pProHN17之蛋白質活性影響之柱狀圖。其中實心黑色柱表示pProHN14之蛋白質；空心白色柱表示pProHN17之蛋白質。Second panel: A bar graph showing the effect of temperature on the protein activity of pProHN14 and pProHN17 at pH 7.0. The solid black column indicates the protein of pProHN14; the hollow white column indicates the protein of pProHN17.
第三圖：係揭露pH8.0時溫度對pProHN14與pProHN17之蛋白質之脯胺酸寡胜肽酶活性影響之柱狀圖。其中實心黑色柱表示pProHN14之蛋白質；空心白色柱表示pProHN17之蛋白質。Figure 3 is a bar graph showing the effect of temperature on the activity of proline oligopeptidase of pProHN14 and pProHN17 proteins at pH 8.0. The solid black column indicates the protein of pProHN14; the hollow white column indicates the protein of pProHN17.
第四圖：係揭露重組脯胺酸寡胜肽酶熱安定性之折線圖。其中「實線」之折線表示pProHN14之蛋白質；「虛線」之折線表示pProHN17之蛋白質。又，「●」符號之折線表示以30℃預熱；「■」符號之折線表示以37℃預熱；「▲」符號之折線表示以45℃預熱；「◆」符號之折線表示以55℃預熱。Figure 4: A line graph showing the thermal stability of recombinant proline oligopeptides. The "solid line" of the line indicates the protein of pProHN14; the "dashed line" of the line indicates the protein of pProHN17. Moreover, the broken line of the "●" symbol indicates preheating at 30 °C; the broken line of the "■" symbol indicates preheating at 37 °C; the broken line of the "▲" symbol indicates preheating at 45 °C; the broken line of the "◆" symbol indicates 55 °C preheating.
1. Abdus Sattar, A. K. M., Yamamoto, N., Yoshimoto, T., and Tsuru, D. 1990. Purification and characterization of an extracellular prolyl endopeptidase fromAgaricus bisporus . J. Biochem. 107: 256-261.1. Abdus Sattar, AKM, Yamamoto, N., Yoshimoto, T., and Tsuru, D. 1990. Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus . J. Biochem. 107: 256-261.
2. Atack, J.R., Suman-Chauhan, N., Dawson, G., and Kulagowski, J. J. 1991.In vitro andin vivo inhibition of prolyl endopeptidase. Eur. J. Pharmacol. 205: 157-163.2. Atack, JR, Suman-Chauhan, N., Dawson, G., and Kulagowski, JJ 1991. In vitro and in vivo inhibition of prolyl endopeptidase. Eur. J. Pharmacol. 205: 157-163.
3. Atta-ur-Rahman, Akhtar, M.N., Choudhary, M.I., Tsuda, Y., Yasin, A., Sener, B., Parvez, M. 2005. New diterpene isopimara-7,15-dien-19-oic acid and its prolyl endopeptidase inhibitory activity. Nat. Prod. Res. 19:13-22.3. Atta-ur-Rahman, Akhtar, MN, Choudhary, MI, Tsuda, Y., Yasin, A., Sener, B., Parvez, M. 2005. New diterpene isopimara-7, 15-dien-19-oic Acid and its prolyl endopeptidase inhibitory activity. Nat. Prod. Res. 19:13-22.
4. Diefenthal, T., Dargatz, H., Witte, V., Reipen, G., and Svendsen, I. 1993. Cloning of proline-specific endopeptidase gene fromFlavobacterium menigosepticum : expression inEscherichia coli and purification of the heterologous protein. Appl. Microbiol. Biotechnol. 40: 90-97.4. Diefenthal, T., Dargatz, H., Witte, V., Reipen, G., and Svendsen, I. 1993. Cloning of proline-specific endopeptidase gene from Flavobacterium menigosepticum : expression in Escherichia coli and purification of the heterologous protein Appl. Microbiol. Biotechnol. 40: 90-97.
5. Gass, J., Ehren, J., Strohmeler, G., Isaacs, I., Khosla, C. 2005. Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy. Biotechnol. Bioeng. 92(6):674-84.5. Gass, J., Ehren, J., Strohmeler, G., Isaacs, I., Khosla, C. 2005. Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy Biotechnol. Bioeng. 92(6): 674-84.
6. Habibi-Najafi, M. B. and Lee, B. H. 1994. Proline-specific peptidases ofLactobacillus casei subspecies. J. Dairy Sci. 77:385-392.6. Habibi-Najafi, MB and Lee, BH 1994. Proline-specific peptidases of Lactobacillus casei subspecies. J. Dairy Sci. 77:385-392.
7. Harwood, V. J. Denson, J. D., Robinson-Bidle, K. A. and Schreier, H. J. 1997. Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic ArchaeonPyrococcus furiosus . J. Bacterol. 179: 3613-3618.7. Harwood, VJ Denson, JD, Robinson-Bidle, KA and Schreier, HJ 1997. Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus . J. Bacterol. 179: 3613-3618.
8. Harwood, V.J. and Schreier, H.J. 2001. Prolyl oligopeptidase from Pyrococcus furiosus. Methods Enzymol. 330: 445-454.8. Harwood, V.J. and Schreier, H.J. 2001. Prolyl oligopeptidase from Pyrococcus furiosus. Methods Enzymol. 330: 445-454.
9. Heinis, C., Alessi, P., and Neri, D. 2004. Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy. Biochemistry 43:6293-6303.9. Heinis, C., Alessi, P., and Neri, D. 2004. Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy. Biochemistry 43:6293-6303.
10. Jarho, E.M., Wallen, E.A., Christiaans, J.A., Forsberg, M.M., Venalainen, J.I., Mannisto, P.T., Gynther, J., and Poso, A. 2005. Dicarboxylic acid azacycle-1-prolyl-pyrrolidine amides as prolyl oligopeptidase inhibitors and three-dimensional quantitative structure-activity relationship of the enzyme-inhibitor interactions. J. Med. Chem. 48: 47772-4782.10. Jarho, EM, Wallen, EA, Christiaans, JA, Forsberg, MM, Venalainen, JI, Mannisto, PT, Gynther, J., and Poso, A. 2005. Dicarboxylic acid azacycle-1-prolyl-pyrrolidine amides as prolyl Oligopeptidase inhibitors and three-dimensional quantitative structure-activity relationship of the enzyme-inhibitor interactions. J. Med. Chem. 48: 47772-4782.
11. Kanatani, A., Yoshimoto, T., Kitazono, A., Kokubo, T., and Tsuru, D. 1993. Prolyl endopeptidase fromAeromonas hydrophila : cloninig, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme. J. Biochem. 113: 790-796.11. Kanatani, A., Yoshimoto, T., Kitazono, A., Kokubo, T., and Tsuru, D. 1993. Prolyl endopeptidase from Aeromonas hydrophila : cloninig, sequencing, and expression of the enzyme gene, and characterization of the Exhibited enzyme. J. Biochem. 113: 790-796.
12. Lee, J.H., Lee, S.Y., Lee, K.S., Jang,H.J., Lee, K.H., Hahn, T.R., and Paik, Y.S. 2004. Prolyl endopeptidase inhibitors from the leaves ofGinkgo biloba . Planta Med. 70:1228-1230.12. Lee, JH, Lee, SY, Lee, KS, Jang, HJ, Lee, KH, Hahn, TR, and Paik, YS 2004. Prolyl endopeptidase inhibitors from the leaves of Ginkgo biloba . Planta Med. 70:1228-1230 .
13. Li, M., Chen, C., Wang, D. [Gene cloning and expression of prolyl endopeptidase from Aeromonas punctata] Wei Sheng Wu Xue Bao. 2000 40(3):277-283. Chinese.13. Li, M., Chen, C., Wang, D. [Gene cloning and expression of prolyl endopeptidase from Aeromonas punctata] Wei Sheng Wu Xue Bao. 2000 40(3): 277-283. Chinese.
14. Marighetto, A., Touzani, K., Etchamendy, N., Torrea, C.C., De Nanteuil, G., Guez, D., Jaffard, R., and Morain, P. 2000. Further evidence for a dissociation between different forms of mnemonic expressions in a mouse model of age-related cognitive decline: effects of tacrine and S 17092, a novel prolyl endopeptidase inhibitor. Learn Mem. 7: 159-169.14. Marighetto, A., Touzani, K., Etchamendy, N., Torrea, CC, De Nanteuil, G., Guez, D., Jaffard, R., and Morain, P. 2000. Further evidence for a dissociation between Different forms of mnemonic expressions in a mouse model of age-related cognitive decline: effects of tacrine and S 17092, a novel prolyl endopeptidase inhibitor. Learn Mem. 7: 159-169.
15. Marti, T., Molberg, O., Li, Q., Gray, G. M., Khosla, C., and Solid, L. M. 2005. Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization. J. Pharmacol. Exp. Ther. 312:19-26.15. Marti, T., Molberg, O., Li, Q., Gray, G. M., Khosla, C., and Solid, L. M. 2005. Prolyl endopeptidase-mediated destruction of T cell Epitopes in whole gluten: chemical and immunological characterization. J. Pharmacol. Exp. Ther. 312:19-26.
16. Matysiak-Budnk, T., Candalh, C., Cellier, C., Dugave, C., Namane, A., Vidal-Martinez, T., Cerf-Bensussan, N., and Heyman, M. 2005. Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac desease. Gastroenterol. 129(3):786-796.16. Matysiak-Budnk, T., Candalh, C., Cellier, C., Dugave, C., Namane, A., Vidal-Martinez, T., Cerf-Bensussan, N., and Heyman, M. 2005. Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac desease. Gastroenterol. 129(3):786-796.
17. Piper, J. L., Gray, G. M., and Khosla, C. 2004. Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J. Pharmacol. Exp. Ther. 311: 213-219.17. Piper, J. L., Gray, G. M., and Khosla, C. 2004. Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J. Pharmacol. Exp. Ther. 311: 213-219.
18. Polgár, L. 1994. Prolyl oligopeptidase. Methods Enzymol. 244: 188-200.18. Polgár, L. 1994. Prolyl oligopeptidase. Methods Enzymol. 244: 188-200.
19. Polgár, L. 2002. The prolyl oligopeptidase family. Cell. Mol. Life Sci. 59: 349-362.19. Polgár, L. 2002. The prolyl oligopeptidase family. Cell. Mol. Life Sci. 59: 349-362.
20. Pyle, G.G., Paaso, B., Anderson, B.E., Allen, D.D., Marti,T., Li, Q., Sieqel,M., Khosla, C., Gray, G.M. 2005. Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue. Clin. Gastroenterol. Hepatol. 3(7): 687-94.20. Pyle, GG, Paaso, B., Anderson, BE, Allen, DD, Marti, T., Li, Q., Sieqel, M., Khosla, C., Gray, GM 2005. Effect of pretreatment of food gluten With prolyl endopeptidase on gluten-induced malabsorption in celiac sprue. Clin. Gastroenterol. Hepatol. 3(7): 687-94.
21. Sorensen, R., Kildal, E., Stepaniak, L., Pripp, A. H., and Sorhaug, T. 2004. Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase. Nahrung 48(1): 53-5621. Sorensen, R., Kildal, E., Stepaniak, L., Pripp, A. H., and Sorhaug, T. 2004. Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase. Nahrung 48(1): 53-56
22. Szwajcer-Dey, E., Rasmussen, J., Meldal, M., and Breddam, K. 1992. Proline-specific endopeptidases from microbial sources: isolation of enzyme from aXanthomonas sp.. J. Baceteriol. 174: 2454-2459.22. Szwajcer-Dey, E., Rasmussen, J., Meldal, M., and Breddam, K. 1992. Proline-specific endopeptidases from microbial sources: isolation of enzyme from a Xanthomonas sp.. J. Baceteriol. 174: 2454 -2459.
23. Tobiassen, R. O., Pripp, A. H., Stepaniak, L., and Srhaug, T. 1996. Purification and characterization of an endopeptidase fromPropionibacterium freudenreichii . J. Dairy Sci. 79:2129-2136.23. Tobiassen, RO, Pripp, AH, Stepaniak, L., and S Rhaug, T. 1996. Purification and characterization of an endopeptidase from Propionibacterium freudenreichii . J. Dairy Sci. 79:2129-2136.
24. Uchiyama, H., Inaoka, T., Ohykuma-Soyejima, T., Togame, Shibanaka, Y., Yoshimoto, T., and Kokubo, T. 2000. Directed evolution to improve the thermostability of prolyl endopeptidase. J. Biochem. 128: 441-447.24. Uchiyama, H., Inaoka, T., Ohykuma-Soyejima, T., Togame, Shibanaka, Y., Yoshimoto, T., and Kokubo, T. 2000. Directed evolution to improve the thermostability of prolyl endopeptidase. J. Biochem. 128: 441-447.
25. Wilk, S. 1983. Prolyl endopeptidase. Life Sci. 33:2149-2157.25. Wilk, S. 1983. Prolyl endopeptidase. Life Sci. 33:2149-2157.
26. Xiu, Z., Li, M., Zhou, S. Dou, H., Zhou, H., Chen, C. 2002. A new method for the preparation of human parahyroid hormone 1-34 peptides. Biotechnol. Appl. Biochem. 36(Pt2): 111-117.26. Xiu, Z., Li, M., Zhou, S. Dou, H., Zhou, H., Chen, C. 2002. A new method for the Preparation of human parahyroid hormone 1-34 peptides. Biotechnol. Appl. Biochem. 36(Pt2): 111-117.
27. Yoshimoto, T., Kado, K., Matsubara, Fl, Koriyama, N., Kaneto, H., and Tsura, D. 1987. Specific inhibitors for prolyl endopeptidase and their anti-amnesic effect. J. Pharmacobio-Dyn. 10: 730-735.27. Yoshimoto, T., Kado, K., Matsubara, Fl, Koriyama, N., Kaneto, H., and Tsura, D. 1987. Specific inhibitors for prolyl endopeptidase and their anti-amnesic effect. J. Pharmacobio-Dyn 10: 730-735.
28. Yoshimoto, T., Walter, R., and Tsuru, D. 1980. Proline-specific endopeptidase fromFlavobacterium . J. Biol. Chem. 255: 4786-4792.28. Yoshimoto, T., Walter, R., and Tsuru, D. 1980. Proline-specific endopeptidase from Flavobacterium . J. Biol. Chem. 255: 4786-4792.
29. Yoshimoto, T., Kabashima, T., Fujii, M. 1998. Prolyl oligopeptidase and gene coding for the same. Japanese patent JP10066570.29. Yoshimoto, T., Kabashima, T., Fujii, M. 1998. Prolyl oligopeptidase and gene coding for the same. Japanese patent JP10066570.
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| Gass et al.,Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy. Biotechnol Bioeng. 2005 Dec 20 92(6):674-84. * |
| Marti et al., Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization. J Pharmacol Exp Ther. 2005 Jan;312(1):19-26. Epub 2004 Sep 9. * |
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