TWI464265B - Production method of a fusion protein in vivo - Google Patents
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本發明關於一種融合蛋白質的製造方法,且特別是攸關一種於細胞內製造融合蛋白質的方法。 The present invention relates to a method for producing a fusion protein, and in particular to a method for producing a fusion protein in a cell.
融合蛋白質(Fusion protein),是指一種連接有至少二個以上多胜肽的蛋白質,可能保有單一多胜肽原有的生物功能,或是呈現不同於單一多胜肽的生物功能。 Fusion protein refers to a protein linked to at least two peptides, which may retain the original biological function of a single polypeptide or exhibit biological functions different from a single multi-peptide.
舉例來說,融合有β-半乳糖苷酶(β-galactosidase)及半乳糖去氫酶(galactose dehydrogenase)的蛋白質,經細胞外實驗後,證實其相較於單一酵素具有酵素動力學上的優勢(請參閱Ljungcrantz.P.,et al.,Biochem.,1989,28:8786-8792)。又舉例來說,融合有海藻糖磷酸合成酶(trehalose-6-phosphate synthetase)及海藻糖磷酸脂酶(trehalose-6-phosphate phosphatase)的蛋白質,經細胞外實驗後,證明其不僅保留單一酵素本身的活性外,更較單一酵素具有酵素動力學上的優勢(請參閱Seo H.S.,et al.,Appl.Environ.Microbiol.,2000,66:2484-2490)。再舉例來說,甜菜醛去氫酶(betaine aldehyde dehydrogenase)-膽鹼去氫酶(chloline dehydrogenase)融合蛋白質於大腸桿菌(Escherichia coli)或菸草(Nicotiana tabacum)內表現時,可以提升這些生物體抵抗逆境的能力(請參閱Yilmaz J.L.,et al.,Biotechnol.Progs.,2002,18:1176-1182)。 For example, a protein fused with β-galactosidase and galactose dehydrogenase has been shown to have an enzyme kinetic advantage over a single enzyme after extracellular experiments. (See Ljungcrantz. P., et al., Biochem., 1989, 28: 8786-8792). For example, a protein fused with trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase, after extracellular experiments, proves that it not only retains a single enzyme itself. In addition to its activity, it has an enzyme kinetic advantage over a single enzyme (see Seo HS, et al., Appl. Environ. Microbiol., 2000, 66: 2484-2490). For example, betaine aldehyde dehydrogenase-chloline dehydrogenase fusion protein can enhance the resistance of these organisms when expressed in Escherichia coli or Nicotiana tabacum . The ability to adversity (see Yilmaz JL, et al., Biotechnol. Progs., 2002, 18: 1176-1182).
融合蛋白質可以自然存在於細胞體內,例如癌症細胞中的bcr-abl融合蛋白質。融合蛋白質也可以有目的地透過人為方式存在於細胞體內,基因 工程已提供一般實驗流程來實現之,如聚合酶連鎖反應(polymerase chain reaction,PCR)、限制酶剪切(enzyme digestion)、核酸接合(DNA ligation)、...等等。詳細地說,須將二個以上各自編碼一個多胜肽的基因片段連接在一起,再送入至細胞體內。接著,於細胞體內表現這些多胜肽而形成融合蛋白質。 Fusion proteins can naturally exist in cells, such as bcr-abl fusion proteins in cancer cells. Fusion proteins can also be purposefully present in cells by humans, genes Engineering has provided general experimental procedures to achieve such as polymerase chain reaction (PCR), enzyme digestion, DNA ligation, and the like. In detail, two or more gene fragments each encoding a multi-peptide are ligated together and fed into a cell. These multi-peptides are then expressed in the cells to form a fusion protein.
就基因工程而言,製備融合蛋白質的方法雖然相當簡單,但是融合蛋白質大多屬於大分子,使得其在空間上的彈性度及自由度受到阻礙,且彼此易相互干擾而無法折疊,造成融合蛋白質失去生物活性,甚至於細胞體內形成不可溶的包涵體(inclusion body)。因此,後續須破壞細胞體取得包涵體,再從包涵體得到失活的融合蛋白質,並於適當溶液中使失活的融合蛋白質折疊而復性。但復性過程畢竟係屬人為操作的,仍不比細胞體內提供的環境,因此在溶液中折疊之融合蛋白質的生物功能仍然有改善空間。 In terms of genetic engineering, although the method for preparing the fusion protein is quite simple, the fusion protein is mostly a macromolecule, so that its spatial elasticity and degree of freedom are hindered, and each other easily interferes with each other and cannot be folded, resulting in loss of the fusion protein. Biological activity, even in the body of the body to form an insoluble inclusion body. Therefore, the cell body is subsequently destroyed to obtain inclusion bodies, and the inactivated fusion protein is obtained from the inclusion body, and the inactivated fusion protein is folded and renatured in a suitable solution. However, the renaturation process is artificially operated after all, and is still no more than the environment provided by the cells in the body. Therefore, the biological function of the folded protein folded in the solution still has room for improvement.
自然界中,厭氧微生物演化出一種相當特殊且可以有效分解纖維素的胞器。厭氧微生物在細胞外形成纖維體(cellulosome),纖維體含有一撐架蛋白質(scaffolding protein),而撐架蛋白質具有一黏附區域(coherin domain),黏附區域結合至一纖維素水解酶(cellulase)的錨定區域(dockerin domain),藉此容許纖維體成為一酵素複合體(enzyme complex)而有效地分解纖維素(請參閱Doi R.H.,et al.,Nature Rev.Microbiol.,2004,2:541-551)。 In nature, anaerobic microorganisms evolve a rather special organelle that can effectively break down cellulose. Anaerobic microorganisms form a cellulosome outside the cell, the fibrous body contains a scaffolding protein, and the scaffold protein has a coherin domain, and the adhesion region binds to a cellulase. The dockerin domain, thereby allowing the fibrous body to become an enzyme complex and efficiently decompose cellulose (see Doi RH, et al., Nature Rev. Microbiol., 2004, 2: 541). -551).
本發明乃是根據黏附區域結合至錨定區域的特性,而發現二各具有黏附區域及錨定區域的融合蛋白質在同一宿主細胞中被表現,黏附區域及錨 定區域不易影響此二融合蛋白質中其他多胜肽的彈性度及自由度,使得此些融合蛋白質可以於宿主細胞內折疊並相互融合,並提供此二融合蛋白質優異的生物功能。 The present invention is based on the fact that the adhesion region binds to the anchor region, and it is found that the two fusion proteins having the adhesion region and the anchor region are expressed in the same host cell, the adhesion region and the anchor. The fixed region does not easily affect the elasticity and degree of freedom of other polypeptides in the two fusion proteins, so that the fusion proteins can be folded and fused in the host cell, and provide excellent biological functions of the two fusion proteins.
因此,在第一方面,本發明揭露一種融合蛋白質的製造方法,其包括下列步驟:(a)構築一編碼一錨定區域及一第一目標胜肽的第一核酸分子;(b)構築一編碼一黏附區域及一第二目標胜肽的第二核酸分子;(c)遞送第一核酸分子及第二核酸分子至一宿主細胞內;以及(d)培養宿主細胞,以表現一第一融合胜肽及一第二融合胜肽,且第一融合胜肽具有錨定區域及第一目標胜肽,第二融合胜肽具有黏附區域及第二目標胜肽,藉此錨定區域連接至黏附區域,以容許第一融合胜肽及第二融合胜肽相互融合。 Therefore, in a first aspect, the present invention discloses a method for producing a fusion protein comprising the steps of: (a) constructing a first nucleic acid molecule encoding an anchor region and a first target peptide; (b) constructing a a second nucleic acid molecule encoding an adhesion region and a second target peptide; (c) delivering the first nucleic acid molecule and the second nucleic acid molecule to a host cell; and (d) cultivating the host cell to express a first fusion a peptide and a second fusion peptide, and the first fusion peptide has an anchor region and a first target peptide, and the second fusion peptide has an adhesion region and a second target peptide, whereby the anchor region is attached to the adhesion a region to allow the first fusion peptide and the second fusion peptide to fuse with each other.
在第二方面,本發明揭露一種宿主細胞,其包括:一第一核酸分子及一第二核酸分子,而第一核酸分子係編碼一錨定區域及一第一目標胜肽,第二核酸分子係編碼一黏附區域及一第二目標胜肽。 In a second aspect, the invention discloses a host cell comprising: a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes an anchor region and a first target peptide, the second nucleic acid molecule The code encodes an adhesion region and a second target peptide.
迄今針對纖維體之黏附區域及錨定區域的相關研究及應用多著重在纖維體於細胞外的製造,並運用得到的纖維體來進行纖維素的分解,但卻未見任何研究文獻或專利文獻將二各具有黏附區域及錨定區域的融合蛋白質表現於同一細胞體中,更無法瞭解到對此些融合蛋白質在結構上及生物功能上的影響。 So far, the related research and application of the adhesion region and the anchoring region of the fibrous body have focused on the extracellular production of the fibrous body, and the obtained fibrous body is used for the decomposition of cellulose, but no research literature or patent literature has been found. The fusion proteins with the adhesion region and the anchor region are expressed in the same cell body, and the influence on the structure and biological function of the fusion proteins is not known.
經本發明人多年努力與研究,發現編碼錨定區域的基因及編碼黏附區域的基因送入到同一細胞體後,細胞體表現的二融合蛋白質除了各具有相互連接的錨定區域及黏附區域,使此些融合蛋白質相互結合外,此些融合 蛋白質也不會形成不可溶的包涵體。而且,此些融合蛋白質可以在細胞體中折疊,因此後續不須將此些融合蛋白質置於溶液中進行復性,則可使用於不同的領域,如疾病治療及/或預防、生質材料及/或能源開發、廢棄物分解、環境保護、...等等。 After years of efforts and research by the present inventors, it has been found that the gene encoding the anchor region and the gene encoding the adhesion region are fed into the same cell body, and the two fusion proteins expressed by the cell body have interconnected anchor regions and adhesion regions, respectively. These fusion proteins are combined with each other, and these fusions Protein also does not form insoluble inclusion bodies. Moreover, such fusion proteins can be folded in the cell body, so that subsequent fusion of the fusion proteins in a solution can be used in different fields such as disease treatment and/or prevention, biomass materials and / or energy development, waste decomposition, environmental protection, ... and so on.
因此,本發明提供之融合蛋白質的製造方法,含有下列步驟:(a)構築一編碼一錨定區域及一第一目標胜肽的第一核酸分子;(b)構築一編碼一黏附區域及一第二目標胜肽的第二核酸分子;(c)遞送第一核酸分子及第二核酸分子至一宿主細胞內;以及(d)培養宿主細胞,以表現一第一融合胜肽及一第二融合胜肽,且第一融合胜肽具有錨定區域及第一目標胜肽,第二融合胜肽具有黏附區域及第二目標胜肽,藉此錨定區域連接於黏附區域,以容許第一融合胜肽及第二融合胜肽相互融合。 Therefore, the method for producing a fusion protein provided by the present invention comprises the steps of: (a) constructing a first nucleic acid molecule encoding an anchor region and a first target peptide; (b) constructing a code-adhesion region and a a second nucleic acid molecule of the second target peptide; (c) delivering the first nucleic acid molecule and the second nucleic acid molecule to a host cell; and (d) cultivating the host cell to express a first fusion peptide and a second a fusion peptide, wherein the first fusion peptide has an anchor region and a first target peptide, and the second fusion peptide has an adhesion region and a second target peptide, whereby the anchor region is attached to the adhesion region to allow the first The fusion peptide and the second fusion peptide are fused to each other.
文中單獨或組合使用的術語「胜肽」,意謂一種由多個胺基酸組成的聚合物,且包含已知的天然存在胺基酸或人造仿效物(artificial chemical mimic)。且本文所用的術語「胜肽」可與「多胜肽」、「蛋白質」等術語交換使用。 The term "peptide" as used herein, alone or in combination, means a polymer composed of a plurality of amino acids and comprises a known naturally occurring amino acid or artificial chemical mimic. And the term "peptide" as used herein may be used interchangeably with terms such as "polypeptide" and "protein".
特別指明的是,本文單獨或組合使用的術語「目標胜肽」,意謂一種選定胜肽,且選定胜肽具有已知的生物功能,如化合物催化、胞內或胞外訊息傳遞、抗原辨識、...等等。 In particular, the term "target peptide" used herein, alone or in combination, means a selected peptide, and the selected peptide has known biological functions, such as compound catalysis, intracellular or extracellular signaling, antigen recognition. ,...and many more.
文中單獨或組合使用的術語「核酸」或「核酸分子」,意謂一種由核苷酸組成的聚合物,可呈單股或雙股,且包含已知的天然存在核苷酸或人造仿效物。且本文所用的術語「核酸」或「核酸分子」可與「基因片段」、「基因」、「DNA」等術語交換使用。 The term "nucleic acid" or "nucleic acid molecule" as used herein, alone or in combination, means a polymer consisting of nucleotides which may be single or double stranded and comprising known naturally occurring nucleotides or artificial imitations. . The term "nucleic acid" or "nucleic acid molecule" as used herein may be used interchangeably with terms such as "gene fragment", "gene", and "DNA".
除非另有指明,本技術領域人士應當瞭解本文列示的核酸序列不限於顯示的特定核苷酸序列,亦涵蓋其互補序列(complementary sequence)。此外,由於核酸序列中的密碼子(由三個核苷酸組成)具有簡併性,不同密碼子能編碼出同一胺基酸,因此針對相同的胜肽,本技術領域人士可透過已知的密碼子偏好表(codon usage table)獲得特定序列的簡併序列(degenerative sequence)。換句話說,文中列示的核酸序列還涵蓋特定序列的簡併序列。 Unless otherwise indicated, those skilled in the art will appreciate that the nucleic acid sequences set forth herein are not limited to the particular nucleotide sequences shown, but also to their complementary sequences. Furthermore, since the codons (consisting of three nucleotides) in the nucleic acid sequence are degenerate, and different codons can encode the same amino acid, those skilled in the art can understand the same peptide. A codon usage table obtains a degenerative sequence of a particular sequence. In other words, the nucleic acid sequences listed herein also encompass degenerate sequences of a particular sequence.
文中單獨或組合使用的術語「宿主細胞」,除了意謂一特定個體的細胞外,還涵蓋其繼代培養的子代或可能的子代。而所有子代可能在後續培養過程中因突變或環境等因素造成基因修飾,使得子代與母代不一致,但子代仍涵蓋於本發明的範圍內。且本文所用的術語「宿主細胞」可與「細胞」、「細胞體」、「生物體」等術語交換使用。 The term "host cell" as used herein, alone or in combination, encompasses subcultures or progeny of its subculture, in addition to the cells of a particular individual. However, all progeny may cause genetic modification during subsequent culture due to mutation or environmental factors, such that the progeny are inconsistent with the mother, but the progeny are still covered by the present invention. And the term "host cell" as used herein may be used interchangeably with terms such as "cell", "cell body", and "organism".
本發明步驟(a)中的錨定區域可衍生自但不限於下列微生物中的一者:Acetivibrio cellulolyticus、Bacteroides cellulosolvens、Butyrivibrio fibrisolvens、Clostridium acetobutylicum、Clostridium cellobioparum、Clostridium cellulolyticum、Clostridium cellulovorans、Clostridium josui、Clostridium papyrosolvens、Clostridium thermocellum、Ruminococcus albus、Ruminococcus flavefaciens、Neocallimastix patriciarum、Orpinomyces joyonii、Orpinomyces PC-2、Piomyces equi、Piomyces E2。 The anchoring region in step (a) of the present invention may be derived from, but not limited to, one of the following microorganisms: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 .
本發明步驟(b)中的黏附區域可衍生自但不限於下列微生物中的一者:Acetivibrio cellulolyticus、Bacteroides cellulosolvens、Butyrivibrio fibrisolvens、Clostridium acetobutylicum、Clostridium cellobioparum、 Clostridium cellulolyticum、Clostridium cellulovorans、Clostridium josui、Clostridium papyrosolvens、Clostridium thermocellum、Ruminococcus albus、Ruminococcus flavefaciens、Neocallimastix patriciarum、Orpinomyces joyonii、Orpinomyces PC-2、Piomyces equi、Piomyces E2。 The adhesion region in step (b) of the present invention may be derived from, but not limited to, one of the following microorganisms: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 .
本發明步驟(a)及步驟(b)的構築過程,已於本技術領域的相關研究文獻及專利文獻中有詳細報導,如Sambrook J.,et al.,2001,Molecular Cloning:a Laboratory Manual,3rd ed.。除非另有指明,關於此二步驟的詳細說明可參考前述文獻。 The construction process of the steps (a) and (b) of the present invention has been reported in detail in related research literature and patent documents in the technical field, such as Sambrook J., et al., 2001, Molecular Cloning: a Laboratory Manual, 3rd ed. Unless otherwise indicated, a detailed description of these two steps can be found in the aforementioned documents.
進行本發明步驟(a)及步驟(b)時,此二步驟的順序並無一定限制。也就是說,先進行步驟(a),後進行步驟(b);先進行步驟(b),後進行步驟(a);或是同時進行步驟(a)和步驟(b)均為允許的。 When the steps (a) and (b) of the present invention are carried out, the order of the two steps is not limited. That is, step (a) is performed first, followed by step (b); step (b) is performed first, then step (a) is performed; or both steps (a) and (b) are allowed at the same time.
依據本發明之一較佳實施例,進行本發明步驟(a)時,可將第一核酸分子植入至一可供宿主細胞表現的載體,而進行本發明步驟(b)時,可將第二核酸分子植入至植有第一核酸分子的載體。 According to a preferred embodiment of the present invention, when the step (a) of the present invention is carried out, the first nucleic acid molecule can be implanted into a vector which can be expressed by the host cell, and when the step (b) of the present invention is carried out, The second nucleic acid molecule is implanted into a vector implanted with the first nucleic acid molecule.
為控制第一核酸分子及第二核酸分子的轉錄,載體更具有一可操作地連接至第一核酸分子及第二核酸分子的啟動子。文中使用的片語「可操作地連接」,意謂二基因片段相當接近,且其中一片段足以影響另一片段。於本較佳例中,啟動子是位於第一核酸分子及第二核酸分子之5’端的位置。 To control transcription of the first nucleic acid molecule and the second nucleic acid molecule, the vector further has a promoter operably linked to the first nucleic acid molecule and the second nucleic acid molecule. The phrase "operably linked" as used herein means that the two gene segments are quite close and one of the fragments is sufficient to affect the other. In the preferred embodiment, the promoter is located at the 5' end of the first nucleic acid molecule and the second nucleic acid molecule.
於本較佳例中,啟動子可以為組合式啟動子(constitutive promoter)或可誘導性啟動子(inducible promoter)。啟動子是依照宿主細胞來決定的,其可衍生自病毒、細菌細胞、酵母菌細胞、真菌細胞、藻類細胞、植物細胞、動物細胞、昆蟲細胞、人類細胞中之一者。來自於細菌細胞的啟動子, 如tac啟動子、T7啟動子、T7 A1啟動子、lac啟動子、trp啟動子、trc啟動子、araBAD啟動子或λPRPL啟動子。來自於植物細胞的啟動子,如35S CaMV啟動子、肌動蛋白啟動子(actin promoter)或泛素啟動子(ubiquitin promoter)。來自於動物細胞的啟動子,如SV40啟動子、RSV啟動子、CMV啟動子或HSV啟動子。 In this preferred embodiment, the promoter may be a constitutive promoter or an inducible promoter. The promoter is determined in accordance with the host cell, and may be derived from one of a virus, a bacterial cell, a yeast cell, a fungal cell, an algal cell, a plant cell, an animal cell, an insect cell, or a human cell. Promoters derived from bacterial cells, such as the tac promoter, the T7 promoter, the T7 A1 promoter, the lac promoter, the trp promoter, the trc promoter, the ara BAD promoter or the lambda P R P L promoter. A promoter derived from a plant cell, such as a 35S CaMV promoter, an actin promoter, or a ubiquitin promoter. A promoter derived from an animal cell, such as the SV40 promoter, the RSV promoter, the CMV promoter or the HSV promoter.
載體另含有其他表現控制要素,以進一步控制第一核酸分子及第二核酸分子的轉錄,如一轉錄起始位置(transcription start site)、一轉錄終止位置(transcription stop site)、一核糖體結合位置(ribosome binding site)、一RNA剪切位置(RNA splicing site)、一聚腺苷酸化位置(polyadenylation site)及一轉譯終止位置(translation stop site)。這些表現控制要素是依照宿主細胞來決定的。 The vector further contains other expression control elements to further control transcription of the first nucleic acid molecule and the second nucleic acid molecule, such as a transcription start site, a transcription stop site, and a ribosome binding site ( A ribosome binding site), an RNA splicing site, a polyadenylation site, and a translation stop site. These performance control factors are determined by the host cell.
載體尚含有一供篩選載體的標記基因(marker gene)或報導基因(reporter gene)。標記基因是依照宿主細胞來決定的,如真核生物細胞的二氫葉酸還原酶(dihydrofolate reductase)基因、抗G418基因或抗新黴素(neomycin)基因、細菌細胞的抗胺苄青黴素(ampicilin)基因、抗鏈黴素(streptomycin)基因或抗康那黴素(kanamycin)基因。報導基因,如綠色螢光蛋白質(green fluorescent protein)基因、螢光素酶(luciferase)基因、β-半乳糖苷酶基因或氯黴素乙醯轉移酶(chloramphenicol acetyltransferase)基因。 The vector also contains a marker gene or reporter gene for screening the vector. The marker gene is determined according to the host cell, such as the dihydrofolate reductase gene of the eukaryotic cell, the anti-G418 gene or the neomycin gene, and the ampicillin of the bacterial cell. Gene, anti-streptomycin gene or anti-kanamycin gene. The gene is reported, such as a green fluorescent protein gene, a luciferase gene, a β-galactosidase gene or a chloramphenicol acetyltransferase gene.
依據本發明之另一較佳實施例,進行本發明步驟(a)時,可將第一核酸分子植入至一可供宿主細胞表現的第一載體,而進行本發明步驟(b)時,可將第二核酸分子植入至一可供宿主細胞表現的第二載體。 According to another preferred embodiment of the present invention, when performing step (a) of the present invention, the first nucleic acid molecule can be implanted into a first carrier which can be expressed by the host cell, and when step (b) of the present invention is carried out, The second nucleic acid molecule can be implanted into a second vector that can be expressed by the host cell.
為控制第一核酸分子及第二核酸分子的轉錄,第一載體更具有一可操作地連接至第一核酸分子的第一啟動子,而第二載體更具有一可操作地連接至第二核酸分子的第二啟動子。於本較佳例中,第一啟動子是位於第一核酸分子之5’端的位置,而第二啟動子是位於第二核酸分子之5’端的位置。 To control transcription of the first nucleic acid molecule and the second nucleic acid molecule, the first vector further has a first promoter operably linked to the first nucleic acid molecule, and the second vector further has an operably linked second nucleic acid The second promoter of the molecule. In the preferred embodiment, the first promoter is located at the 5' end of the first nucleic acid molecule and the second promoter is located at the 5' end of the second nucleic acid molecule.
於本較佳例中,第一啟動子及第二啟動子可分別為組合式啟動子或可誘導性啟動子。第一啟動子及第二啟動子是依照宿主細胞來決定的,其可衍生自病毒、細菌細胞、酵母菌細胞、真菌細胞、藻類細胞、植物細胞、動物細胞、昆蟲細胞、人類細胞中之一者。來自於細菌細胞的啟動子,如tac啟動子、T7啟動子、T7 A1啟動子、lac啟動子、trp啟動子、trc啟動子、araBAD啟動子或λPRPL啟動子。來自於植物細胞的啟動子,如35S CaMV啟動子、肌動蛋白啟動子或泛素啟動子。來自於動物細胞的啟動子,如SV40啟動子、RSV啟動子、CMV啟動子或HSV啟動子。 In the preferred embodiment, the first promoter and the second promoter may each be a combinatorial promoter or an inducible promoter. The first promoter and the second promoter are determined according to the host cell, and may be derived from one of a virus, a bacterial cell, a yeast cell, a fungal cell, an algal cell, a plant cell, an animal cell, an insect cell, and a human cell. By. A promoter derived from a bacterial cell, such as a tac promoter, a T7 promoter, a T7 A1 promoter, a lac promoter, a trp promoter, a trc promoter, an ara BAD promoter, or a lambda P R P L promoter. A promoter derived from a plant cell, such as the 35S CaMV promoter, an actin promoter or a ubiquitin promoter. A promoter derived from an animal cell, such as the SV40 promoter, the RSV promoter, the CMV promoter or the HSV promoter.
第一載體及第二載體還各自含有其他表現控制要素,以進一步控制第一核酸分子及第二核酸分子的轉錄,如一轉錄起始位置、一轉錄終止位置、一核糖體結合位置、一RNA剪切位置、一聚腺苷酸化位置及一轉譯終止位置。這些表現控制要素是依照宿主細胞來決定的。 The first vector and the second vector each further contain other expression control elements to further control transcription of the first nucleic acid molecule and the second nucleic acid molecule, such as a transcription start position, a transcription termination position, a ribosome binding position, and an RNA cutter. The cleavage position, a polyadenylation position, and a translation termination position. These performance control factors are determined by the host cell.
第一載體及第二載體另各自含有一供篩選這些載體的標記基因或報導基因。標記基因是依照宿主細胞來決定的,如真核生物細胞的二氫葉酸還原酶基因、抗G418基因或抗新黴素基因、細菌細胞的抗胺苄青黴素基因、抗鏈黴素基因或抗康那黴素基因。報導基因,如綠色螢光蛋白質基因、螢光素酶基因、β-半乳糖苷酶基因或氯黴素乙醯轉移酶基因。 The first vector and the second vector each additionally contain a marker gene or reporter gene for screening these vectors. The marker gene is determined according to the host cell, such as the dihydrofolate reductase gene of the eukaryotic cell, the anti-G418 gene or the anti-neomycin gene, the anti-ampicillin gene of the bacterial cell, the anti-streptomycin gene or the anti-Kang Natamycin gene. Reporting genes, such as the green fluorescent protein gene, the luciferase gene, the β-galactosidase gene, or the chloramphenicol acetyltransferase gene.
本發明步驟(c)中的宿主細胞可以為原核生物細胞或真核生物細胞。 原核生物細胞的例子,可以為但不限於細菌、藍綠藻或放射菌,其中細菌的代表例為大腸桿菌、枯草桿菌(Bacillus subtilis)、乳桿菌屬(Lactobacillus sp.)、鏈黴菌屬(Streptomyces sp.)或沙門氏傷寒桿菌(Salmonella typhimurium)。 The host cell in step (c) of the present invention may be a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells may be, but are not limited to, bacteria, blue-green algae or radiobacteria, and representative examples of the bacteria are Escherichia coli, Bacillus subtilis , Lactobacillus sp. , Streptomyces. Sp. ) or Salmonella typhimurium .
真核生物細胞的例子,可以為但不限於真菌細胞、原生動物細胞、植物細胞、動物細胞、昆蟲細胞或人類細胞。真菌細胞的代表例為酵母菌細胞,如麵包酵母菌或嗜甲醇酵母菌。植物細胞可取自於裸子植物或被子植物。而且植物細胞可分離自上述植物的根、莖、葉或分生組織等部位,並以原生質體或癒合組織的形式培養。昆蟲細胞的代表例為果蠅S2細胞或秋行軍蟲Sf21細胞或Sf9細胞。動物細胞不限於培養過的細胞或活體內的細胞,較佳地為取自於脊椎動物,更佳地為取自於哺乳動物。而且動物細胞可分離自腎臟、肝臟、肺臟、卵巢、***、皮膚、骨骼、肌肉或皮膚等器官或組織,其例子可以為CHO、COS、BHK、HEK-293、Hela、NIH3T3、VERO、MDCK、MOLT-4、Jurkat、K562或HepG2。 Examples of eukaryotic cells can be, but are not limited to, fungal cells, protozoan cells, plant cells, animal cells, insect cells, or human cells. Representative examples of fungal cells are yeast cells such as baker's yeast or methanolophilus yeast. Plant cells can be obtained from gymnosperms or angiosperms. Further, the plant cell can be isolated from the root, stem, leaf or meristematic tissue of the above plant and cultured in the form of a protoplast or a healing tissue. Representative examples of insect cells are Drosophila S2 cells or S. cerevisiae Sf21 cells or Sf9 cells. The animal cells are not limited to cultured cells or cells in vivo, preferably from vertebrate animals, and more preferably from mammals. Moreover, the animal cells can be isolated from organs or tissues such as kidney, liver, lung, ovary, breast, skin, bone, muscle or skin, and examples thereof can be CHO, COS, BHK, HEK-293, Hela, NIH3T3, VERO, MDCK, MOLT-4, Jurkat, K562 or HepG2.
一般來說,當宿主細胞為原核生物細胞時,本發明步驟(c)的遞送過程稱為「轉形(transformation)」。本技術領域人士理應瞭解如何實施轉形,如利用碳酸鈣、氯化鈣為媒介、基因槍、噬菌體感染,故不再贅述。 Generally, when the host cell is a prokaryotic cell, the delivery process of step (c) of the present invention is referred to as "transformation." Those skilled in the art should understand how to implement transformation, such as using calcium carbonate, calcium chloride as a medium, gene gun, phage infection, and therefore will not be described again.
一般來說,當宿主細胞為真核生物細胞時,本發明步驟(c)的遞送過程稱為「轉染(transfection)」。本技術領域人士理應瞭解如何實施轉染,如利用微脂體(liposome)為媒介、電穿孔法(electroporation)、微注射法(microiniection)、動、植物病毒感染,故不再贅述。 Generally, when the host cell is a eukaryotic cell, the delivery process of step (c) of the present invention is referred to as "transfection." Those skilled in the art will understand how to perform transfection, such as the use of liposome as a medium, electroporation, microiniection, animal and plant viral infections, and therefore will not be described again.
本發明步驟(d)的培養過程,乃為本技術領域人士依選用的宿主細胞 使用合適的培養基及培養條件則可輕易實現的。舉例來說,當宿主細胞為大腸桿菌時,宿主細胞是培養於LB培養基中,並以搖晃方式置於30℃培養箱中。 The culture process of step (d) of the present invention is a host cell selected by those skilled in the art. This can be easily accomplished using suitable media and culture conditions. For example, when the host cell is Escherichia coli, the host cell is cultured in LB medium and placed in a 30 ° C incubator in a shaking manner.
於本發明揭露範圍內,亦提供一種宿主細胞,其包含:一編碼一錨定區域及一第一目標胜肽的第一核酸分子;以及一編碼一黏附區域及一第二目標胜肽的第二核酸分子。 Also included within the scope of the present invention is a host cell comprising: a first nucleic acid molecule encoding an anchor region and a first target peptide; and a first encoding an adhesion region and a second target peptide Two nucleic acid molecules.
特別指明的是,此處提及的宿主細胞、錨定區域及黏附區域係與前述製造方法中所提及者相同,故不再贅述。 It is specifically noted that the host cells, anchoring regions, and adhesion regions mentioned herein are the same as those mentioned in the foregoing manufacturing methods, and therefore will not be described again.
茲以下述實施例,以進一步例示說明本發明。 The invention is further illustrated by the following examples.
下述實施例採用的方法主要是參考本技術領域人士熟悉的教科書:Sambrook J.,et al.,2001,Molecular Cloning:a Laboratory Manual,3rd ed.。 The methods employed in the following examples are primarily based on textbooks familiar to those skilled in the art: Sambrook J., et al., 2001, Molecular Cloning: a Laboratory Manual, 3rd ed.
細菌培養液濃度是使用分光光度計(V530,Jasco)測得的,使用的波長為550nm,測得的吸光值記錄為OD550。總蛋白質濃度是使用蛋白質分析試劑(BioRad Co.)測得的。標的蛋白質濃度是利用影像分析儀(AlphaImager EP,Alpha Innotech Co.)分析電泳分離之蛋白質測得的。 The bacterial culture solution concentration was measured using a spectrophotometer (V530, Jasco) using a wavelength of 550 nm, and the measured absorbance was recorded as OD 550 . The total protein concentration was measured using a protein assay reagent (BioRad Co.). The target protein concentration was determined by analyzing the electrophoretically separated protein using an image analyzer (AlphaImager EP, Alpha Innotech Co.).
細菌染色體(chromosome)、質體(plasmid)和DNA的純化是分別使用Wizard Genomic DNA Purification Kit(Promega Co.)、High-Speed Plasmid Mini Kit(Geneaid Co.)及Gel/PCR DNA Fragments Extraction Kit(Geneaid Co.)等商業套組。限制酶是購自於New England Biolabs及Fermentas Life Science。T4接合酶及Pfu DNA聚合酶是購自於Promega Co.。用於聚合酶連鎖反應的引子是委由明欣生物科技公司(台北)及源資生物科技公司(台 北)合成的。 The purification of bacterial chromosomes, plasmids and DNA was performed using Wizard Genomic DNA Purification Kit (Promega Co.), High-Speed Plasmid Mini Kit (Geneaid Co.) and Gel/PCR DNA Fragments Extraction Kit (Geneaid, respectively). Co.) and other commercial groups. Restriction enzymes were purchased from New England Biolabs and Fermentas Life Science. T4 ligase and Pfu DNA polymerase were purchased from Promega Co. The primer used for the polymerase chain reaction was commissioned by Mingxin Biotechnology Co., Ltd. (Taipei) and Yuanzi Biotech Co., Ltd. North) synthetic.
DNA轉殖是採用化學轉形法完成的,其詳細過程如下:從LB固態培養基中選取單一菌落至LB液態培養基,並於適當溫度下,以150rpm水平震盪培養12至16小時。將菌液接種到新鮮LB液態培養基,其起始濃度為OD550=0.08,並在適當溫度下,以150rpm水平震盪培養。直到菌液濃度OD550=0.3-0.5,取出4mL的菌液置至無菌試管中,冰浴10分鐘,然後以4,000rpm離心2分鐘並移除上清液。將離心剩下的菌體與2mL的0.1M MgCl2均勻混合後,冰浴5分鐘,再以4,000rpm離心2分鐘並移除上清液。將離心剩餘的菌體與1.5mL的0.05M CaCl2均勻混合後,冰浴20分鐘,再以4,000rpm離心2分鐘並移除上清液。加入300μL的0.05M CaCl2與離心殘存的菌體均勻混合後,則製備完成勝任細胞(competent cell)。接著,取2ng/mL的質體與100μL的勝任細胞加入至無菌試管中並混合均勻。冰浴30分鐘後,將無菌試管移至42℃恆溫水浴槽中2分鐘,再冰浴5分鐘。最後加入1mL的新鮮LB液態培養基(4℃)至勝任細胞菌液後,置於適當溫度中培養2小時,再以4,000rpm離心10分鐘並移除上清液。將殘存的LB液態培養基與離心下來的勝任細胞菌體混合均勻後,吸取適量的勝任細胞菌液,均勻塗佈在含有抗生素的LB固態培養基,並將其放置於適當溫度的恆溫培養箱中,隔夜培養至長出菌落。 DNA transfer was performed by chemical transformation, and the detailed procedure was as follows: Single colonies were selected from LB solid medium to LB liquid medium, and cultured at a suitable temperature for 12 to 16 hours at 150 rpm. The bacterial solution was inoculated into fresh LB liquid medium at an initial concentration of OD 550 = 0.08, and cultured at a suitable temperature at 150 rpm. Until the bacterial concentration OD 550 = 0.3-0.5, 4 mL of the bacterial solution was taken out into a sterile test tube, ice-bathed for 10 minutes, then centrifuged at 4,000 rpm for 2 minutes and the supernatant was removed. The cells remaining after centrifugation were uniformly mixed with 2 mL of 0.1 M MgCl 2 , and then ice-bathed for 5 minutes, centrifuged at 4,000 rpm for 2 minutes, and the supernatant was removed. The cells remaining after centrifugation were uniformly mixed with 1.5 mL of 0.05 M CaCl 2 , and then ice-bathed for 20 minutes, centrifuged at 4,000 rpm for 2 minutes, and the supernatant was removed. After 300 μL of 0.05 M CaCl 2 was added and uniformly mixed with the centrifuged cells, a competent cell was prepared. Next, 2 ng/mL of plastids and 100 μL of competent cells were added to a sterile test tube and mixed well. After 30 minutes in an ice bath, the sterile test tube was transferred to a 42 ° C constant temperature water bath for 2 minutes and then ice bathed for 5 minutes. Finally, 1 mL of fresh LB liquid medium (4 ° C) was added to the competent cell bacteria solution, and the cells were incubated at an appropriate temperature for 2 hours, centrifuged at 4,000 rpm for 10 minutes, and the supernatant was removed. After mixing the remaining LB liquid medium with the centrifuged competent cell bacteria, take appropriate amount of competent cell bacteria solution, uniformly apply it to the LB solid medium containing antibiotics, and place it in a constant temperature incubator at an appropriate temperature. Cultivate overnight to grow colonies.
酵素活性是採用高效率液相層析儀(High-pressure liquid chromatography,HPLC)測得的。Agrobacterium radiobacter B11291 D型胺甲醯基水解酶(D-carbamoylase,AHL)的活性測試是將樣品與反應溶液(最終濃度:20mM CpHPG及10mM磷酸鈉緩衝液,pH6.6-6.8)混合,並於40 ℃中反應30分鐘。100℃加熱反應混合液10分鐘終止反應後,以12,000rpm離心10分鐘,再取上清液進行HPLC分析。HPLC分析使用的管柱為C18管柱(250mm×4.6mm,Bos Hypersil),使用的移動相為2mM醋酸銨(pH3.2):甲醇=91:9體積比的溶液,並以波長280nm UV進行訊號偵測。Agrobacterium radiobacter B11291 D型乙內醯酶(D-hydantoinase,HDT)的活性測試與AHL相似,惟其所用的反應溶液為最終濃度:10mM HPH,0.1M Tris buffer,pH8.0及0.5mM MnCl2的溶液。酵素活性定義為每單位酵素活性(U)=產物生成量(μmole)/反應時間(min)。 The enzyme activity was measured by high-pressure liquid chromatography (HPLC). The activity test of Agrobacterium radiobacter B11291 D-carbamoylase (AHL) is to mix the sample with the reaction solution (final concentration: 20 mM CpHPG and 10 mM sodium phosphate buffer, pH 6.6-6.8). The reaction was carried out at 40 ° C for 30 minutes. The reaction mixture was heated at 100 ° C for 10 minutes to terminate the reaction, and then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken for HPLC analysis. The column used for HPLC analysis was a C18 column (250 mm × 4.6 mm, Bos Hypersil), and the mobile phase used was a solution of 2 mM ammonium acetate (pH 3.2): methanol = 91:9 by volume, and was carried out at a wavelength of 280 nm UV. Signal detection. The activity test of Agrobacterium radiobacter B11291 D-hydantoinase (HDT) is similar to that of AHL except that the reaction solution used is a final concentration: 10 mM HPH, 0.1 M Tris buffer, pH 8.0 and 0.5 mM MnCl 2 solution. . Enzyme activity is defined as enzyme activity per unit (U) = product production (μmole) / reaction time (min).
如圖1所示,質體pETW-A2HCh含有T7啟動子、受啟動子驅動來編碼Agrobacterium radiobacter B11291 AHL及HDT(AHL-HDT)融合蛋白質的基因片段、大腸桿菌的ColE1複製起始點(replication origin)、抗胺苄青黴素基因。其構築流程如下,首先依據美國國家生物科技資訊中心(National Center for Biotechnology Information,NCBI)基因體資料庫中Agrobacterium radiobacter B11291 AHL的核酸序列(登記號:U59376)來設計引子:順向引子:5’-tttgtgatatcgctaccaccaccaccggtcaggccaagccaagcttg-3’(SEQ ID NO:1);反向引子:5’-taacttctagaaggagatatacatatgac-3’(SEQ ID NO:2)。 As shown in Fig. 1, the plastid pETW-A2HCh contains a T7 promoter, a gene fragment driven by a promoter to encode Agrobacterium radiobacter B11291 AHL and HDT (AHL-HDT) fusion protein, and a ColE1 origin of replication of Escherichia coli (replication origin) ), anti-ampicillin gene. The construction process is as follows. First, according to the nucleic acid sequence of Agrobacterium radiobacter B11291 AHL (registration number: U59376) in the National Center for Biotechnology Information (NCBI) genomic database, the primer is introduced: Forward introduction: 5' -tttgt gatatc gctaccaccaccaccggtcaggccaagccaagcttg-3' (SEQ ID NO: 1); reverse primer: 5'-taact tctaga aggagatatacatatgac-3' (SEQ ID NO: 2).
順向引子設計有限制酶EcoRV的剪切位置,反向引子設計有XbaI的剪切位置(如底線所標示者)。以質體pChA203(請參閱Chiang C.J.et al.,J.Agri.Food Chem.,2008,56:6348-6354)為模板,並使用此二引子進行PCR反應,增幅出一編碼AHL的基因片段(約0.96kb)。利用Gel/PCR DNA Fragments Extraction Kit純化增幅得到的基因片段後,使用限制酶EcoRV及XbaI剪切增幅得到的基因片段,再利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的基因片段。另一方面,利用High-Speed Plasmid Mini kit純化質體pHDT(請參閱Chem J.T. et al.,J.Biotechnol.,2005,117:267-275)。使用限制酶EcoRV及XbaI剪切質體pHDT後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pHDT。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pETW-A2HCh,其編碼AHL-HDT融合蛋白質的基因片段則列示於SEQ ID NO:3。 The forward primer is designed with the shear position of the restriction enzyme EcoR V, and the reverse primer is designed with the shear position of Xba I (as indicated by the bottom line). Using plastid pChA203 (see Chiang CJ et al., J. Agri. Food Chem., 2008, 56: 6348-6354) as a template, and using this two primers for PCR reaction, a gene fragment encoding AHL was amplified. 0.96kb). After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by cleavage of the restriction enzymes EcoR V and Xba I was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plastid pHDT was purified using a High-Speed Plasmid Mini kit (see Chem JT et al., J. Biotechnol., 2005, 117: 267-275). After the plastid pHDT was sheared using the restriction enzymes EcoR V and Xba I, the sheared plastid pHDT was recovered using a Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product is sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pETW-A2HCh, which encodes AHL-HDT. A gene fragment of the fusion protein is shown in SEQ ID NO: 3.
如圖2所示,質體pTH-ChA203含有T7啟動子、受啟動子驅動來編碼Agrobacterium radiobacter B11291 AHL及Bacillus circulans WL-12幾丁質結合區域(chitin binding domain,ChBD)(AHL-ChBD)融合蛋白質的基因片段、大腸桿菌的pSC101複製起始點、抗康那黴素基因。其構築流程如下,首先依據美國國家生物科技資訊中心基因體資料庫中pTH18Kr的核酸序列(登記號:AB019603)來設計引子:順向引子:5’-taaccagatctgattagaaaaactcatcg-3’(SEQ ID NO:5);反向引子:5’-agaacctgcagtcagatccttccgtatttagc-3’(SEQ ID NO:6)。 As shown in Figure 2, plastid pTH-ChA203 contains a T7 promoter, which is driven by a promoter to encode Agrobacterium radiobacter B11291 AHL and Bacillus circulans WL-12 chitin binding domain (ChBD) (AHL-ChBD) fusion. A gene fragment of the protein, a pSC101 origin of replication of E. coli, and an anti-connamycin gene. The construction process is as follows. First, the primer is designed according to the nucleic acid sequence of pTH18Kr in the genomic database of the National Center for Biotechnology Information (registration number: AB019603): forward primer: 5'-taacc agatct gattagaaaaactcatcg-3' (SEQ ID NO: 5); reverse primer: 5'-agaac ctgcag tcagatccttccgtatttagc-3' (SEQ ID NO: 6).
順向引子設計有限制酶BglII的剪切位置,反向引子設計有PstI的剪切位置(如底線所標示者)。以質體pTH18Kr為模板,並使用此二引子進行PCR反應,增幅出一有pSC101複製起始點及抗康那黴素基因的基因片段(約2.5kb)。利用Gel/PCR DNA Fragments Extraction Kit純化增幅得到的基 因片段後,使用限制酶BglII及PstI剪切增幅得到的基因片段,再利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的基因片段。另一方面,利用High-Speed Plasmid Mini kit純化質體pChA203。使用限制酶BglII及PstI剪切質體pChA203後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pChA203。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pTH-ChA203,其編碼AHL-ChBD融合蛋白質的基因片段則列示於SEQ ID NO:7。 The forward primer is designed with the restriction position of the restriction enzyme Bgl II, and the reverse primer is designed with the shear position of Pst I (as indicated by the bottom line). The plastid pTH18Kr was used as a template, and the PCR reaction was carried out using the two primers, and a gene fragment (about 2.5 kb) having a pSC101 origin of replication and an anti-connamycin gene was amplified. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by cleavage of the restriction enzymes Bgl II and Pst I was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pChA203 was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pChA203 using the restriction enzymes Bgl II and Pst I, the spliced plastid pChA203 was recovered using a Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product is sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTH-ChA203, which encodes AHL-ChBD. A gene fragment of the fusion protein is shown in SEQ ID NO: 7.
如圖3所示,質體pTH-AL203Coh含有T7啟動子、受啟動子驅動來編碼Agrobacterium radiobacter B11291 AHL及Clostridium thermocellum黏附區域CohI(AHL-CohI)融合蛋白質的基因片段、大腸桿菌的pSC101複製起始點、抗康那黴素基因。其構築流程如下,首先依據美國國家生物科技資訊中心基因體資料庫中Clostridium thermocellum cipA基因的核酸序列(登記號:X67406)來設計引子:順向引子:5’-atcatctcgagttatgcggccgcaagctttggtg-3’(SEQ ID NO:9);反向引子:5’-attagaattcagatctcagccaaatgttcc-3’(SEQ ID NO:10)。 As shown in Figure 3, the plastid pTH-AL203Coh contains a T7 promoter, a gene fragment driven by a promoter to encode the Agrobacterium radiobacter B11291 AHL and Clostridium thermocellum adhesion region CohI (AHL-CohI) fusion protein, and the pSC101 replication initiation of Escherichia coli Point, anti-connamycin gene. The construction process is as follows. First, according to the nucleic acid sequence of Clostridium thermocellum cipA gene in the genomic database of the National Center for Biotechnology Information (registration number: X67406), the primer is introduced: forward primer: 5'-atcat ctcgag ttatgcggccgcaagctttggtg-3' (SEQ ID NO: 9); reverse primer: 5'-atta gaattc agatctcagccaaatgttcc-3' (SEQ ID NO: 10).
順向引子設計有限制酶XhoI的剪切位置,反向引子設計有EcoRI的剪切位置(如底線所標示者)。利用質體Clostridium thermocellum DSM1237(取自Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH)染色體為模板,並使用此二引子進行PCR反應,增幅出一編碼黏附區域CohI的基因片段(約0.5kb)。利用Gel/PCR DNA Fragments Extraction Kit純化增 幅得到的基因片段後,使用限制酶XhoI及EcoRI剪切增幅得到的基因片段,再利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的基因片段。另一方面,利用High-Speed Plasmid Mini kit純化質體pTH-ChA203。使用限制酶XhoI及EcoRI剪切質體pTH-ChA203後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pTH-ChA203。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pTH-AL203Coh,其編碼AHL-CohI融合蛋白質的基因片段則列示於SEQ ID NO:11。 The forward primer is designed with the shear position of the restriction enzyme Xho I, and the reverse primer is designed with the shear position of EcoR I (as indicated by the bottom line). The chromosome of the plastid Clostridium thermocellum DSM1237 (taken from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) was used as a template, and the PCR reaction was carried out using the two primers to increase a gene fragment (about 0.5 kb) encoding the adhesion region CohI. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the amplified gene fragment was amplified by restriction enzymes Xho I and EcoR I, and the fragmented gene fragment was recovered using a Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plastid pTH-ChA203 was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pTH-ChA203 using the restriction enzymes Xho I and EcoR I, the spliced plastid pTH-ChA203 was recovered using a Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTH-AL203Coh, which encodes AHL-CohI. A gene fragment of the fusion protein is shown in SEQ ID NO:11.
如圖4所示,質體pTac-HDTDoCh含有Tac啟動子、受啟動子驅動來編碼硫還原蛋白質(thioredoxin,TrxA)、Agrobacterium radiobacter B11291 HDT、Clostridium thermocellum錨定區域Doc1及Bacillus circulans WL-12 ChBD(TrxA-HDT-Doc1-ChBD)融合蛋白質的基因片段、大腸桿菌的ColE1複製起始點、抗胺苄青黴素基因。其構築流程如下,首先依據質體pET32a(Novagen Co.)的核酸序列來設計引子:順向引子:5’-ggtgaataattttatcgctcatctgtatatctccttctagaggg-3’(SEQ ID NO:13);反向引子:5’-ccctctagaaggagatatacagatgagcgataaaattattcacc-3’(SEQ ID NO:14)。 As shown in Figure 4, the plastid pTac-HDTDoCh contains a Tac promoter, which is driven by a promoter to encode a thioredoxin (TrxA), an Agrobacterium radiobacter B11291 HDT, a Clostridium thermocellum anchor region Doc1, and a Bacillus circulans WL-12 ChBD ( TrxA-HDT-Doc1-ChBD) gene fragment of fusion protein, ColE1 origin of replication of Escherichia coli, and ampicillin resistance gene. The construction process is as follows. First, the primer is designed according to the nucleic acid sequence of plastid pET32a (Novagen Co.): forward primer: 5'-ggtgaataattttatcgctcat c tgtatatctccttctagaggg-3' (SEQ ID NO: 13); reverse primer: 5'- Ccctctagaaggagatatacagatgagcgataaaattattcacc-3' (SEQ ID NO: 14).
此二引子均設計有突變限制酶NdeI的剪切位置(如底線所標示者)。以質體pET32a為模板,並使用QuickChange Site-Directed Mutagenesis Kit(Strategene Co.)及此二引子進行PCR反應,增幅出一有T7啟動子下游第 1個NdeI剪切位置突變之質體pET32a的基因片段。利用Gel/PCR DNA Fragments Extraction Kit純化增幅得到的基因片段後,以上述「化學轉形法」將純化後的DNA產物送至大腸桿菌DH5α中,而得到一質體pET32-N。接著,使用限制酶XhoI及NdeI剪切質體pET32-N後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pET32-N。另一方面,利用High-Speed Plasmid Mini kit純化質體pChHDT(請參閱Chem J.T. et al.,J.Biotechnol.,2005,117:267-275)。使用限制酶XhoI及NdeI剪切質體pChHDT後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pChHDT。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pET-TrHDTCh。 Both primers were designed with the cleavage position of the mutant restriction enzyme Nde I (as indicated by the bottom line). The plastid pET32a was used as a template, and the PCR reaction was carried out using the QuickChange Site-Directed Mutagenesis Kit (Strategene Co.) and the two primers to increase the plastid pET32a with the first Nde I cleavage position mutation downstream of the T7 promoter. Gene fragment. After the amplified gene fragment was purified by Gel/PCR DNA Fragments Extraction Kit, the purified DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain a plastid pET32-N. Next, after cleavage of the plastid pET32-N using the restriction enzymes Xho I and Nde I, the spliced plastid pET32-N was recovered using a Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plastid pChHDT was purified using a High-Speed Plasmid Mini kit (see Chem JT et al., J. Biotechnol., 2005, 117: 267-275). After cleavage of the plastid pChHDT using the restriction enzymes Xho I and Nde I, the spliced plastid pChHDT was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments with T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pET-TrHDTCh.
其次,依據美國國家生物科技資訊中心基因體資料庫中Clostridium thermocellum cipA基因的核酸序列(登記號:X67406)來設計引子:順向引子:5’-tgtgactcgagttagagctcgttcttgtacggcaatgtatc-3’(SEQ ID NO:15);反向引子:5’-gcatgaagcttaaagtacctggtactccttc-3’(SEQ ID NO:16)。 Secondly, according to the nucleic acid sequence of Clostridium thermocellum cipA gene in the genomic database of the National Center for Biotechnology Information (registration number: X67406), the primer was introduced: forward primer: 5'-tgtga ctcgag ttagagctcgttcttgtacggcaatgtatc-3' (SEQ ID NO: 15) Reverse primer: 5'-gcatg aagctt aaagtacctggtactccttc-3' (SEQ ID NO: 16).
順向引子設計有限制酶XhoI的剪切位置,反向引子設計有HindIII的剪切位置(如底線所標示者)。以質體Clostridium thermocellum DSM1237染色體為模板,並使用此二引子進行PCR反應,增幅出一編碼錨定區域Doc1的基因片段(約0.23kb)。利用Gel/PCR DNA Fragments Extraction Kit純化增幅得到的基因片段後,使用限制酶XhoI及HindIII剪切增幅得到的基因片段,再利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的基因 片段。另一方面,利用High-Speed Plasmid Mini kit純化質體pET-TrHDTCh。使用限制酶XhoI及HindIII剪切質體pET-TrHDTCh後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pET-TrHDTCh。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pET-TrHDTDo。 The forward primer is designed with the shear position of the restriction enzyme Xho I, and the reverse primer is designed with the shear position of Hind III (as indicated by the bottom line). Using the chromosome Clostridium thermocellum DSM1237 as a template, and using the two primers for PCR reaction, a gene fragment encoding the anchor region Doc1 (about 0.23 kb) was amplified. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by restriction amplification with restriction enzymes Xho I and Hind III was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pET-TrHDTCh was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pET-TrHDTCh using the restriction enzymes Xho I and Hind III, the spliced plastid pET-TrHDTCh was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments with T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pET-TrHDTDo.
然後,依據質體pET-TrHDTCh的核酸序列來設計引子:順向引子:5’-agatgcttttctgtgactgg-3’(SEQ ID NO:17);反向引子:5’-agcatgagctcggcctgaccggtctgaac-3’(SEQ ID NO:18)。 Then, the primer was designed according to the nucleic acid sequence of the plastid pET-TrHDTCh: forward primer: 5'-agatgcttttctgtgactgg-3' (SEQ ID NO: 17); reverse primer: 5'-agcat gagctc ggcctgaccggtctgaac-3' (SEQ ID NO: 18).
反向引子設計有SacI的剪切位置(如底線所標示者)。以質體pET-TrHDTCh為模板,並使用此二引子進行PCR反應,增幅出一編碼ChBD的基因片段(約1.3kb)。利用Gel/PCR DNA Fragments Extraction Kit純化增幅得到的基因片段後,使用限制酶SacI剪切增幅得到的基因片段,再利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的基因片段。另一方面,利用High-Speed Plasmid Mini kit純化質體pET-TrHDTDo。使用限制酶SacI剪切質體pET-TrHDTDo後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pET-TrHDTDo。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pET-HDTDoCh。 The reverse primer is designed with the Sic I shear position (as indicated by the bottom line). Using the plastid pET-TrHDTCh as a template, and using the two primers for PCR reaction, a gene fragment encoding ChBD (about 1.3 kb) was amplified. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the amplified gene fragment was cleaved using restriction enzyme Sac I, and the fragmented gene fragment was recovered using a Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pET-TrHDTDo was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pET-TrHDTDo using the restriction enzyme Sac I, the spliced plastid pET-TrHDTDo was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments with T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pET-HDTDoCh.
接著,依據質體pJF-Trxfus(請參閱Chao Y.P.,Appl.Microbiol.Biotechnol.,2000,54:348-353)的核酸序列來設計引子:順向引子:5’-gaatttctagacctgtgtgaaattgttatcc-3’(SEQ ID NO:19);反向引子:5’-gagtgagatatttatgccag-3’(SEQ ID NO:20)。 Next, the primer was designed according to the nucleic acid sequence of the plastid pJF-Trxfus (see Chao YP, Appl. Microbiol. Biotechnol., 2000, 54: 348-353): forward primer: 5'-gaatt tctaga cctgtgtgaaattgttatcc-3' ( SEQ ID NO: 19); reverse primer: 5'-gagtgagatatttatgccag-3' (SEQ ID NO: 20).
順向引子設計有XbaI的剪切位置(如底線所標示者)。以質體pJF-Trxfus為模板,並使用此二引子進行PCR反應,增幅出一有Tac啟動子的基因片段(約1kb)。利用Gel/PCR DNA Fragments Extraction Kit純化增幅得到的基因片段後,使用限制酶XbaI及ApaI剪切增幅得到的基因片段,再利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的基因片段。另一方面,利用High-Speed Plasmid Mini kit純化質體pET32-N。使用限制酶XbaI及ApaI剪切質體pET32-N後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pET32-N。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pTac-N。 The forward guide is designed with the Xba I cut position (as indicated by the bottom line). The plastid pJF-Trxfus was used as a template, and the PCR reaction was carried out using the two primers to increase a gene fragment (about 1 kb) having a Tac promoter. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by cleavage of the restriction enzymes Xba I and Apa I was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pET32-N was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pET32-N using the restriction enzymes Xba I and Apa I, the spliced plastid pET32-N was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTac-N.
最後,使用限制酶XbaI及PstI剪切質體pTac-N後,利用Gel/PCR DNA Fragments Extraction Kit回收經剪切的質體pTac-N。另一部分,使用限制酶XbaI及PstI剪切質體pET-HDTDoCh,以去除質體pET-HDTDoCh的T7啟動子後,利用Gel/PCR DNA Fragments Extraction Kit回收剪切剩餘的基因片段。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pTac-HDTDoCh,其編碼TrxA-HDT-Doc1-ChBD融合蛋白質的基因片段則列示於SEQ ID NO:21。 Finally, after cleavage of the plastid pTac-N using the restriction enzymes Xba I and Pst I, the spliced plastid pTac-N was recovered using the Gel/PCR DNA Fragments Extraction Kit. In another part, the restriction enzyme Xba I and Pst I were used to cleave the plastid pET-HDTDoCh to remove the T7 promoter of the plastid pET-HDTDoCh, and the remaining gene fragments were recovered by the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain a plastid pTac-HDTDoCh encoding TrxA-HDT. A gene fragment of the -Doc1-ChBD fusion protein is shown in SEQ ID NO:21.
如圖5所示,質體pTac-TrChHDT含有Tac啟動子、受啟動子驅動來編碼TrxA、Agrobacterium radiobacter B11291 HDT、Bacillus circulans WL-12 ChBD(TrxA-HDT-ChBD)融合蛋白質的基因片段、大腸桿菌的ColE1複 製起始點、抗胺苄青黴素基因。其構築流程如下,使用限制酶XbaI及PstI剪切質體pTac-HDTDoCh,以保留質體pTac-HDTDoCh的Tac啟動子後,利用Gel/PCR DNA Fragments Extraction Kit回收剪切剩餘的基因片段。另一方面,使用限制酶XbaI及PstI剪切質體pET-TrHDTCh,以去除質體pET-TrHDTCh的T7啟動子後,利用Gel/PCR DNA Fragments Extraction Kit回收剪切剩餘的基因片段。利用T4接合酶黏接上述二回收後的基因片段後,以上述「化學轉形法」將黏接後的DNA產物送至大腸桿菌DH5α中,而得到質體pTac-TrChHDT,其編碼TrxA-HDT-ChBD融合蛋白質的基因片段則列示於SEQ ID NO:23。 As shown in Fig. 5, the plastid pTac-TrChHDT contains a Tac promoter, a gene fragment driven by a promoter to encode a TrxA, Agrobacterium radiobacter B11291 HDT, Bacillus circulans WL-12 ChBD (TrxA-HDT-ChBD) fusion protein, and Escherichia coli. The ColE1 replication initiation point, the ampicillin resistance gene. The construction procedure was as follows. The restriction enzymes Xba I and Pst I were used to cleave the plastid pTac-HDTDoCh to retain the Tac promoter of the plastid pTac-HDTDoCh, and the remaining gene fragments were recovered by the Gel/PCR DNA Fragments Extraction Kit. On the other hand, after the plastid pET-TrHDTCh was cleaved with the restriction enzymes Xba I and Pst I to remove the T7 promoter of the plastid pET-TrHDTCh, the remaining gene fragments were recovered by the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTac-TrChHDT, which encodes TrxA-HDT. A gene fragment of the -ChBD fusion protein is shown in SEQ ID NO:23.
以上述「化學轉形法」,將質體pETW-A2HCh送至大腸桿菌BL21(DE3)得到重組菌株BL21(DE3)/pETW-A2HCh。從LB固態培養基選取重組菌株的單一菌落至含有50μg/mL胺苄青黴素的LB培養液(5mL)後,在30℃及150rpm的恆溫震盪箱中培養16小時,再接種至新鮮含有50μg/mL胺苄青黴素的LB培養液(25mL),使得其起始濃度達OD550=0.08。接著,重組菌株菌液繼續在30℃及150rpm的恆溫震盪箱中培養,直到其濃度達OD550=0.3,再加入50μM IPTG(Isopropyl β-D-1-thiogalactopyranoside)誘導重組菌株生產蛋白質。當繼續培養重組菌株4小時後,收集重組菌株並以0.01M磷酸鈉緩衝液(pH7.5)清洗細菌。最後,將細菌懸浮在1mL的磷酸鈉緩衝液中,使得細菌濃度達OD550=10,再利用超音波打破細菌,並使用非原態硫酸十二酯鈉-聚丙烯醯胺電泳分析生產的蛋白質。如圖6所示,IPTG誘導後,重組菌株BL21(DE3)/pETW-A2HCh可大量生產AHL-HDT融合蛋白質, 惟其形成不可溶的包涵體(圖6的徑5)。 The plastid pETW-A2HCh was sent to Escherichia coli BL21 (DE3) by the above "chemical transformation method" to obtain a recombinant strain BL21(DE3)/pETW-A2HCh. A single colony of the recombinant strain was selected from LB solid medium to LB medium (5 mL) containing 50 μg/mL ampicillin, and cultured in a constant temperature shaking box at 30 ° C and 150 rpm for 16 hours, and then inoculated to freshly containing 50 μg / mL of amine. LB broth (25 mL) of benzylpenicillin was brought to a starting concentration of OD 550 = 0.08. Next, the recombinant strain was continuously cultured in a constant temperature shaking box at 30 ° C and 150 rpm until its concentration reached OD 550 = 0.3, and 50 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to induce recombinant strain to produce protein. After continuing to culture the recombinant strain for 4 hours, the recombinant strain was collected and the bacteria were washed with 0.01 M sodium phosphate buffer (pH 7.5). Finally, the bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD 550 = 10, and the bacteria were broken by ultrasonic waves, and the produced protein was analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide. . As shown in Fig. 6, after IPTG induction, the recombinant strain BL21(DE3)/pETW-A2HCh produced a large amount of the AHL-HDT fusion protein, but it formed an insoluble inclusion body (diameter 5 of Fig. 6).
以上述「化學轉形法」,將質體pTH-ChA203及pTac-TrChHDT同時送至大腸桿菌BAD-5(請參閱Mang Z.W.et al.,J.Agri.Food Chem.,2011,59:6534-6542)得到重組菌株BAD-5/pTH-ChA203/pTac-TrChHDT。從LB固態培養基選取重組菌株的單一菌落至含有50μg/mL胺苄青黴素及50μg/mL康那黴素的LB培養液(5mL)後,在30℃及150rpm的恆溫震盪箱中培養16小時,再接種至新鮮含有50μg/mL胺苄青黴素及50μg/mL康那黴素的LB培養液(25mL),使得其起始濃度達OD550=0.08。接著,重組菌株菌液繼續在30℃及150rpm的恆溫震盪箱中培養,直到其濃度達OD550=0.3,再加入30μM L型***糖誘導重組菌株生產蛋白質。當繼續培養重組菌株4小時後,收集重組菌株並以0.01M磷酸鈉緩衝液(pH7.5)清洗細菌。最後,將細菌懸浮在1mL的磷酸鈉緩衝液中,使得細菌濃度達OD550=10,再利用超音波打破細菌,並使用非原態硫酸十二酯鈉-聚丙烯醯胺電泳分析生產的蛋白質。如圖7所示,***糖誘導後,重組菌株BAD-5/pTH-ChA203/pTac-TrChHDT可大量生產可溶的AHL-ChBD融合蛋白質、TrxA-HDT-ChBD融合蛋白質(圖7的徑2)。 The plastids pTH-ChA203 and pTac-TrChHDT were simultaneously sent to E. coli BAD-5 by the above-mentioned "chemical transformation method" (see Mang ZW et al., J. Agri. Food Chem., 2011, 59:6534-6542). The recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT was obtained. A single colony of the recombinant strain was selected from LB solid medium to LB medium (5 mL) containing 50 μg/mL ampicillin and 50 μg/mL connamycin, and then cultured in a constant temperature shaking box at 30 ° C and 150 rpm for 16 hours. LB medium (25 mL) freshly containing 50 μg/mL ampicillin and 50 μg/mL connamycin was inoculated to a starting concentration of OD 550 = 0.08. Next, the recombinant strain was continuously cultured in a constant temperature shaking box at 30 ° C and 150 rpm until its concentration reached OD 550 = 0.3, and 30 μM L-arabinose was added to induce recombinant strain to produce protein. After continuing to culture the recombinant strain for 4 hours, the recombinant strain was collected and the bacteria were washed with 0.01 M sodium phosphate buffer (pH 7.5). Finally, the bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD 550 = 10, and the bacteria were broken by ultrasonic waves, and the produced protein was analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide. . As shown in Fig. 7, after arabinose induction, the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT can produce a large amount of soluble AHL-ChBD fusion protein and TrxA-HDT-ChBD fusion protein (diameter 2 of Fig. 7). .
以上述「化學轉形法」,將質體pTH-AL203Coh及pTac-HDTDoCh同時送至大腸桿菌BAD-5得到重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh。 從LB固態培養基選取重組菌株的單一菌落至含有50μg/mL胺苄青黴素及50μg/mL康那黴素的LB培養液(5mL)後,在30℃及150rpm的恆溫震盪箱中培養16小時,再接種至新鮮含有50μg/mL胺苄青黴素及50μg/mL康那黴素的LB培養液(25mL),使得其起始濃度達OD550=0.08。接著,重組菌株菌液繼續在30℃及150rpm的恆溫震盪箱中培養,直到其濃度達OD550=0.3,再加入30μM L型***糖誘導重組菌株生產蛋白質。當繼續培養重組菌株4小時後,收集重組菌株並以0.01M磷酸鈉緩衝液(pH7.5)清洗細菌。最後,將細菌懸浮在1mL的磷酸鈉緩衝液中,使得細菌濃度達OD550=10,再利用超音波打破細菌,並使用非原態硫酸十二酯鈉-聚丙烯醯胺電泳分析生產的蛋白質。如圖7所示,***糖誘導後,重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh可大量生產可溶的AHL-ChoI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質(圖7的徑3)。 The plastids pTH-AL203Coh and pTac-HDTDoCh were simultaneously sent to E. coli BAD-5 by the above-mentioned "chemical transformation method" to obtain a recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh. A single colony of the recombinant strain was selected from LB solid medium to LB medium (5 mL) containing 50 μg/mL ampicillin and 50 μg/mL connamycin, and then cultured in a constant temperature shaking box at 30 ° C and 150 rpm for 16 hours. LB medium (25 mL) freshly containing 50 μg/mL ampicillin and 50 μg/mL connamycin was inoculated to a starting concentration of OD 550 = 0.08. Next, the recombinant strain was continuously cultured in a constant temperature shaking box at 30 ° C and 150 rpm until its concentration reached OD 550 = 0.3, and 30 μM L-arabinose was added to induce recombinant strain to produce protein. After continuing to culture the recombinant strain for 4 hours, the recombinant strain was collected and the bacteria were washed with 0.01 M sodium phosphate buffer (pH 7.5). Finally, the bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD 550 = 10, and the bacteria were broken by ultrasonic waves, and the produced protein was analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide. . As shown in Fig. 7, after arabinose induction, the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh can produce a large amount of soluble AHL-ChoI fusion protein and TrxA-HDT-Doc1-ChBD fusion protein (Fig. 7). 3).
以上述「化學轉形法」,將質體pTH-AL203Coh及pTac-TrChHDT同時送至大腸桿菌BAD-5得到重組菌株BAD-5/pTH-AL203Coh/pTac-TrChHDT。依實施例7或8的方法,培養重組菌株生產蛋白質。 The plastids pTH-AL203Coh and pTac-TrChHDT were simultaneously sent to E. coli BAD-5 by the above-mentioned "chemical transformation method" to obtain a recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT. The recombinant strain was cultured to produce a protein according to the method of Example 7 or 8.
過去咸知Bacillus circulans WL-12幾丁質分解酵素(chitinase A1)的ChBD與幾丁質的結合非常專一且穩定,並可在廣泛的pH範圍中進行結合(請參閱Hashimoto et al.,J.Bacteriol.,2000,182:3045-3054;及Chern J.T.et al.,J.Biotechnol.,2005,117:267-275)。本實施例為利用幾丁質球吸附融合蛋白質,其操作過程如下所述:將離心下來的細菌,以0.01M磷酸鈉緩衝液(pH7.5)清洗細菌二次。將細菌懸浮在1mL的磷酸鈉緩衝液中,使得細菌 濃度達OD550=10,再利用超音波打破細菌,離心並回收上清液,而得到細胞萃取液。另一部分,取0.5g的幾丁質球(New England Biolabs)至1.5mL的離心管內。以1mL的0.01M磷酸鈉緩衝液(pH7.5)清洗幾丁質球後,靜待幾丁質球沉降於離心管底部並移去上清液,重複上述步驟3次。接著,將含有50μg之蛋白質的細胞萃取液加入清洗後的幾丁質球中,於4℃下進行吸附反應。反應12小時後,先靜待幾丁質球沉降於離心管底部,再移除上清液並同時以1mL的0.01M磷酸鈉緩衝液(pH7.5)清洗2次,以洗去未吸附於幾丁質球的蛋白質。在吸附於幾丁質球的蛋白質中加入2%SDS溶液,並於100℃加熱5至10分鐘,使吸附的蛋白質與幾丁質球分離後,使用非原態硫酸十二酯鈉-聚丙烯醯胺電泳分析吸附的蛋白質。 In the past, the combination of ChBD and chitin in Bacillus circulans WL-12 chitinase A1 was very specific and stable, and could be combined in a wide pH range (see Hashimoto et al., J. Bacteriol., 2000, 182: 3045-3054; and Chern JT et al., J. Biotechnol., 2005, 117: 267-275). In this example, the fusion protein was adsorbed by chitin beads, and the operation procedure was as follows: the bacteria were centrifuged, and the bacteria were washed twice with 0.01 M sodium phosphate buffer (pH 7.5). The bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD 550 = 10, and the bacteria were broken by ultrasonic waves, centrifuged, and the supernatant was recovered to obtain a cell extract. For the other part, 0.5 g of chitin spheres (New England Biolabs) were taken into a 1.5 mL centrifuge tube. After washing the chitin balls in 1 mL of 0.01 M sodium phosphate buffer (pH 7.5), the chitin balls were allowed to settle at the bottom of the centrifuge tube and the supernatant was removed, and the above procedure was repeated three times. Next, the cell extract containing 50 μg of the protein was added to the washed chitin spheres, and the adsorption reaction was carried out at 4 °C. After reacting for 12 hours, wait for the chitin ball to settle at the bottom of the centrifuge tube, then remove the supernatant and wash it twice with 1 mL of 0.01 M sodium phosphate buffer (pH 7.5) to wash away without adsorption. The protein of chitin balls. Adding a 2% SDS solution to the protein adsorbed to the chitin spheres and heating at 100 ° C for 5 to 10 minutes to separate the adsorbed protein from the chitin spheres, using the non-organic sodium dodecyl sulfate-polypropylene The adsorbed protein was analyzed by guanamine electrophoresis.
如圖8(A)所示,於吸附反應後,重組菌株BAD-5/pTH-AL203Coh/pTac-TrChHDT生產的TrxA-HDT-ChBD融合蛋白質殘留在細胞萃取液中的量明顯變少(徑2、3)。表示說TrxA-HDT-ChBD融合蛋白質因有ChBD而可被幾丁質球吸附。反過來說,重組菌株BAD-5/pTH-AL203Coh/pTac-TrChHDT生產的AHL-CohI融合蛋白質,於吸附反應前後,其殘留在細胞萃取液中的量並無明顯改變(徑2、3)。表示說AHL-CohI融合蛋白質因未有ChBD,故無法被幾丁質球吸附。另外,加熱使吸附的蛋白質與幾丁質球分離後,僅得到TrxA-HDT-ChBD融合蛋白質(徑6),表示說TrxA-HDT-ChBD融合蛋白質與AHL-CohI融合蛋白質之間並無任何作用力,因此只有TrxA-HDT-ChBD融合蛋白質可被幾丁質球吸附。 As shown in Fig. 8(A), after the adsorption reaction, the amount of TrxA-HDT-ChBD fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT was significantly reduced in the cell extract (path 2). 3). It is said that the TrxA-HDT-ChBD fusion protein can be adsorbed by chitin spheres due to ChBD. Conversely, the amount of AHL-CohI fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT did not change significantly in the cell extract before and after the adsorption reaction (paths 2 and 3). It is said that the AHL-CohI fusion protein cannot be adsorbed by chitin balls because it has no ChBD. In addition, after heating to separate the adsorbed protein from the chitin spheres, only the TrxA-HDT-ChBD fusion protein (path 6) was obtained, indicating that there was no effect between the TrxA-HDT-ChBD fusion protein and the AHL-CohI fusion protein. Therefore, only the TrxA-HDT-ChBD fusion protein can be adsorbed by chitin spheres.
如圖8(B)所示,於吸附反應後,重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh生產的AHL-CohI融合蛋白質、 TrxA-HDT-Doc1-ChBD融合蛋白質殘留在細胞萃取液中的量均明顯變少(徑2、3)。這代表著AHL-CohI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質均可被幾丁質球吸附。然而,值得注意的是,AHL-CohI融合蛋白質未含有ChBD,但卻可被幾丁質球吸附。加熱使吸附的蛋白質與幾丁質球分離後,得到AHL-CohI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質(徑6),表示說AHL-CohI融合蛋白質含有的黏附區域CohI以及TrxA-HDT-Doc1-ChBD融合蛋白質含有的錨定區域Doc1之間存在著結合力,因此透過TrxA-HDT-Doc1-ChBD融合蛋白質含有的ChBD被幾丁質球吸附,而間接地使AHL-CohI融合蛋白質亦被幾丁質球吸附。 As shown in Fig. 8(B), after the adsorption reaction, the AHL-CohI fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh, The amount of TrxA-HDT-Doc1-ChBD fusion protein residues in the cell extract was significantly reduced (both 2, 3). This means that the AHL-CohI fusion protein and the TrxA-HDT-Doc1-ChBD fusion protein can be adsorbed by chitin spheres. However, it is worth noting that the AHL-CohI fusion protein does not contain ChBD but is adsorbed by chitin spheres. After heating to separate the adsorbed protein from the chitin sphere, the AHL-CohI fusion protein and the TrxA-HDT-Doc1-ChBD fusion protein (path 6) were obtained, indicating that the adhesion region CohI and TrxA-HDT contained in the AHL-CohI fusion protein were expressed. - The adhesion between the anchor region Doc1 contained in the -Doc1-ChBD fusion protein is such that the ChBD contained in the TrxA-HDT-Doc1-ChBD fusion protein is adsorbed by the chitin sphere, and the AHL-CohI fusion protein is indirectly Adsorbed by chitin balls.
於實施例7的重組菌株BAD-5/pTH-ChA203/pTac-TrChHDT及實施例8的重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh培養完成後,先以4℃離心收集菌體。於使用去離子水將細菌濃度調整至OD550=10時,利用超音波打破細菌,再以4℃及12,000rpm離心並取上清液,而得到細胞萃取液。 After the culture of the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT of Example 7 and the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh of Example 8 was completed, the cells were collected by centrifugation at 4 °C. When the bacterial concentration was adjusted to OD 550 = 10 using deionized water, the bacteria were disrupted by ultrasonic waves, centrifuged at 4 ° C and 12,000 rpm, and the supernatant was taken to obtain a cell extract.
以上述「酵素活性」的檢測,分別測量AHL及HDT的酵素活性。首先,加入含有5μg之蛋白質的細胞萃取液至1mL的反應液中反應,於反應後,使用高效率液相層析儀分析溶液中的反應物濃度。另一部分,使用非原態硫酸十二酯鈉-聚丙烯醯胺電泳分析細胞萃取液中的個別蛋白質,並搭配影像分析儀判定個別蛋白質的AHL及HDT酵素活性。再一部分,取含有15μg之蛋白質的細胞萃取液至1mL之含有20mM對羥基苯海因(DL-hydroxylphenyl hydantoin,DL-HPH)及0.5mM磷酸鈉緩衝液(pH7.5)的反應液中,並於40℃下使細胞萃取液中的AHL及HDT進行酵素串聯的全 反應,而生成D型對羥苯基甘胺酸(D-HPG)。使用高效率液相層析儀分析溶液中生成的D-HPG濃度(mM),並將濃度除以反應時間,而獲得全反應速率。 The enzyme activities of AHL and HDT were measured by the above-mentioned "enzyme activity" test. First, a cell extract containing 5 μg of protein was added to 1 mL of the reaction solution, and after the reaction, the concentration of the reactant in the solution was analyzed using a high-performance liquid chromatograph. In another part, individual proteins in the cell extracts were analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide, and the AHL and HDT enzyme activities of individual proteins were determined by an image analyzer. In a further portion, a cell extract containing 15 μg of protein was taken into a 1 mL reaction solution containing 20 mM DL-hydroxylphenyl hydantoin (DL-HPH) and 0.5 mM sodium phosphate buffer (pH 7.5), and Enzymatic tandem connection of AHL and HDT in cell extract at 40 °C The reaction produces D-p-hydroxyphenylglycine (D-HPG). The D-HPG concentration (mM) generated in the solution was analyzed using a high-performance liquid chromatograph, and the concentration was divided by the reaction time to obtain a total reaction rate.
如表1所示,實施例7生產的AHL-ChBD融合蛋白質、TrxA-HDT-ChBD融合蛋白質各別表現出優於實施例8生產之AHL-CohI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質的HDT活性及AHL活性。但實施例8生產的AHL-CohI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質反而具有高於實施例7生產之AHL-ChBD融合蛋白質、TrxA-HDT-ChBD融合蛋白質1.6倍的全反應速率。這結果表示說,實施例8生產的AHL-CohI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質會透過黏附區域CohI及錨定區域Doc1,而互相結合來形成酵素複合體,而造成所謂的「代謝物輸送道(metabolite channeling)」現象,有效地提升反應速率。 As shown in Table 1, the AHL-ChBD fusion protein and the TrxA-HDT-ChBD fusion protein produced in Example 7 each exhibited an AHL-CohI fusion protein, TrxA-HDT-Doc1-ChBD fusion protein which was superior to that produced in Example 8. HDT activity and AHL activity. However, the AHL-CohI fusion protein produced in Example 8 and the TrxA-HDT-Doc1-ChBD fusion protein had a total reaction rate 1.6 times higher than that of the AHL-ChBD fusion protein produced in Example 7, and the TrxA-HDT-ChBD fusion protein. This result indicates that the AHL-CohI fusion protein produced in Example 8 and the TrxA-HDT-Doc1-ChBD fusion protein are combined with each other to form an enzyme complex through the adhesion region CohI and the anchor region Doc1, resulting in a so-called " The metabolite channeling phenomenon effectively increases the reaction rate.
本實施例是利用實施例7的重組菌株BAD-5/pTH-ChA203/pTac-TrChHDT及實施例8的重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh進行生物轉化反應,主要係催化D-HPG的生成。 In this embodiment, the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT of Example 7 and the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh of Example 8 were used for biotransformation reaction, mainly catalyzing D- HPG generation.
於實施例7的重組菌株及實施例8的重組菌株培養完成後,先以4℃離心收集菌體,再使用0.01M磷酸鈉緩衝液(pH7.5)清洗菌體2次。將菌體懸浮於1mL的磷酸鈉緩衝液(pH7.5)中後,將菌體加到10mL之含有100mM DL-HPH及0.5mM磷酸鈉緩衝液(pH7.5)的反應液中,使最後的細菌濃度為OD550=30,並於40℃下進行10小時的轉化反應,而生成D-HPG。於反應結束後,於常溫下以12,000離心反應液,並取出上層液進行HPLC分析。另使用0.01M磷酸鈉緩衝液(pH7.5)清洗離心下來的菌體2次後,再次離心清洗過的菌體,並加至10mL的反應液中,而進行新的轉化反應,並多次重複上述步驟。 After the recombinant strain of Example 7 and the recombinant strain of Example 8 were cultured, the cells were collected by centrifugation at 4 ° C, and the cells were washed twice with 0.01 M sodium phosphate buffer (pH 7.5). After the cells were suspended in 1 mL of sodium phosphate buffer (pH 7.5), the cells were added to 10 mL of a reaction solution containing 100 mM DL-HPH and 0.5 mM sodium phosphate buffer (pH 7.5) to finally The bacterial concentration was OD 550 = 30, and a conversion reaction was carried out at 40 ° C for 10 hours to form D-HPG. After completion of the reaction, the reaction solution was centrifuged at 12,000 at normal temperature, and the supernatant liquid was taken out for HPLC analysis. After washing the centrifuged cells twice with 0.01 M sodium phosphate buffer (pH 7.5), the washed cells were again centrifuged and added to 10 mL of the reaction solution to carry out a new transformation reaction, and several times. Repeat the above steps.
如圖9(A)所示,實施例7的重組菌株僅可使用1次來達到100%的D-HPG轉化率;而當其重複地使用到第2次後,D-HPG轉化率則降低至80%左右。如圖9(B)所示,實施例8的重組菌株可重複地使用4次來達到100%的D-HPG轉化率;而當其重複地使用到第5次時,D-HPG轉化率仍維持在80%左右。這結果表示說,重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh生產的AHL-CohI融合蛋白質、TrxA-HDT-Doc1-ChBD融合蛋白質會透過黏附區域CohI及錨定區域Doc1,而在細胞內互相結合來形成酵素複合體,相較於不會互相結合的個別蛋白質(如重組菌株BAD-5/pTH-ChA203/pTac-TrChHDT生產的AHL-ChBD融合蛋白質、TrxA-HDT-ChBD融合蛋白質),酵素複合體顯然更具有較高的穩定性,而提升生物轉化反應效率。 As shown in Fig. 9(A), the recombinant strain of Example 7 can be used only once to achieve 100% D-HPG conversion; and when it is repeatedly used for the second time, D-HPG conversion is lowered. Up to 80%. As shown in Fig. 9(B), the recombinant strain of Example 8 can be used repeatedly for 4 times to achieve 100% D-HPG conversion; and when it is repeatedly used for the 5th time, the D-HPG conversion rate is still Maintain at around 80%. This result indicates that the AHL-CohI fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh, the TrxA-HDT-Doc1-ChBD fusion protein will pass through the adhesion region CohI and the anchor region Doc1, but within the cell. Combining with each other to form an enzyme complex, compared to individual proteins that do not bind to each other (such as the AHL-ChBD fusion protein produced by the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT, TrxA-HDT-ChBD fusion protein), The enzyme complex apparently has a higher stability and enhances the efficiency of the biotransformation reaction.
顯然地,依照上面實施例中的描述,本發明可能有許多的修正與差異。因此需要在其附加的權利要求項之範圍內加以理解,除了上述詳細的描述外,本發明還可以廣泛地在其他的實施例中施行。上述僅為本發明之較佳 實施例,並非用以限定本發明之申請專利範圍;凡其它未脫離本發明所揭示之精神下所完成的等效改變或修飾,均應包含在下述申請專利範圍內。 Obviously, many modifications and differences may be made to the invention in light of the above description. It is therefore to be understood that within the scope of the appended claims, the invention may be The above is only preferred of the present invention. The examples are not intended to limit the scope of the invention, and all equivalent modifications or modifications may be made without departing from the spirit of the invention.
圖1為質體pETW-A2HCh的圖譜,圖中簡寫符號:Ap,抗胺苄青黴素基因;T7 promoter,T7啟動子;AHL,編碼Agrobacterium radiobacter B11291 AHL的基因;HDT,編碼Agrobacterium radiobacter B11291 HDT的基因;ColE1,大腸桿菌的ColE1複製起始點。 Figure 1 is a map of plastid pETW-A2HCh, abbreviated symbol: Ap, anti-ampicillin gene; T7 promoter, T7 promoter; AHL, gene encoding Agrobacterium radiobacter B11291 AHL; HDT, gene encoding Agrobacterium radiobacter B11291 HDT ; ColE1, the starting point of ColE1 replication in E. coli.
圖2為質體pTH-ChA203的圖譜,圖中簡寫符號:Km,抗康那黴素基因;T7 promoter,T7啟動子;AHL,編碼Agrobacterium radiobacter B11291 AHL的基因;ChBD,編碼Agrobacterium radiobacter WL-12 ChBD的基因;pSC101,大腸桿菌的pSC101複製起始點。 Figure 2 is a map of plastid pTH-ChA203, abbreviated symbol: Km, anti-connamycin gene; T7 promoter, T7 promoter; AHL, gene encoding Agrobacterium radiobacter B11291 AHL; ChBD, encoding Agrobacterium radiobacter WL-12 The gene for ChBD; pSC101, the origin of pSC101 replication of E. coli.
圖3為質體pTH-AL203Coh的圖譜,圖中簡寫符號:Km,抗康那黴素基因;T7 promoter,T7啟動子;AHL,編碼Agrobacterium radiobacter B11291 AHL的基因;CohI,編碼Clostridium thermocellum黏附區域的基因;pSC101,大腸桿菌的pSC101複製起始點。 Figure 3 is a map of the plastid pTH-AL203Coh, abbreviated in the figure: Km, anti-connamycin gene; T7 promoter, T7 promoter; AHL, gene encoding Agrobacterium radiobacter B11291 AHL; CohI, encoding Clostridium thermocellum adhesion region Gene; pSC101, the origin of pSC101 replication in E. coli.
圖4為質體pTac-HDTDoCh的圖譜,圖中簡寫符號:Ap,抗胺苄青黴素基因;TrxA,編碼TrxA的基因;HDT,編碼Agrobacterium radiobacter B11291 HDT的基因;Doc1,編碼Clostridium thermocellum錨定區域的基因;ColE1,大腸桿菌的ColE1複製起始點。 Figure 4 is a map of the plastid pTac-HDTDoCh, abbreviated in the figure: Ap, anti-ampicillin gene; TrxA, a gene encoding TrxA; HDT, a gene encoding Agrobacterium radiobacter B11291 HDT; Doc1, encoding the anchor region of Clostridium thermocellum Gene; ColE1, the origin of ColE1 replication in E. coli.
圖5為質體pTac-TrChHDT的圖譜,圖中簡寫符號:Ap,抗胺苄青黴素基因;Tac,Tac啟動子;TrxA,編碼TrxA的基因;HDT,編碼Agrobacterium radiobacter B11291 HDT的基因;ChBD,編碼Bacillus circulans WL-12 ChBD 的基因;ColE1,大腸桿菌的ColE1複製起始點。 Figure 5 is a map of the plastid pTac-TrChHDT, abbreviated in the figure: Ap, anti-ampicillin gene; Tac, Tac promoter; TrxA, gene encoding TrxA; HDT, gene encoding Agrobacterium radiobacter B11291 HDT; ChBD, coding Bacillus circulans WL-12 ChBD gene; ColE1, ColE1 replication origin of E. coli.
圖6為非原態硫酸十二酯鈉-聚丙烯醯胺電泳膠圖,以說明重組菌株BL21(DE3)/pETW-A2HCh生產的蛋白質分布;其中,徑1:蛋白質標準物;徑2:未經誘導之重組菌株生產的可溶性蛋白質;徑3:經誘導之重組菌株生產的可溶性蛋白質;徑4:未經誘導之重組菌株生產的不可溶性蛋白質;徑5:經誘導之重組菌株生產的不可溶性蛋白質;箭頭:AHL-HDT的融合蛋白質。 Figure 6 is a non-original sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the protein distribution of recombinant strain BL21(DE3)/pETW-A2HCh; wherein, diameter 1: protein standard; diameter 2: not Soluble protein produced by the induced recombinant strain; diameter 3: soluble protein produced by the induced recombinant strain; diameter 4: insoluble protein produced by the uninduced recombinant strain; run 5: insoluble by the induced recombinant strain Protein; arrow: fusion protein of AHL-HDT.
圖7為非原態硫酸十二酯鈉-聚丙烯醯胺電泳膠圖,以說明重組菌株BAD-5/pTH-ChA203/pTac-TrChHDTh及BAD-5/pTH-AL203Coh/pTac-HDTDoCh生產的蛋白質分布;其中,徑1:蛋白質標準物;徑2:經誘導之重組菌株BAD-5/pTH-ChA203/pTac-TrChHDTh生產的可溶性蛋白質;徑3:經誘導之重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh生產的可溶性蛋白質;徑4:經誘導之菌株BAD-5生產的可溶性蛋白質(作為對照組);箭頭由上至下依序為:TrxA-HDT-Doc1-ChBD融合蛋白質、TrxA-HDT-ChBD融合蛋白質、AHL-CohI融合蛋白質、AHL-ChBD融合蛋白質。 Figure 7 is a diagram showing the non-original sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the proteins produced by the recombinant strains BAD-5/pTH-ChA203/pTac-TrChHDTh and BAD-5/pTH-AL203Coh/pTac-HDTDoCh. Distribution; wherein, diameter 1: protein standard; diameter 2: soluble protein produced by the induced recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDTh; run 3: induced recombinant strain BAD-5/pTH-AL203Coh Soluble protein produced by /pTac-HDTDoCh; Path 4: Soluble protein produced by induced strain BAD-5 (as a control); arrow from top to bottom in order: TrxA-HDT-Doc1-ChBD fusion protein, TrxA- HDT-ChBD fusion protein, AHL-CohI fusion protein, AHL-ChBD fusion protein.
圖8(A)為非原態硫酸十二酯鈉-聚丙烯醯胺電泳膠圖,以說明重組菌株BAD-5/pTH-AL203Coh/pTac-TrChHDT生產的蛋白質與幾丁質球吸附的情況;其中,徑1:蛋白質標準物;徑2:吸附前的細胞萃取液;徑3:吸附後的細胞萃取液;徑4:經吸附後第1次清洗的清洗液;徑5:經吸附後第2次清洗的清洗液;徑6:加熱分離的吸附蛋白質;箭頭由上至下依序為:TrxA-HDT-ChBD融合蛋白質、AHL-CohI融合蛋白質。 Figure 8 (A) is a non-associated sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the adsorption of proteins and chitin spheres produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT; Among them, diameter 1: protein standard; diameter 2: cell extract before adsorption; diameter 3: cell extract after adsorption; diameter 4: cleaning solution after first adsorption after adsorption; diameter 5: after adsorption Washing solution with 2 washes; Path 6: Heating and separating the adsorbed proteins; arrows from top to bottom in order: TrxA-HDT-ChBD fusion protein, AHL-CohI fusion protein.
圖8(B)為非原態硫酸十二酯鈉-聚丙烯醯胺電泳膠圖,以說明重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh生產的蛋白質與幾丁質球吸附的情況;其中,徑1:蛋白質標準物;徑2:吸附前的細胞萃取液;徑3:吸附後的細胞萃取液;徑4:經吸附後第1次清洗的清洗液;徑5:經吸附後第2次清洗的清洗液;徑6:加熱分離的吸附蛋白質;箭頭由上至下依序為:TrxA-HDT-Doc1-ChBD融合蛋白質、AHL-CohI融合蛋白質。 Figure 8 (B) is a non-primary sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the adsorption of proteins and chitin spheres produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh; Among them, diameter 1: protein standard; diameter 2: cell extract before adsorption; diameter 3: cell extract after adsorption; diameter 4: cleaning solution after first adsorption after adsorption; diameter 5: after adsorption Washing solution with 2 washes; Path 6: Heating and separating the adsorbed proteins; arrows from top to bottom are: TrxA-HDT-Doc1-ChBD fusion protein, AHL-CohI fusion protein.
圖9(A)為重組菌株BAD-5/pTH-ChA203/pTac-TrChHDT之D-HPG轉化率的結果圖;其中,圖中數據取自於3次獨立實驗的平均值及標準差。 Figure 9 (A) is a graph showing the results of D-HPG conversion of the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT; wherein the data in the graph were taken from the mean and standard deviation of 3 independent experiments.
圖9(B)為重組菌株BAD-5/pTH-AL203Coh/pTac-HDTDoCh之D-HPG轉化率的結果圖;其中,圖中數據取自於3次獨立實驗的平均值及標準差。 Figure 9 (B) is a graph showing the results of D-HPG conversion of the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh; wherein the data in the figure were taken from the mean and standard deviation of 3 independent experiments.
<110> 逢甲大學 <110> Feng Chia University
<120> 於細胞內製造融合蛋白質的方法 <120> Method for producing a fusion protein in a cell
<160> 24 <160> 24
<210> 1 <210> 1
<211> 47 <211> 47
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 1 <400> 1
<210> 2 <210> 2
<211> 29 <211> 29
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 2 <400> 2
<210> 3 <210> 3
<211> 2310 <211> 2310
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 編碼AHL-HDT融合蛋白質的基因片段 <223> Gene fragment encoding the AHL-HDT fusion protein
<400> 3 <400> 3
<210> 4 <210> 4
<211> 770 <211> 770
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> AHL-HDT融合蛋白質 <223> AHL-HDT fusion protein
<400> 4 <400> 4
<210> 5 <210> 5
<211> 29 <211> 29
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 5 <400> 5
<210> 6 <210> 6
<211> 32 <211> 32
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 6 <400> 6
<210> 7 <210> 7
<211> 1104 <211> 1104
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 編碼AHL-ChBD融合蛋白質的基因片段 <223> Gene fragment encoding the AHL-ChBD fusion protein
<400> 7 <400> 7
<210> 8 <210> 8
<211> 368 <211> 368
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> AHL-ChBD融合蛋白質 <223> AHL-ChBD fusion protein
<400> 8 <400> 8
<210> 9 <210> 9
<211> 34 <211> 34
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 9 <400> 9
<210> 10 <210> 10
<211> 30 <211> 30
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 10 <400> 10
<210> 11 <210> 11
<211> 1386 <211> 1386
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 編碼AHL-CohI融合蛋白質的基因片段 <223> Gene fragment encoding the AHL-CohI fusion protein
<400> 11 <400> 11
<210> 12 <210> 12
<211> 462 <211> 462
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> AHL-CohI融合蛋白質 <223> AHL-CohI fusion protein
<400> 12 <400> 12
<210> 13 <210> 13
<211> 44 <211> 44
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 13 <400> 13
<210> 14 <210> 14
<211> 44 <211> 44
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 14 <400> 14
<210> 15 <210> 15
<211> 41 <211> 41
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 15 <400> 15
<210> 16 <210> 16
<211> 31 <211> 31
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 16 <400> 16
<210> 17 <210> 17
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 17 <400> 17
<210> 18 <210> 18
<211> 29 <211> 29
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 18 <400> 18
<210> 19 <210> 19
<211> 31 <211> 31
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 順向引子 <223> Forward introduction
<400> 19 <400> 19
<210> 20 <210> 20
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 20 <400> 20
<210> 21 <210> 21
<211> 2301 <211> 2301
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 編碼TrxA-HDT-Doc1-ChBD融合蛋白質的基因片段 <223> Gene fragment encoding the TrxA-HDT-Doc1-ChBD fusion protein
<400> 21 <400> 21
<210> 22 <210> 22
<211> 767 <211> 767
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> TrxA-HDT-Doc1-ChBD融合蛋白質 <223> TrxA-HDT-Doc1-ChBD fusion protein
<400> 22 <400> 22
<210> 23 <210> 23
<211> 2070 <211> 2070
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 編碼TrxA-HDT-ChBD融合蛋白質的基因片段 <223> Gene fragment encoding the TrxA-HDT-ChBD fusion protein
<400> 23 <400> 23
<210> 24 <210> 24
<211> 690 <211> 690
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> TrxA-HDT-ChBD融合蛋白質 <223> TrxA-HDT-ChBD fusion protein
<400> 24 <400> 24
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