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公开(公告)号：KR100658943B1
公开(公告)日：2006-12-19
申请号：KR1020050062664
申请日：2005-07-12
Applicant: 한국과학기술원
Abstract: A method for selecting microorganisms cloned with a target gene by using photochemical reaction is provided to remove undesired microorganism clones using simple photo illumination prior to the desired gene screening, so that the gene cloned with a desired gene fragment or its library is able to be simply screened. The method for selecting microorganisms cloned with a target gene by using photochemical reaction comprises the steps of: preparing a recombinant vector by inserting a target gene or its library and a promoter of the target gene into a recombinant plasmid containing origin, antibiotic makers, restriction enzyme sites and ferrochelatase gene(hemH); transforming a microorganism with the recombinant vector; culturing the transformed microorganism in a medium containing antibiotics with photo illumination; and validating the cloning of microorganism when the transformed microorganism is grown, wherein the transformed microorganism is obtained by inserting the ferrochelatase gene(hemH) into a photosensitive mutant microorganism obtained by deleting the ferrochelatase gene(hemH); the recombinant plasmid is p19hemH; and the microorganism is Escherichia coli.
Abstract translation: 提供了一种通过使用光化学反应来选择与目标基因克隆的微生物的方法，以便在期望的基因筛选之前使用简单的光照照射除去不需要的微生物克隆，从而使用所需的基因片段或其文库克隆的基因能够简单 筛选。 选择使用光化学反应克隆靶基因的微生物的方法包括以下步骤：通过将靶基因或其文库和靶基因的启动子插入含有来源的重组质粒，抗生素制造者，限制酶 位点和铁螯合酶基因（hemH）; 用重组载体转化微生物; 在含光合照射抗生素的培养基中培养转化的微生物; 并验证转化微生物生长时微生物的克隆，其中通过将铁螯合酶基因（hemH）插入到通过缺失铁螯合酶基因（hemH）而获得的光敏突变体微生物中来获得转化微生物; 重组质粒为p19hemH; 而微生物是大肠杆菌。
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公开(公告)号：KR100557338B1
公开(公告)日：2006-03-06
申请号：KR1020030084888
申请日：2003-11-27
Applicant: 한국과학기술원
CPC classification number: B82Y30/00 , G01N33/544
Abstract: 본 발명은 자기조립(self-assembly)물질을 탄소나노튜브 상에 자기조립시키는 것을 특징으로 하는 자기조립물질로 랩핑(wrapping)된 수용성 탄소나노튜브의 제조방법 및 자기조립물질로 랩핑되어 있는 수용성 탄소나노튜브에 관한 것이다.
본 발명에 따른, 자기조립물질로 랩핑된 탄소나노튜브는 수용성 성질을 나타내므로 일반 탄소나노튜브와 비교하여 월등히 우수한 응용성을 가지게 된다. 특히, 상기 자기조립물질로 랩핑된 탄소나노튜브에 표적 바이오물질 혹은 유기화합물과 결합하는 리셉터를 선택적으로 부착하여 바이오센서를 제작하는 것이 가능하다.
자기조립, 탄소나노튜브, 자기조립, 수용성, 랩핑(wrapping), 바이오센서-
3.发明公开
公开(公告)号：KR1020000034279A
公开(公告)日：2000-06-15
申请号：KR1019980051563
申请日：1998-11-28
Applicant: 한국과학기술원
Abstract: PURPOSE: Recombinant plasmids having secretion signal sequence of endoxylanase from Bacillus are provided which express and secret foreign protein in transformed E. coli therewith. CONSTITUTION: A secretion signal sequence of endoxylanase fragment from plasmid, pKJX4, containing endoxylase gene from Bacillus is amplified by PCR. Recombinant plasmid, pJS101, is constructed by cloning PCR fragments into plasmid, pTrc99A, having strong trc promoter. Plasmid pJS101 contains signal sequence of endoxylanase and endoxylanase joined with PstI site. 3' end of endoxylanase has restriction sites of BamHI, XbaI, SalI, PstI and HindIII for the cloning of foreign protein. Plasmid pJS101DeltaP is constructed by removing PstI site of 3' end of endoxylanase gene for the easy introduction of foreign protein. T7 promoter containing vector, pJS301, is constructed by joining of NdeI and BamHI digested fragment from pJS101DeltaP and pET21c. Induction experiment with IPTG shows that the yield of secreted protein is at least 6% of total protein. Alkaline phosphatase is over expressed and constitutes 6.9% of total protein.
Abstract translation: 目的：提供具有来自芽孢杆菌的内切木聚糖酶的分泌信号序列的重组质粒，其在转化的大肠杆菌中表达和分泌外源蛋白。 构成：通过PCR扩增来自质粒pKJX4的含有来自芽孢杆菌的内切酶基因的内切木聚糖酶片段的分泌信号序列。 通过将PCR片段克隆到具有强trc启动子的质粒pTrc99A中构建重组质粒pJS101。 质粒pJS101含有与PstI位点连接的内切木聚糖酶和内切木聚糖酶的信号序列。 3'末端的木聚糖酶具有BamHI，XbaI，SalI，PstI和HindIII的限制性位点，用于克隆外来蛋白。 通过除去3'末端的内切木聚糖酶基因的PstI位点构建质粒pJS101DeltaP，便于引入外源蛋白。 通过从pJS101DeltaP和pET21c连接NdeI和BamHI消化的片段构建含有T7启动子的载体pJS301。 IPTG的诱导实验表明，分泌蛋白的产量至少占总蛋白的6％。 碱性磷酸酶过表达，占总蛋白的6.9％。
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4.发明授权
公开(公告)号：KR100844992B1
公开(公告)日：2008-07-08
申请号：KR1020070000177
申请日：2007-01-02
Applicant: 한국과학기술원
Abstract: 본 발명은 인체 유래 상피세포성장인자(human epidermal growth factor: hEGF)를 세포 배양액으로 분비·생산하는 방법에 관한 것으로, 보다 구체적으로는, 대장균 유래 세포외막에 단백질 F (
OmpF ) 유전자를 함유하는 재조합 발현벡터와 세포 배양액으로 분비하고자 하는 인체 유래 상피세포성장인자를 함유하는 재조합 발현벡터로 동시에 형질전환된 미생물 및 상기 미생물을 배양하여 인체 유래 상피세포성장인자를 세포배양액으로 분비·생산하는 방법에 관한 것이다.
본 발명에 따르면, 고농도의 인체 유래 상피세포성장인자를 순수한 상태로 세포 배양액으로 분비시킬 수 있어, 매우 효율적인 분리·정제가 가능하다.
대장균, 세포외막 단백질 F (OmpF), 인체 유래 상피세포성장인자(human epidermal growth factor: hEGF), 세포외 분비생산-
公开(公告)号：KR1020000056440A
公开(公告)日：2000-09-15
申请号：KR1019990005773
申请日：1999-02-22
Applicant: 한국과학기술원
CPC classification number: C12N15/70 , C07K14/245 , C07K2319/00
Abstract: PURPOSE: An expression vector containing OmpC gene as a cell surface anchoring motif, organisms transformed therewith and protein expression method on the cell surface with the transformed organism are provided. The vector is used such as to remove heavy metals by expression of poly-histidine peptide truncated with OmpC gene. CONSTITUTION: Chromosome DNA of E. coli MC4100 is used as a template to amplify OmpC gene from E. coli. 1.1 kb of PCR product is isolated from agarose gel and cloned into pTrc99A having strong trc promoter. The resulting plasmid pTrcC has two Pst1I at 7th loop and C-terminal. PstI site of C-terminal is removed to make cloning easily and the constructed plasmid is named as pTCdP. The resulting plasmid is transformed into E. coli MC4100 and E. coli MC 4100/pTCdP (KCYC0576BP) is obtained. Poly-histidine linker peptide (poly-His) consisted 6 histidine is amplified by PCR and cloned into recombinant expression vector pTCdP. pTCHP1 is obtained. Other expression vectors having 2,3 and 6 unit of poly-His are constructed. SDS-PAGE analysis shows that poly-His protein consists 30-40% of outer membrane protein.
Abstract translation: 目的：提供含有OmpC基因作为细胞表面锚定基序的表达载体，其转化的生物体和转化的生物体在细胞表面上的蛋白质表达方法。 使用载体，通过用OmpC基因截短的多组氨酸肽的表达来除去重金属。 构成：大肠杆菌MC4100的染色体DNA用作扩增大肠杆菌OmpC基因的模板。 从琼脂糖凝胶中分离1.1kb的PCR产物，并克隆到具有强trc启动子的pTrc99A中。 得到的质粒pTrcC在第7个环和C末端有两个PstII。 去除C末端的PstI位点以使其易于克隆，将构建的质粒命名为pTCdP。 将得到的质粒转化到大肠杆菌MC4100中，得到大肠杆菌MC 4100 / pTCdP（KCYC0576BP）。 通过PCR扩增多组氨酸接头肽（poly-His）组成的6个组氨酸，并克隆到重组表达载体pTCdP中。 获得pTCHP1。 构建了具有2,3和6单位的poly-His的其他表达载体。 SDS-PAGE分析显示多聚蛋白质蛋白占30-40％的外膜蛋白。
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Patent Agency Ranking
公开(公告)号：KR1020090039027A
公开(公告)日：2009-04-22
申请号：KR1020070104418
申请日：2007-10-17
Applicant: 한국과학기술원
Abstract: A new promoter form expressing and secreting a target protein is provided to produce pure target protein by extracellularly expressing the target protein. Gene constructs comprise: a secretion promoting motif gene selected among a group of a coding gene of arginine transporter subunit(ArtJ), a coding gene of hyperosmotically inducible periplasmic protein(OsmY) and a coding gene of peptidyl-prolyl cis-trans isomerase(FkpA) and a gene coding a target protein. The gene constructs are characterized in intracelluarly expressing in a fusion form of the secretion promoting motif selected and the target protein and extracelluarly secreting. Method of extracellular secretion and production of the target protein comprises: a step of culturing recombinant microorganism containing the gene constructs to extracellularly secret the target protein; and a step of collecting the target protein in culture media.
Abstract translation: 提供表达和分泌靶蛋白的新的启动子形式，通过细胞外表达靶蛋白来产生纯的靶蛋白。 基因构建体包含：选自精氨酸转运子亚基（ArtJ）的编码基因，高肌诱导周质蛋白（OsmY）的编码基因和肽基 - 脯氨酰顺反异构酶（FkpA）的编码基因的分泌促进基序基因 ）和编码靶蛋白的基因。 基因构建体的特征在于以选择的分泌促进基序的融合形式和靶蛋白质和细胞外分泌物进行脑内表达。 细胞外分泌方法和靶蛋白的产生包括：培养含有基因构建体的重组微生物以细胞外分泌靶蛋白的步骤; 以及在培养基中收集靶蛋白的步骤。
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公开(公告)号：KR100381381B1
公开(公告)日：2003-12-31
申请号：KR1019980051563
申请日：1998-11-28
Applicant: 한국과학기술원
Abstract: PURPOSE: Recombinant plasmids having secretion signal sequence of endoxylanase from Bacillus are provided which express and secret foreign protein in transformed E. coli therewith. CONSTITUTION: A secretion signal sequence of endoxylanase fragment from plasmid, pKJX4, containing endoxylase gene from Bacillus is amplified by PCR. Recombinant plasmid, pJS101, is constructed by cloning PCR fragments into plasmid, pTrc99A, having strong trc promoter. Plasmid pJS101 contains signal sequence of endoxylanase and endoxylanase joined with PstI site. 3' end of endoxylanase has restriction sites of BamHI, XbaI, SalI, PstI and HindIII for the cloning of foreign protein. Plasmid pJS101DeltaP is constructed by removing PstI site of 3' end of endoxylanase gene for the easy introduction of foreign protein. T7 promoter containing vector, pJS301, is constructed by joining of NdeI and BamHI digested fragment from pJS101DeltaP and pET21c. Induction experiment with IPTG shows that the yield of secreted protein is at least 6% of total protein. Alkaline phosphatase is over expressed and constitutes 6.9% of total protein.
Abstract translation: 目的：提供具有来自芽孢杆菌属的木聚糖内切酶分泌信号序列的重组质粒，其在转化的大肠杆菌中表达并分泌外源蛋白质。 构成：通过PCR扩增来自质粒pKJX4的含有来自杆菌的内切酶基因的木聚糖内切酶片段的分泌信号序列。 通过将PCR片段克隆到具有强trc启动子的质粒pTrc99A中构建重组质粒pJS101。 质粒pJS101含有内切木聚糖酶的信号序列和与PstI位点连接的木聚糖内切酶。 内切木聚糖酶的3'末端具有BamHI，XbaI，SalI，PstI和HindIII的限制位点用于克隆外源蛋白。 质粒pJS101DeltaP是通过去除内切木聚糖酶基因的3'末端的PstI位点以便于引入外来蛋白质而构建的。 含有T7启动子的载体pJS301通过连接来自pJS101DeltaP和pET21c的NdeI和BamHI消化的片段构建。 用IPTG诱导实验显示分泌蛋白的产量至少为总蛋白的6％。 碱性磷酸酶过度表达并占总蛋白的6.9％。
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公开(公告)号：KR1020030081548A
公开(公告)日：2003-10-22
申请号：KR1020020019820
申请日：2002-04-11
Applicant: 한국과학기술원
IPC: C12N15/11
CPC classification number: C12N15/70 , C12N9/20 , C12P41/003 , C12Y301/01003
Abstract: PURPOSE: A gene for cell-surface expression of foreign proteins or peptides is provided, thereby expressing the foreign proteins or peptides having normal function on the cell surface. CONSTITUTION: A gene for cell-surface expression of foreign proteins or peptides comprises the nucleotide sequence encoding the amino acid sequence from the N-terminal to 5th loop of outermembrane protein C(OmpC). A cell-surface expression vector of green fluorescent protein(GFP), pT7KSC-GFP comprises a gene for cell-surface expression encoding the amino acid sequence from the N-terminal to 7th loop of OmpC, T7 promoter, kanamycin resistant gene and GFP gene. A cell-surface expression vector of lipase, placKSC-lip comprises a gene for cell-surface expression encoding the amino acid sequence from the N-terminal to 7th loop of OmpC, lac promoter, kanamycin resistant gene and lipase gene. Transformed microorganisms, Escherichia coli BL21(DE3)/pT7KSC-GPF(KCTC-0897BP) and Escherichia coli MC4100/placKSC-lip are provided. The cell-surface expression proteins are obtained by culturing the transformed microorganisms and collecting proteins expressed on the cell surface.
Abstract translation: 目的：提供外源蛋白或肽的细胞表面表达基因，从而表达在细胞表面具有正常功能的外源蛋白或肽。 构成：用于外源蛋白质或肽的细胞表面表达的基因包含编码外膜蛋白C（OmpC）的N末端至第五环的氨基酸序列的核苷酸序列。 绿色荧光蛋白（GFP），pT7KSC-GFP的细胞表面表达载体包含编码OmpC，T7启动子，卡那霉素抗性基因和GFP基因的N末端至第七环的氨基酸序列的细胞表面表达基因 。 脂肪酶的细胞表面表达载体placKSC-lip包含编码OmpC，lac启动子，卡那霉素抗性基因和脂肪酶基因的N末端至第七环的氨基酸序列的细胞表面表达基因。 提供转化的微生物，大肠杆菌BL21（DE3）/ pT7KSC-GPF（KCTC-0897BP）和大肠杆菌MC4100 / placKSC-唇。 通过培养转化的微生物并收集在细胞表面表达的蛋白质来获得细胞表面表达蛋白质。
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公开(公告)号：KR100313135B1
公开(公告)日：2001-11-05
申请号：KR1019990005773
申请日：1999-02-22
Applicant: 한국과학기술원
CPC classification number: C12N15/70 , C07K14/245 , C07K2319/00
Abstract: 본발명은미생물세포표면에외래단백질을효율적으로발현시킬수 있도록표면발현모체로서대장균에서유래한세포외막단백질 C(OmpC)의유전자를포함하는재조합발현벡터와원하는외래단백질의유전자를삽입시킬수 있도록구성된전기발현벡터에의하여형질전환된형질전환체및 전기발현벡터에외래단백질을코딩하는유전자를삽입한발현벡터로형질전환체를제조하여배양하고, 발현을유도하여세포표면에발현시키는공정을포함하는외래단백질의세포표면발현방법에관한것이다. 또한, 본발명은세포표면발현모체인대장균유래 OmpC의유전자및 폴리-히스티딘링커펩타이드를코딩하는유전자를포함하는발현벡터에의하여형질전환되어, 세포표면에폴리-히스티딘을발현하는형질전환체를유효성분으로포함하는중금속제거제를제공하는것이다. 본발명의발현벡터를이용하여세포표면에외래단백질을발현시키면, 삽입되는외래유전자에따라, 재래식백신보다지속적이고강력하게면역반응을나타낼수 있는재조합생백신, 중금속제거나폐수처리에응용이가능한전세포흡착제, 생물학적전환에이용되는효소를세포표면에발현시켜지속적으로촉매의활성의감소없이안정되게사용할수 있는전세포생물전환등의목적에유용하게사용할수 있다.
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公开(公告)号：KR100283677B1
公开(公告)日：2001-03-02
申请号：KR1019980058891
申请日：1998-12-26
Applicant: 한국과학기술원
IPC: C12N15/70
Abstract: 본 발명은 합성 분비 신호서열(artificial secretory signal sequence) 및 그를 이용한 외래 단백질의 제조방법에 관한 것이다. 좀 더 구체적으로, 본 발명은 23개의 아미노산으로 구성된 신규의 합성 분비 신호서열, 그를 포함한 재조합 플라스미드 및 전기 플라스미드에 원하는 외래 단백질의 유전자를 삽입하고 그를 대장균(Escherichia coli)에 도입하여 형질전환체를 배양하고 발현을 유도함으로써, 재조합 대장균의 세포질 밖으로 효율적으로 분비된 외래 단백질의 제조방법에 관한 것이다. 본 발명의 재조합 플라스미드는 여러 대장균 및 여러 발현 시스템에서 매우 높은 단백질 분비효율을 나타내는 바, 이러한 점을 이용하여 특정 외래 단백질을 대량생산한다면, 주변 세포질으로의 분비가 가능하기 때문에 분리 및 정제 과정에서의 경비와 시간을 상당히 줄이는 효과를 얻을 수 있어, 산업적인 측면에서 재조합 단백질의 경제적인 생산이 가능하게 된다. 또한, 본 발명의 재조합 플라스미드는 합성 분비 신호서열과 분비하고자 하는 외래 단백질의 유전자를 링커(linker) 없이 바로 연결할 수 있으므로, 분비되는 외래 단백질에 불필요한 아미노산이 삽입되지 않아 의료용 단백질 생산에 매우 유리할 것이다.
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