• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.83-86
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• 2002

Thermus caldophilus로부터 TDP-glucose 4,6-degydratase (TDPDH) 유전자를 분리하고 염기서열을 결정하고, 결정된 염기서열에 해당하는 유전자를 발현벡터에 삽입하고, 발현벡터를 대장균에 형질전환시켜 배양함으로써 TDP-glucose 4,6-dehydratase를 대량 생산하였다. 본 연구의 TDP-glucose 4,6-dehydratase는 주반응으로 TDP-4-keto-6-glucose를 합성하고, 여러 가지 당을 디옥시당으로 합성하는 효과가 있다.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2006.02a
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• pp.38-44
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• 2006

프로테오믹스는 생물체 안에 포함되어 있는 단백질을 통합적으로 연구하는 학문이다. 단백질을 동정(Protein identification)하고, 단백질의 상태를 분석(Protein characterization)하며, 단백질의 양적 변화를 관찰(Protein quantitation)한다. 유전자로부터 mRNA 로 복제되고 codon 의 규칙에 따라 합성되는 단백질이 세포 내에 얼만큼 존재하는가라는 단백질의 양적인 변화는 세포 내의 환경에 따라 시시각각 변화할 수 있으며, 이러한 변화의 추적은 단백질의 기능을 밝히는 기초자료로서 중요성을 가진다. 특히 질병의 조기 진단을 위한 바이오마커를 발굴하기 위한 스크리닝 역할로서, 단백질의 발현 양상을 비교하는 프로테오믹스는 기대를 모으고 있다. 단백질에 대한 분석, 특히 질량분석기에 의해 초고속으로 대량의 단백질 데이터를 생산하는 프로테오믹스의 연구는 정량적인 단백질 발현양상 분석의 정확도를 높이기 위해 다양한 실험기법과 데이터 분석기법을 동원하고 있다. 이번 발표에서는 프로테오믹스에서 단백질의 양을 측정하기 위한 실험 방법들과 그에 따른 데이터 분석 방법들을 소개하고자 한다. 프로테오믹스 연구의 초창기부터 사용되어온 2차원 전기영동법에 의해 생성되는 2D-gel image 에서의 spot 분석법으로부터, 탄뎀 질량분석기를 사용하는 ICAT, iTRAQ 등의 labeling 방법에 의한 정량분석, 그리고 질량분석기의 정확도를 최대한으로 활용하는 label-free 방법에 대한 기본 개념을 살펴보고 데이터 분석 기술의 적용 방법을 알아본다.

• Proceedings of the Korean Information Science Society Conference
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• 2004.10a
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• pp.205-207
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• 2004

유전자 발현 분석 시스템에 있어서 microarray 기술의 발전은 유전 질환 진단의 정확성과 신뢰도를 향상시키는 데에 큰 기여를 하였다. 다양한 microarray기술을 통해 얻은 대량의 유전자 발현 정보는 기계 학습분류기를 이용한 암의 분류와 진단, 예측 분야에도 효과적으로 이용될 수 있다. 이 과정에서 종류에 따른 암의 정확한 분류를 위해서는 되도록 해당 암 클래스와의 직접적인 연관이 있는 유전자만을 선택하여 활용하는 것이 효과적이다. 본 논문에서는 이러한 정보력 있는 유전자(informative gene)를 효과적으로 선택 할 수 있는 유전자 선택 방법을 제시하고, 이를 이용하여 세 가지 벤치마크 암 데이터에 대하여 체계적인 실험을 하였다. 그 결과 향상된 분류 성능을 확인할 수 있었다.

• Journal of Life Science
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• v.21 no.1
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• pp.119-126
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• 2011

There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

• Korean Journal Plant Pathology
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• v.12 no.3
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• pp.363-367
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• 1996

배나무잎 이상반점증상의 이병여부를 조기에 판별할 수 있는 가장 간편한 검정방법을 개발코자 시험하였다. 접목시기는 늦어질수록 병징발현율이 감소하였다. 접목방법은 2중절접, 2중삭아절접 순으로 양호하였으나 숙련된 기술이 필요한 이중절접방법보다 간편한 2중삭아절접 방법이 대량검정에 적합하였다. 이상반점증상의 전염에 필요한 최소 접촉시간은 1일 이상이었고 칼루스가 형성되어 접목부위가 활착된 21일 이후에서 발병이 가장 높았다.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2003.05a
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• pp.248-249
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• 2003

경골어류의 뇌하수체에서는 두 종류의 생식선자극호르몬 (GTHs; FSH와 LH)이 생산된다. 이들 호르몬들은 공통적인 $\alpha$ 쇄와 특이적인 $\beta$ 쇄를 가진다 (Querat, 1995). 연어과 어종들에서, FSH는 난황형성과 정자형성의 역할을 하며, LH는 배우자의 최종성숙을 유도한다 (Blazquez et al., 1997; Swanson and Dittman, 1997). 냉수성 고유어종인 열목어의 멸종을 방지하고 번식시키기 위한 생명공학적 연구방법을 검토하기 위하여 GTH$\alpha$, FSH$\beta$와 LH$\beta$ 쇄를 발현벡터에 cloning하여 염기서열을 결정하였다. (중략)

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.305-311
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• 2002

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ$\alpha$C-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOXI promoter and connected downstream of mating factor u-1 signal sequence. The plasmid linearized by Sacl was integrated into the 5'AOXI region of the chromosomal DNA of P. pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/1 and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/1 of AXEase protein was produced in the extracellular medium.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.274-281
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• 2006

A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.91-97
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• 1990

Complementary DNA (cDNA) coding for human cytoplasmic superoxide dismutase (SOD1) (superoxide: superoxide oxidoreductase E.C.1.15.1.1) was isolated from human liver cDNA library of $\lambda$gt11 by in situ plaque hybridization. The insery cDNA gas the 5' untranslational region (UTR) and 3'UTR of SOD1 gene. Polymerase Chain Reaction (PCR) method was used fro subcloning of SOD1 structural gene. Using synthetic sense strand primer (24mer) containing a start codon and antisense strand primer (24mer), SOD1 structural gene was selectively amplified. Amplified DNA was directly cloned into the HincII site of pUC19 plasmid. Insery cDNA was subcloned into M13 mp19 and sequenced by dideowy chain termination method with Sequenase. The nucleotide sequence of insert cDNA had an open reading frame (ORF) coding for 153 amino acid residues. The structural gene of cytoplasmic SOD was placed under the control of bacteriophage $\lambda P_{L}$ regulatory sequences, generating a highly efficient expression plasmid. The production of human SOD1 in E. coli cells was about 7% of total cellular proteins and recombinant human SOD1 possessed its own enzymatic acitivity.

• Journal of Life Science
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• v.21 no.12
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• pp.1726-1731
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• 2011

Human Stem Cell Factor (hSCF) is a cytokine that binds to the c-Kit receptor and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. To produce the human Stem Cell Factor (hSCF) recombinant protein, we constructed a germline transgenic silkworm using the piggyback vector. The expression of the hSCF gene was driven by the Drosophila heat shock protein 70 (dHsp70) promoter. 3XP3 promotor-driven EGFP was used as a marker which allowed us to rapidly distinguish the transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,020 eggs of bivoltin silkworms, Keomokjam. We obtained approximately 22 G1 broods that were EGFP-positive. The expression of the hSCF gene in the transgenic silkworm was analyzed by SDS-PAGE and immunoblotting. Also, analysis of insertion sites into the silkworm genome using inverse PCR showed that exogenous DNA was inserted into the transgenic silkworm genome. These results show that successfully constructed transgenic silkworm expresses the hSCF recombinant protein.