• 한국균학회소식:학술대회논문집
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• 한국균학회 2018년도 춘계학술대회 및 임시총회
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• pp.51-51
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• 2018

The application of recombinant DNA technology has been remarkable and nearly replaced commonly used traditional methods. Traditional industrial microbiology long depended on the discovery of valuable strains and mutagenesis of such strains to improve its secretion capacity of enzymes and secondary metabolites on the industrial scale. Commodities included industrial enzymes and biopharmaceuticals. The purpose of genome manipulation by the crossing of different strains or genetic recombination of naked DNA to the genome is of increased production of valuable metabolites. We optimized a transformation method to either for removal of innate genes, introduction of heterologous genes, or combination of both. We have been used selected whole or partial genes to manipulate target fungi toward the development of strains overproducing invaluable proteins. We have also used the whole genome sequence information of fungal genomes in public databases and functional genomics approach to select genes to manipulate and eventually contributing greatly to the development of overproducing industrial strains overproducing proteins or secondary metabolites. I will briefly review 1) filamentous fungi as a host for production of recombinant proteins and secondary metabolites, 2) markets of industrial metabolites, 3) a new approach to manipulate up to five genes at the same time in the system that ProxEnrem uses.

• BMB Reports
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• 제40권1호
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• pp.7-14
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• 2007

Helicases are ubiquitous enzymes, which utilize the energy liberated during nucleotide triphosphate hydrolysis to separate double-stranded nucleic acids into single strands. These enzymes are very attractive targets for the development of new antibacterial compounds. The PcrA DNA helicase from Staphylococcus aureus is a good candidate for drug discovery. This enzyme is unique in the genome of S. aureus and essential for this bacterium. Furthermore, it has recently been published that it is possible to identify inhibitors of DNA helicases such as PcrA. In this report, we study the properties of recombinant PcrA from S. aureus purified from Escherichia coli to develop ATPase and helicase assays to screen for inhibitors.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2003년도 추계학술대회
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• pp.89-90
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• 2003

Biological products are composed of vaccines, antitoxin, blood products, DNA recombinant protein drugs, monoclonal antibody, cell therapy and gene therapy. Biological products are divided into traditional (i.e. recombinant proteins and monoclonal antibodies) and novel biological products (gene and cell therapy) and will require a similar re-evaluation of the approaches taken during each development program.(omitted)

• 한국미생물·생명공학회지
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• 제40권4호
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• pp.348-355
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• 2012

본 연구에서는 S. cerevisiae와 P. pastoris에서 bovine pancreatic (bp-) DNase I의 과발현과 재조합 DNase I의 특성을 조사하였다. bp-DNase I 유전자는 GAL10 promoter, $MF{\alpha}$, GAL7 terminator 사이에 삽입하여 재조합 plasmid인 pGAL-$MF{\alpha}$-DNaseI (6.4 kb)를 구축하였다. 그리고 bp-DNase I 유전자를 AOX1 promoter, $MF{\alpha}$, AOX1 terminator 에 삽입하여 재조합 plasmid인 pPEXI (8.8 kb)를 구축하였다. 재조합 plasmid인 pGAL-$MF{\alpha}$-DNaseI과 pPEXI를 각각 S. cerevisiae와 P. pastoris 숙주세포에 형질전환시켰다. 형질전환된 효모세포들을 galactose와 methanol 배지에서 $30^{\circ}C$, 48시간 배양하면 bp-DNase I은 대부분이 배양 상등액으로 과발현되었다. P. pastoris 형질전환체는 배양 상등액에서 45.5 unit/mL의 DNase I 활성을 보였으며, 반면에 S. cerevisiae 형질전환체는 37.7 unit/mL의 DNase I 활성을 보였다. 또한 DNA 분해 특성을 조사한 결과, P. pastoris 재조합 DNase I으로 기질 DNA(calf thymus)를 처리하였을 때 1분 이내 DNA가 분해되는 것을 확인할 수 있었으며 이는 상업용 bp-DNase I과 S. cerevisiae 재조합 DNase I으로 처리했을 때보다 빠른 분해 패턴을 보였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.391-391
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• 2003

• 약학회지
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• 제29권6호
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• pp.162-163
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• 1985

• 생명과학회지
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• 제27권5호
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• pp.524-529
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• 2017

본 연구에서는 zebrafish에 인간의 KCNE1 유전자가 삽입된 형광단백질 vector를 microinjection하고, 그 발현 여부를 확인하고자 하였다. 먼저 양 말단에 제한효소(EcoRΙ, BamHΙ) site를 넣어 제작한 primer들로 genomic DNA에서 KCNE1 유전자를 분리하였다. 그 결과는 약 402 bp 크기의 DNA band였고 이 PCR 산물을 형광단백질 vector인 pPB-CMVp-EF1-GreenPuro 속에 클로닝하여 pPB-CMVp-hKCNE1-EF1-GreenPuro plasmid를 제작하였다. 이렇게 준비된 형광 vector를 zebrafish 수정란에 microinjection하였고, 부화된 치어에서 RT-PCR과 DNA sequencing을 통해 GFP 및 hKCNE1의 발현을 최종 확인하였다. 본 연구는 향후 QT 연장증후군(LQTs)에 대한 동물 모델로써 신경자극 전도, 유전자 치료, 유용 유전자 클로닝을 위한 기술 개발에 응용될 수 있을 것으로 기대된다.

• Journal of Periodontal and Implant Science
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• 제29권3호
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• pp.663-676
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• 1999

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$ , and screened for recombinant clones with hemin-binding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa hemin-binding protein.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.470-475
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• 2000

Genetic chracterstics of the structural gene of guamerin (a novel elastase inhibitor from Korean leech), integrated into the HIS4 locus of chromosomal DNA of Pichia pastoris along with the $\alpha$-factor leader sequence, were investigated. In the selected clone from candidates, two copies of the integration cassette including the structural gene copies of the integration cassette including the structural gene of guamerin were found in the integration site of the chromosomal DNA of P.pastoris. It was demonstrated that the integrated structural gene of guamerin was stable up to about 70 generations in the relay flask culture. Then, a high-cell-density culture could be fulfilled easily by DO-stat fed-batch culture, in which the cell growth and the recombinant guamerin production reached about 250 of OD600nm and 260 mg/l, respectively. Finally, it was revealed that the DNA sequence of the integrated structural gene of guamerin in P. pastoris was maintained correctly in the end of production cells of relay flask culture and high-cell-density culture.

• 한국미생물·생명공학회지
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• 제16권4호
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• pp.316-319
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• 1988

토양으로부터 분리한 알카리내성 Bacillus sp. YA-14의 Pectate lyase(PL) 유전자를 E. coli에 cloning하여 제조한 재조합 plasmid pYPC29는 삽입 된 1.6kb 단편내에 PL 유전자를 함유하고 있었으며, 이 외래 DNA가 Bacillus sp. YA-14의 chromosomal DNA에서 유래된 것임을 Southern hybridization을 통하여 확인하였다. 재조합 plasmid pYPC29는 E. coli내에서 안정하게 존재하였으며 이를 함유한 재조합체의 전체 PL 활성 중 약 70%가 periplasmic space에 존재하였다.