Optimization of Homopolymer Tailing

이현환,공운영,배종찬 한국유전학회 1984 Genes & Genomics Vol.6 No.3

Homopolymer tailing is one of the earliest and still widely used method of constructing plasmid chimeras by hybridizing of complementary nucleotide tails synthesized enzymatically. Several parameters including time, temperature were explored to increase the tailing and transformation efficiency of various E. coli strains with pBR 322-SV 40 virus DNA chimera formed via d(A)·d(T) and d(G)·d(C) homopolymer tails. The adequate number of tail length for the chimera is about 20 and 40 in case of d(G), d(C) tailing respectively and 30 minute is sufficient for the tailing. The optimum temperature for the tailing and ligation with the tailed DNA were also studied. Now the transformation efficiency of various strains of E. coli is also under study with the tailed chimera DNA.

Staphylococcus epidermidis로부터 단백질 분해효소 유전자의 Cloning과 동정

이현환 한국외국어대학교 외국학종합연구센터 부설 기초과학연구소 1996 기초과학연구 Vol.4 No.-

사람의 피부로부터 Staphylococcus epidermidis를 분리하고 동정하였다. 이 균을 skim milk를 함유한 배지에 배양 하였을 때 colony 주위에 투명한 환(clear zone)을 보임으로서 체외 분비성 단백질 분해효소를 생성하는 것으로 밝혀졌다. 이 protease의 최적 pH가 7.0인 것으로 보아, neutral protease였다. 한편 이 균은 tetracycline에 대한 내성을 보이는데, 이는 7-8 Kb의 cryptic plasmid에 있는 내성 유전자에 기인하는 것으로 밝혀졌다. 이 균으로부터 단백질 분해효소 유전자를 shot-gun 방법에 의해 cloning하고 그 유전자를 동정하였다. 그 결과 3개의 재조합 플라스미드(recombinant plasmid)를 선별하였으며, 이들을 각각 pMG301, pMG302, pMG303으로 명명하였다. 이들 plasmid들은 각각 2.6Kb, 1.8Kb and 4.4Kb의 절편을 가지고 있었다. 한편 이 재조합 플라스미드들의 유전자 지도를 작성하였으며, 이로부터 단백질 분해효소를 coding하는 유전자의 정확한 위치를 찾기 위하여 subcloning을 시도하였다. The strain Staphylococcus epidermidis, isolated from the human skin in our laboratory, was characterized. The strain showed a clear zone on the agar medium containg 1% skim milk. The fact revealed that the strain produced some protease into the medium. The protease production was closely related with the growth of the cells. The optimal activity of the protease was found at pH 7.0, indicating the protease is neutral. The strain has cryptic plasmid of 7∼8 Kb long. The cryptic plasmid conferred the resistance against the tetracycline. To study the role of protease of the S. epidermidis in the infection process, three protease gene was cloned and characterized. 3 transformants which showed clear zone around the colonies were screened and selected. Each plasmid isolated from the colonies, named a pMG301, pMG302 and pMG303, has the 2.6Kb, 1.8Kb and 4.4Kb of foreign DNA fragmant, respectively. To determine the exact position of protease gene, the gene was deleted. subcloned and characterized.

초유에서 천연의 사람 lactoferrin의 분리와 이를 이용한 항바이러스성 연구

이현환 한국외국어대학교 외국학종합연구센터 부설 기초과학연구소 2003 기초과학연구 Vol.15 No.-

사람 lactoferrin이 다량 함유된 초유에서 천연의 사람 lactoferrin을 얻기 위하여, 양이온 크로마토그래피의 수행으로 천연의 사람 lactoferrin 분리한 후, 이를 이용하여 천연 lactoferrin의 Vero cell에 대한 EMCV 바이러스의 cytophatic effect 저해 여부를 알아보았다. 결과 천연의 사람 lactoferrin은 EMCV 바이러스의 cytophatic effect를 저해하였고, 이러한 lactoferrin의 항바이러스성은 EMCV 바이러스의 adsorption에 필요한 cell membrane와 lactoferrin의 결합에 의한 바이러스 attachment 저해에 기초한 것이라 예상할 수 있다.

Cloning and Expression of Human lactoferrin cDNA in Pichia pastoris

이현환 한국외국어대학교 외국학종합연구센터 부설 기초과학연구소 1999 기초과학연구 Vol.7 No.-

향균성 당 단백질인 lactoferrin을 효모인 Pichia pastoris에서 발현하기 위해 Human breast cDNA library로부터 중합효소 연쇄반응(PCR)법을 이용하여 lactoferrin을 형성하는 유전자를 선별적으로 cloning하였다. 이 유전자를 Pichia pastoris에 형질 전환시켜 chromosomal DNA에 integration시켰으며 이를 Southern blotting에 의해 확인하였다. 이 재조합 Pichia pastoris를 배양하여 human lactoferrin을 발현시켰으며 Northern blotting과 Western blotting에 의해 확인하였다. In order to express the bacteriocidal protein, human lactoferrin(hLf), in yeast Pichia pastoris, the cDNA encoding the hLf was specifically amplified from human breast cDNA library by polymerase chain reaction. The cDNA was cloned and transformed into Pichia pastoris by integration into the chromosomal DNA. The transformant containing hLf cDNA was selected and identified by Southern blotting and sequencing. The recombinant Pichia pastoris expressed hLf and secreted it into the medium. The expression was confirmed by Nothern blotting and Western blotting.

Threonine operon의 leader sequence의 mRNA 2차 구조가 threonine operon의 발현에 미치는 영향

이현환 한국외국어대학교 외국학종합연구센터 부설 기초과학연구소 1999 기초과학연구 Vol.9 No.-

L-threonine 은 필수아미노산 중 하나이며 분자량은 119.12이고 무취의 백색 결정 분말로 단맛을 지닌다. 또한 E. coli의 threonine operon은 thrA, thrB, thrC인 3개의 structural gene 과 thrA 앞에 regulatory region이 존재한다. 각각의 structural gene은aspartokinase I-homoserine dehydrogenase I, homoserine kinase, threonine synthase를 coding 한다. E. coli내에서 threonine 합성에 관여하는 효소의 발현은 transcriptional attenuation에 의해 조절 받는다. Threonine 합성에 관여하는 효소들의 발현을 조절하는 attenuator regio을 일부 제거하고, 이로 인해 형성되는 attenuator region의 mRNA의 2차구조가 변화함으로서 threonine 생성량에 어떤 영향을 미치는가를 알아보았다. 그 결과 attenuator 의 secondary structure형성이 파괴되어 threonine 생성량이 증가되거나 감소된 mutnt들을 얻었다. 이는 leader sequence의 attenuator에 있는 terminator의 2차구조가 변형되면서 threonine operon이 constituticve expression 되거나, 낮은 수준으로 발현되기 때문인 것이로 밝혀졌다. L-threonine is an essential amino acid which has the MW of 119.12. E coli thr operon consists of thrA, thrB and thrC and a regulatory region. Each structural gene codes for the aspartokinase I -homoscrine dehydrogenase I, homoserine kinase, and threonine synthase, respectively. The biosynthesis of threonine is regulated by the repression with threonine and isoleucine and the the expression of the gene is also regulated by attenuation. The effect of secondry structure of mRNA in the leader sequence on the expression of threonine operon was studied by deletion of the attenuator with Ba131. The plasmids which caused the increase or decrease in the productivity of threonine in E. coli were obtained. The changes in the productivity were due to the destruction of secondary structure of mRNA around the attenuator in the leader sequence. Among them, plasmid pE2, pA4 and pA11 showed constitutive expression of thr operon by the disruption of transcription terminator. Another plasmid pE1 and pE16 showed decrease in the productivity by the too much deletion of the leader sequence. These results indicated that the change in the secondary structure of mRNA in the leader sequence in thr operon affected the productivity of threonine in E. coli.

PDMS-tethered Siloxane Hybrid with Improved Elongation for Flexible Protective Coating

이현환,이융,강승모,강현석,배병수 한국고분자학회 2021 한국고분자학회 학술대회 연구논문 초록집 Vol.46 No.2

With the advent of foldable devices, flexible protective coating is indispensable to keep the device from failure. To obtain the reliable protective coating, sufficiently hard, but coating material having high elongation property is required. However, both characteristics in polymeric materials are mutually exclusive. In this study, we developed unique material for surface protective coating by tethering elastic PDMS to a rigid epoxy-siloxane network. PDMS-tethered epoxy-siloxane hybrid (P-t-ESH) material was fabricated by in situ sol-gel reaction between PDMS and epoxy-functionalized silane precursor. The flexible backbone in PDMS enhanced the strain at fracture, while tensile strength also improved through chemical crosslinking between PDMS and epoxy-siloxane network. As a result, P-t-ESH endured under repetitive folding test (bending radius: 5mm, for 100,000 cycles). Moreover, due to the hydrophobic nature of PDMS, P-t-ESH also endured an abrasion test.

Recombinant baculovirus를 이용한 곤충세포에서 human protease Inhibitor Nexin-1의 발현

이현환 한국외국어대학교 2001 한국외국어대학교 논문집 Vol.33 No.-

Homopolymer - tail 의 E . coli 형질 전환에 미치는 영향

이현환,공운영,현형환,유무영 한국유전학회 1986 Genes & Genomics Vol.8 No.3

Effect of parameters such as time, temperature and tail-length on the efficiency of tailing and transformation into E. coli strains using pBR322-SV40 DNA chimera formed via d(A)·d(T) or d(G)·d(C) homopolymer tailing were explored. The tail-length optimal for the construction was about 20 and 15 nucleotides in case of d(G) and d(C), respectively. The optimal temperature and time-length for tailing were 37℃ and 35min, respectively. The transformation efficiencies of recA^+ strains were slightly higher than those of recA^- strains when they were transformed with the DNA formed via homopolymer tailing. These facts indicated that some recA proteins may be involved in transformation step in E. coli.

Staphylococcus epidermidis로부터 새로운 단백질 분해효소 유전자의 cloning과 sequencing I: Subcloning for sequencing

하상덕,전성후,이현환 한국외국어대학교 외국학종합연구센터 부설 기초과학연구소 1996 기초과학연구 Vol.5 No.-

사람의 피부로부터 분리된 Staphylococcus epidermidis 균으로부터 새로운 단백질 분해효소 유전자들을 Cloning하고 동정하였다. 이들 protease 유전자들은 각각 0.7Kb와 1.2Kb의 크기였으며, 각각 pSS1과 pSS2로 명명하고 이들을 가진 재조합 plasmid를 대장균에 도입하였을 때 colony 주위에 투명한 환을 보였다. 이들 유전자들은 이미 보고된(J. Basic Sci., HUFS, 4, 1996, pp.211-221)것들과 크기나 유전자 지도가 달랐다. 이들 유전자들을 sequencing하기 위하여 몇 개의 subclone을 만들고 이들로부터 염기서열을 부분 결정하였으며, 정확한 유전자 지도를 작성하였다. Novel protease genes from Staphylococcus epidermidis, which was isolated from Human skin, were cloned and characterized. The insert size of these protease gene is 0.7Kb and 1.2Kb, respectively. The E. coli cell which harboring these plasmids showed a clear halo around the colony. The size and restriction map were different from those of previously reported pMG301, pMG302 and pMG303(J. Basic. Sci. HUFS. 4. 1996. pp.211-221). To determine the nucleotide sequence of the protease gene from pSS1, several subclones were made and characterized. Using the subclones, partial nucleotide sequence and fine restriction map were determined. The searching for complete ORF is now understudied.

中學校 混成學級 運營에 對한 敎師와 學生의 態度 比較 硏究

손충기,이현환 원광대학교 교과교육연구소 1999 교과교육연구 Vol.1 No.2