• Food Science and Biotechnology
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• 제18권4호
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• pp.954-958
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• 2009

Effects of controlled- and uncontrolled-pH operations on phenylalanine ammonia lyase (PAL) production by a recombinant Escherichia coli strain were investigated at uncontrolled-pH ($pH_{UC}$) and controlled-pH ($pH_C$) of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 in bioreactor systems. The results showed that the recombinant PAL activity was improved significantly by controlled pH strategy. Among the $pH_C$ operations, the highest PAL activities were obtained under $pH_C$ 7.5 strategy where cell mass ($OD_{600\;nm}$) and PAL activity was 1.3 and 1.8 fold higher than those of $pH_{UC}$, respectively. The maximum PAL activity reached 123 U/g. The $pH_C$ 7.5 strategy made recombinant plasmid more stable and therefore allowed easier expression of PAL recombinant plasmid, which increased PAL production. It was indicated that the new approach (controlled-pH strategy) obtained in this work possessed a high potential for the industrial production of PAL, especially in the biosynthesis of L-phenylalanine.

• 미생물학회지
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• 제28권2호
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• pp.91-97
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• 1990

생체내의 유해산소를 제거하는 superoxide dismutase (superoxide : superoxide oxidoreductase E.C.1.15.1.1) 중 세포질내에서 그 활성을 지니는 인체의 세포질 superoxide dismuta~ie (SODl) 유전자를 사람의 간 cDNA library로부터 동위원소로 표지된 oligonucleotide probe를 이용, in situ plaque hybridization 방법으로 선별 분리하여 내장균 벡터로 클로닝하였다. 이 클론은 SOD1 유전자의 5"L"TR과 3’UTR을 포함한 1.6 kb 정도의 cDNA였다 SOD1 구조유전자만을 선택적으로 분리하기 위해서 ATG를 포함하는 sense strand primer와 3’UTR 부위의 antisense strand primer를 이용하여 중합효소연쇄반응(Polymerase Chain Reaction) 방법을 써서 SOD1 구조유전자 부위만을 선택적으로 증폭시켰다. Taq DNA polymerase에 의해 증폭된 DNA를 벡터 pUCl9의 multiple cloning site (MCS) 내의 Hinc II 위치에 넣였으며 이 insert DNA를 M13 mp19으로 옮겨 dideoxy chain termination 방법으로 sequenase를 사용하여 염기서열을 결정하였다. 클론닝된 cDNA는 153개의 아미노산을 포함하고 있는 하나의 open reading frame (ORF)을 가셨다. 중합효소연쇄반응에 의해 이때 증폭된 SOD1 구조유전자를 $\lambda P_{L}$ 프로모터를 포함하고 있는 발현 벡터 pUPL에 옮긴 후 대장균에서 대량으로 발현시켰다. 이때 발현된 단백질 SOD1은 고유의 효소활성을 가지고 있었다.

• 생명과학회지
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• 제14권1호
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• pp.26-31
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• 2004

본 연구는 redox regulator로 알려 진 Ref-1 (Redox factor-1)과 결합하는 새로운 단백질을protein-protein interaction의 원리를 이용한 방법인 yeast two-hybrid assay로 검색, 동정하고, 검색된 단백질의 in vitro, in vivo 기능을 규명하는 데 그 목적을 두고, mouse 11-day Embryo cNA library를 prey로, full length REF-1을 bait로 하여 yeast strain 인 HF7C에 cotransformatiom시킨 후 histidine, leucine, tryptophan이 결핍된 SD plate에서 키워 자란 yeast transformants를 $\beta$-galactosidaseassay하여 screening하여 분리한 세 개의 clone중 한 clone이 DNA sequencing으로 확인한 결과 mouse thioredoxin임을 확인하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.749-754
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• 2000

알려진 dTDP-D-glucose 4,6-dehydratase의 amino acid 서열로 부터 primer를 제작하여 내열성 균주인 Thermus caldophilus GK24에서 colony hybridization 과정을 거쳐 dTDP-D-glucose 4,6-dehydratase를 포함하는 cosmid DNA를 얻었다. 유전자 분석을 위해 cosmid DNA를 subclone 하여 작은 크기로 분리하였다. 분리된 cosmid를 pSMTC-1 으로 명명하고 pSMTC-1를 BamHI으로 반응시켜 BamHI 단편 모두를 pGEM 7(+)를 이용하여 subclone 하였다. 각각의 이름은 크기에 따라 pKCB10(1.2kb-BamHI), pKCB20(1.6kb-BamHI), pKCB30(2.Ikb-BamHI), pKCB40(2.5kb-BamHI), pKCB50(2.5kb-BamHI), pKCB60(2.7kb-BamHI), pKCB70(3.4kb-BamHI), pKCB80(4.4kb-BamHI), pKCB90(7.0kb-BomHI) 으로 명명하였다. 각각의 subclone된 유전자를 분석하기 위해 Erase-a-base 방법을 이용하여 template를 준비하였고 이를 자동 염기서열 분석기를 이용하여 염기서열을 분석하였다. 염기서열분석 결과 pKCB80(4.2kb)에 dTDP-D-glucose synthase(orfA) 유전자를 비롯하여 dTDP-D-glucose 4,6-dehydratase(orfB), orfC (dTDP-4-keto-L-rhamnose reductase) 그리고 orfD(dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase)와 유사한 유전자들이 있음이 확인 되었고 dTDP-L-rhamnose의 생합성 과정을 예상할 수 있었다.

• 미생물학회지
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• 제30권4호
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• pp.246-251
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• 1992

대두 (Glycine max) 뿌리혹의 질소고정 공생균주 Bradyrhizobium japonicum SNU001 의 nod 유전자를 클로닝하였다. Rhizobium meliloti 의 4.5 kb EcoRI/ HindIII 절편을 탐침으로 한 게놈 혼성화 반응을 분리균주의 게놈상에 nod 유전자가 존재함을 확인하고, lambda EMBL3-BamHI vector 를 이용하여 genomic library 를 작성하였다. 작성된 library 로부터 1, 2 차 선별과정을 통해 nod 유전자가 있는 클론 1-5 를 선별하고, 클론 2로부터 nod DABC 탐침과 lambda DNA 탐침을 사용한 혼성화반응을 수행하여 삽입된 genomic DNA 에 대한 부분적인 제한효소 지도를 작성하였다. nod DABC 탐침과 가장 강한 혼성화반응을 보인 phage 클론 lambda CNS-1 의 3.9 kb BamHI 절편을 pBS KS(+) vector 에 subcloning 하고 동일한 탐침을 이용한 혼성화반응을 통해 subclone pBjCNS-1 을 선별하였다. 이 subclone 에 대한 부분적인 제한효소 지도를 작성하여 nod DABC 가 1.8 KpnI/SacI 절편에 존재함을 확인하였다.

• The Plant Pathology Journal
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• 제30권1호
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• pp.58-67
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• 2014

The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast ${\beta}$-ATPase. However, chloroplast ${\beta}$-ATPase interacts only with $TGB1_{L88}$, and not with weak silencing suppressor $TGB1_{L88}$. This selective interaction indicates that chloroplast ${\beta}$-ATPase is not required for AltMV movement and replication; however, TRV silencing of chloroplast ${\beta}$-ATPase in Nicotiana benthamiana induced severe tissue necrosis when plants were infected by AltMV $TGB1_{L88}$ but not AltMV $TGB1_{L88}$, suggesting that ${\beta}$-ATPase selectively responded to $TGB1_{L88}$ to induce defense responses.

• The Plant Pathology Journal
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• 제16권1호
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• pp.9-18
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• 2000

Using differential display techniques, a new acidic pathogenesis-related (PR) protein-1 cDNA (GMPRla) gene was isolated from a cDNA library of soybean (Glycinemax L.Merr, cultivar Jangyup) hypocotyls infected by Phytophthora sojae f. sp. glycines. The 741 bp of fulllength GMPRla clone contains an open reading frame of 525 nucleotides encoding 174 amino acid residues (pI 4.23) with a putative signal peptide of 27 amino acids in the N-terminus. Predicted molecular weight of the protein is 18,767 Da. The deduced amino acid sequence of GMPRla has a high level of identity with PR-1 proteins from Brassica napus, Nicotiana tabacum, and Sambucus nigra. The GMPRla mRNA was more strongly expressed in the incompatible than the compatible interaction. The transcript accumulation was induced in the soybbean hypocotyls by treatment with ethephon or DL-$\beta$-amino-n-butyric acid, but not by wounding. In situ hybridization data showed that GMPRIa mRNAs were usually localized in the vascular bundle of hypocotyl tissues, especially phloem tissue. Differences between compatible and incompatible interactions in the timing of GMPRla mRNA accumulation were remarkable, but the spatial distribution of GMPRla mRNA was similar in both interactions. However, more GMPRla mRNA was accumulated in soybean hypocotyls at 6 and 24 h after inoculation.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.449-456
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• 2008

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a $Ni^{2+}$-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately $40^{\circ}C$. The enzyme maintained approximately 70% of its maximum activity even at $60^{\circ}C$, whereas its activity rapidly decreased at below $37^{\circ}C$. The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.

• 대한의생명과학회지
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• 제10권3호
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• pp.211-217
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• 2004

For discrimination of ductal and ascinar cells, we isolated a single-chain variable domain fragment (scFv) antibody against ductal cells of salivary gland using phage display technique. From the spleen of a mouse immunized with ductal cell lysate, total RNA was prepared and used as a template for cDNA synthesis of antibody genes. The scFv genes were constructed with variable domain genes of heavy and light chain and were introduced into pCANTAB5E to construct phage scFv library. The phage particles specific for acinar cells were screened by subtraction using immunotubes coated with acinar and ductal cell lysate and enzyme-linked immunoabsorbance assay (ELISA). The characteristics of the scFv were determined by immunohistochemistry (IHC) and the result indicated that the isolated scFv has the specificity against ductal cells of salivary glands and tubules of kidney. And the scFv has an unique binding activity specific for Hashimoto's thyroiditis. The nucleotide sequence of isolated scFv gene was determined and revealed that V/sub H/ belongs to the mouse H-chain family subgroup IB and V/sub L/ to the mouse L-chain family subgroup III.