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Huh, Tae-Lin,Koh, Suk-Hoon,Lee, Se-Yong The Korean Society for Microbiology and Biotechnol 1985 한국미생물·생명공학회지 Vol.13 No.4
Plasmid pBR322와 runaway Plasmid pSY343을 vector로 사용하여 E. stearothermophilus IAM 11062내의 $\alpha$-amylase 유전자를 E. coli내에 클로닝 하였다. 이때 얻어진 $\alpha$-amylase유전자는 제한효소 Hind III의 말단을 갖고 있는 4.7kb의 크기였으며 E. coli내에서 이들 유전자는 비교적 안정적 있게 유지되고 발현되었다. 재조합 $\alpha$-amylase유전자가 클로닝된 E. coli는 B. stearothermophilus IAM 11062보다 3배의 $\alpha$-amylase를 더 많이 생성하였다. EDTA를 사용한 osmotic shock 방법에 의하여 E. coli내에서 생성된 $\alpha$-amylase는 그 효소 생성량의 75%정도가 periplasm에 존재함이 밝혀졌다. 재조합된 $\alpha$-amylase 유전자에 의해서 E. coli에서 생성된 $\alpha$-amylase는 최적 작용온도가 55$^{\circ}C$로서 이들의 열안정성과 분자량(61,000)도 B. stearothermophilus IAM 11062의 $\alpha$-amylase와 거의 동일하게 나타나 E. coli와 B. stearothermophilus IAM 11062에서 생성된 $\alpha$-amylase는 효소학적 성질이 같음을 보여주었다. A 4.7 kb Hind III fragment containing $\alpha$-amylase gene of Bacillus stearothermophilus IAM 11062 was cloned in Escherichia coil HB101, using plasmid pBR322 and runaway plasmid pSY343 as a vector. The cloned gene was stably maintained and expressed In E.coli. The constructed strain of E. coli have at least 3 times higher amylase activity than the donor strain, of B. stearothermophilus. About 75% of the $\alpha$-amylase produced by the constructed strain of E. coli was localized in the periplasm and it was found that the enzymes can be released by an osmotic shock using EDTA. The enzymatic properties of L-amylase produced in E. coli were very similar to those produced by B. stearothermophilus in terms of optimum temperature, heat stability and molecular weight.
Huh, Tae Lin,PARK, HEE SUNG,Jeng, Jiingjau,Kim, Young Ou,Oh, II Ung,Song, Byoung J. 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1
A 0.6 kb cDNA fragment encoding the human NAD^+-specific isocitrate dehydrogenase α-subunit (H-IDHα) was amplified by PCR using oligonucleotide primers synthesized on the basis of pig tryptic peptide sequences [Huang and Colman (1990) Biochemistry 29, 8266-8273]. With the amplified cDNA as a probe, cDNA clones for IDHα were isolated from a human heart λgt11 cDNA library. The deduced protein sequence of the largest cDNA clone (2628 bp) rendered a precursor protein of 366 amino acids (39591 Da) and a mature protein of 339 amino acids (36640 Da). The deduced H-IDHα protein sequence is highly similar to the partial peptide sequences of the pig enzyme. It is 55, 43 and 44 % identical with yeast NAD^+-specific IDH2, yeast NAD^+-specific IDH1 and monkey NAD^+-specific IDH γ-subunit (IDHγ) respectively. However, it has less similarity (about 30%) to NADP^+-specific IDH from Escherichia coli and bovine mitochondria. These results indicate that the structure of IDHα closely resembles that of IDH2, the catalytic subunit of the yeast enzyme. Structural analysis of the deduced H-IDHα protein revealed that the amino acids responsible for the binding of isocitrate, Mg^(2+) and NAD^+ are highly conserved. It also has two conserved motifs for the binding sites of ATP and ADP, but a canonical Ca^(2+)-binding motif was not recognized. Unusual penta- (ATTTA) and tri-(TAA or ATT) nucleotides which are respectively believed to interact with RNA-binding proteins and be near the endonuclease cleavage sites were frequently recognized in its 3' untranslated region, indicating the possibility of an additional method of regulation of this enzyme. Northern-blot analysis suggests that one mRNA transcript (2.8 kb) exists in cultured HeLa cells. Genomic DNA Southern-blot analysis indicates that the IDHα gene is not closely related to that of the other IDH isoenzymes, and IDHα appears to be encoded by a single gene.
α - Pheny - N - t - butylnitrone Protects Oxidative Damage to HepG2 Cells
Huh, Tae Lin,Park, Jeen Woo,Kim, Sun Yee,Kim, Ryung Hyo 생화학분자생물학회 2002 BMB Reports Vol.34 No.1
α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. Recently, there has been considerable interest in the antioxidant nature of PBN on degenerative diseases, presumably related to oxidative stress. In the present study, the protective effect of PBN on the HepG2 cell line under oxidative stress was investigated. When the HepG2 cells were exposed to oxidant, such as hydrogen peroxide, menadione, or ethanol, the protective role of PBN was manifested as a reduction in trypan blue uptake and a decrease in the endogenous production of oxidants, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin. The modulation of activity of major antioxidant enzymes, such as superoxide dismutase and catalase, was not significantly dittferent either in the presence or in the absence of PBN. This indicates that PBN acts as a direct scavenger of reactive oxygen species.
Huh, Tae Lin,Kim, Yong Ou,Kim, Suk Hyung 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1
A 1.5 kb cDNA fragment encoding for the human NAD^+-specific isocitrate dehydrogenase γ subunit (H-IDHγ) was isolated from a human heart λgt11 cDNA library. The deduced protein sequence of the cDNA clone (1,506 bp) rendered a precursor protein of 393 amino acids (42,794 Da) containing a mature protein of 354 amino acids (38,814 Da). The deduced H-IDHγ protein sequence is 48%, 37% and 42% identical to yeast NAD^+-specific IDH1, yeast NAD^+-specific IDH2, and human NAD^+-specific IDHα subunit (IDHα), respectively. However, H-IDHγ shares less similarity ($lt; 30%) to NADP^+-specific IDHs from the E. coli, bovine mitochondria and rat cytosol. Structural analysis of the deduced H-IDHγ protein revealed that it lacked three of ten amino acids responsible for the binding of either isocitrate or Mg^(2+)-isocitrate in E. coli NADP^+-specific IDH. These results indicate that the structure of IDHγ closely resembles that of IDH1, the regulatory subunit of the yeast enzyme, thus suggest that IDHγ might be a regulatory subunit rather than a catalytic subunit for IDH. Genomic DNA Southern-blot analysis indicates that IDHγ gene does not closely relate to that of the other IDH isoenzymes and appears to be encoded by a single gene.
Cellulases of trichoderma viride ( 2 ) : Induction of Cellulases by Avicel and Their Mode of Action
Tae Lin Huh,Eun Kyong Song,Se Young Lee 생화학분자생물학회 1981 BMB Reports Vol.14 No.4
In order to investigate the characteristics of cellulases induced by Avicel, Trichoderma viride QM 1414 was cultured on Avicel. The culture filtrates were concentrated by (NH₄)₂SO, fractionation and used as enzyme sources, The enzyme preparations were further purified by gel filtration and DEAE-cellulose chromatogaphy and assayed for cotton activity, CMC activity and β-glucosidase activity. The following results were obtained 1. The optimum pH of the cotton activity and CMC activity was between pH 4.8∼ 2. The optimum temperature of cotton activity was 50℃ and of CMC activity 55℃. 5.2. 3. Cellulases induced by Avicel contained low β-glucosidase activity induced by CMC and cellobiose. 4. Cellulase induced by Avicel contained high cotton activity compared with cellulase induced by CMC and cellobiose, 5. A low molecular weight CMCase activity was separated from the rest by Bio Gel P-150 column chromatography. From these, a cellulase that could only degrade CMC woas obtained. The remaining CMCase activity and cotton activity were separated by DEAE-cellulose chromatography, 6. Trichoderma cellulases induced by Avicel could hydrolyze cotton and CMC into glucose in the absence of β-glucosidase.
Expression of Cholesteryl Ester Transfer Protein cDNA using Recombinant Vaccinia Viruses
Huh, Tae Lin,Bok, Song Hae,Park, Yong Bok,Ahn, Byung Yoon,Jang, Moon Kyoo 생화학분자생물학회 1977 BMB Reports Vol.28 No.3
cDNA for human cholesteryl ester transfer protein (CETP), a potent atherogenic plasma protein that redistributes the neutral lipids among lipoproteins, was expressed in recombinant vaccinia virus-infected cells (CV-1). Two insertion vectors regulated by different promoters were constructed. The vectors were introduced into human thymidine kinase-negative (TK^-) 143B cells infected with wild-type vaccinia virus (WR strain). Recombinant viruses were selected with 5-bromodeoxyuridine (BUdR) and X-gal and identified with DNA dot blot analysis (vSC11-CETP and vTM1-CETP). The CETP cDNA insert in the recombinant vaccinia virus genome was identified by Southern blot analysis. Transcription of CETP cDNA in CV-1 cells infected with recombinant vaccinia virus was monitored by Northern blot analysis using the CETP cDNA as a probe. Positive signals were detected at 1.8 kb in cells infected with vSC11-CETP and at 2.3 kb in cells infected with vTM1-CETP. The recombinant vaccinia virus-infected CV-1 cells were shown to produce functional CETP when the culture medium was subjected to the CETP assay.