• Journal of Industrial Technology
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• v.30 no.B
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• pp.61-68
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• 2010

Three chitinase genes (chiA, chiB, and chiC) were cloned into E. coli by PCR amplification from Serratia marcescens KFRI314. The sizes of cloned chitinase genes were 1692 bp, 1500 bp, and 1443 bp which correspond to 563, 499, and 480 amino acids, respectively. Recombinant chitinases were overexpressed using pHCEIA expression vector and purified to homogenity. The molecular weights of chitinases were about 60kDa, 50 kDa, 52 kDa, respectively. Optimum pHs were around pH 5~6 and optimum temperatures were $45{\sim}50^{\circ}C$ while 90% of enzyme activities were stable up to $50^{\circ}C$. The specific activities of ChiA, ChiB, and ChiC were 233.1, 278.8, $111.3{\mu}mol\;(min)^{-1}\;mg^{-1}$ against colloidal chitin. From experiments using TLC and fluorescent substrate analogues, it was demonstrated that ChiA was endo-chitinase while ChiB and ChiC were chitobiosidase.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

• BMB Reports
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• v.42 no.5
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• pp.299-303
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• 2009

In this study, we use promoter analysis to show that interaction between Jab1 and p53 induces suppression of p53 activation in U2OS and H1299 cells. Interaction between p53 and Jab1 was further confirmed by immunoprecipitation and immunofluorescent analyses. In particular, Jab1 was able to induce nuclear export of p53 as previously reported. When Jab1 was overexpressed in U2OS cells followed by etoposide or hydrogen peroxide ($H_2O_2$), cell death induced by such stresses was protected against. On the contrary, when the level of Jab1 was suppressed in U2OS cells, cytotoxicity imposed by etoposide and $H_2O_2$ was dramatically increased, suggesting a cell protective role for Jab1. These results indicate that Jab1 is a negative regulator of p53 and a plausible oncogene.

• BMB Reports
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• v.41 no.5
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• pp.363-368
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• 2008

Two genes of temperate mycobacteriophage L5, namely, gp63 and gp64, were hypothesized to be toxic to M. smegmatis. An identical L5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage L1 and characterized at length here. As expected, hlg1 affected the growth of M. smegmatis when overexpressed from a resident plasmid. HLG1 (the protein encoded by hlg1) in fact caused growth retardation of M. smegmatis and the region encompassing its 57-114 C-terminal amino acid residues was found indispensable for its growthretardation activity. Both nucleic acid and protein biosynthesis were severely impaired in M. smegmatis expressing HLG1. Interestingly, HLG1 also affected E. coli almost similarly. This putative delayed early lipoprotein did not participate in the lytic growth of L1.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.62-65
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• 2002

We have previously shown that the addition of silkworm hemolymph to a culture medium increases the longevity of insect and mammalian cells by inhibiting apoptosis. This indicates that the component which inhibits apoptosis is contained in the silkworm hemolymph, The apoptosis-inhibiting component was isolated from silkwonn hemolymph and characterized in our previous study. A database search using the N-terminal amino acid sequence of this component as a template resulted in a 95% homology with a low molecular weight lipoprotein, the so called ’30K protein' of unknown function. In this study, the 30K protein gene was expressed in mammalian and insect cells to confirm the apoptosis-inhibiting effect. The overexpression of 30K protein in mammalian cell inhibited the staurosporin-induced apoptosis by the prevention of the activation of caspase 3. Using an Autographa californicanuclear polyhedrosis virus (AcNPV) system, the 30K protein was overexpressed also in insect cells. The expression of the 30K protein increased the longevity of baculovirus-infected insect cells by inhibiting apoptosis. These results suggest that the 30K protein is a novel anti-apoptotic protein.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.681-684
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• 2007

An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE3l-bar) and efficiently purified by $Ni^{2+}$ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: $0.01{\mu}g/ml$) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [$4.9{\pm}0.4{\mu}g/g$ of sample (n=6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.178-181
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• 2004

A gene encoding an extracellular nuclease was cloned from Aeromonas hydrophila strain ATCC14715. The gene was overexpressed and the enzyme was purified by fusing to maltose binding protein. It was shown that the protein possessed DNase activity on both single-stranded and double-stranded DNAs. It exhibited both endo- and exonuclease activities. It was also shown that the protein had an RNase activity. Possible roles of this extracellular enzyme in the A. hydrophila life cycle are discussed.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.76-76
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• 1995

Virus-encoded HIV-1 reverse transcriptase (RTase) is one of the major targets for the development of drugs for HIV-1 since it is an essential enzyme-for the replication cycle of HIV-1. We cloned the entire reverse trancriptase gene into an inducible expression vector with tac promotor= RTase was stably overexpressed and induced by IPTG and the highly-expressed RTase was purified partially by use of DEAE cellulose and Mono Q column. The partially purified enzyme (663kDa, 51kDa) as exhibited by SDS-PAGE showed the high specific activity (16,570U/mg) when the assay for the RTase activity was carried out using $^3$H-dTTP and poly(rA): oligo(dT)12-18 as the substrate.

• Bulletin of the Korean Chemical Society
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• v.28 no.9
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• pp.1549-1552
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• 2007

The cytoplasmic domain of syndecan-4, a type I transmembrane heparan sulfate proteoglycan, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF and NMR spectroscopy. The yield of the peptide was 3.0-1.5 mg per liter in LB or minimal medium, respectively. The recombinant expression and purification of this domain will enable us its structural and functional studies using multidimensional NMR spectroscopy.

• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.240-245
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• 2000

Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.