• Applied Biological Chemistry
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• v.41 no.3
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• pp.219-223
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• 1998

We have established a transformation system for entomopathogenic fungus, Metarhizium anisopliae, in order to develop mycoinsecticide by recombinant DNA techniques. Protoplasts of M. anisopliae would be transformed to a benomyl-resistant by introducing pBRG-4 plasmid DNA, which contains a ${\beta}-tubulin$ gene of Aspergillus flavus conferring resistance to benomyl and a pyr4 gene of Neurospora crassa, in the presence of 5% polyethylene glycol and 10 mM calcium chloride. Transformants occuring at a frequency of 10 colonies per $50\;{\mu}g$ pBRG-4 DNA grew on the $5\;{\mu}g/ml$ concentrations of benamyl, while the wild type was inhibited by $2.5\;{\mu}g/ml$. From the Southern analysis using genomic DNAs isolated from M. anisopliae transformants, the positive signals suggested that the ${\beta}-tubulin$ gene had integrated in the M. anisopliae genome by homologous recombination.

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.55-55
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• 1999

Imperatoxin A (IpTx$_{a}$), a 3.7 kDa peptide from the African scorpion Pandinus imperator, has been known as an agonist of skeletal ryanodine receptor (RyR). In order to study the structure and function of the toxins on RyR, the IpTx$_{a}$ cDNA was PCR-amplified using 3 pairs of primers and the toxin was expressed in E. coli expression system.(omitted)ted)

• Applied Biological Chemistry
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• v.38 no.1
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• pp.7-12
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• 1995

The purpose of this study was the development of promoter for the lacZ' gene. Two heterologous promoter I and II of lacZ' gene were isolated from chromosomal DNA Bam HI fragment of yeast. The size of the promoter I was estimated to be 2.5 kb and ${\beta}-galactosidase$ activity was 124.6 U/mg protein, and the size of the promoter II was 4.0 kb and its ${\beta}-galactosidase$ activity was 168.8 U/mg protein, respectively. The stability of the recombinant YEp plasmid in the transformant was from 52.7 to 67.4% at minimal medium. YIp plasmid was constructed from YEp plasmid, and expressed both in E. coli and yeast. The promoter I aid II iso-lated from yeast chromosomal DNA can be used for promoter of plasmid YEp and YIp.

• BMB Reports
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• v.40 no.1
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• pp.22-28
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• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• Toxicological Research
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• v.17
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• pp.167-171
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• 2001

The introduction of genetically modified crops has raised concerns regarding safety issues over the insertion of foreign genes into plant genomes using recombinant DNA technology. Since 1991 in Japan, 29 foods and 6 food additives have been evaluated, based on the "Guideline for Safety Assessment", before these foods were marketed. The MHW, however, decided that safety assessment of such foods and food additives should be legally imposed. because soon such foods and food additives are expected to circulate globally and a new system for assessing safety of such foods and food additives at a pre-market stage is necessary, in order to avoid the distribution of any genetically modified foods that have had no safety assessment. The MHW published relevant announcements to amend existing regulations on 1 May 2000. "Standards for safety assessment of seed plant" is established based on a concept of substantial equivalence, and applicable to the products which are regarded as equivalent to the existing products used as foods and food additives. The characterization of the food products entails consideration of the molecular characterization. phenotypic and compositional characteristics, key nutrients and toxicants, and toxicity and allergenicity of the introduced proteins, and if there are indications of unintended effects of the modification, whether further safety testing (animal studies etc.) is needed should be considered. Safety and wholesomeness studies with whole foods should be care fully designed in order to avoid nutritional imbalances causing artifacts and uninterpretable results as was the case of Dr. Pusztaiis report. A case study of genetically modified soybeans (glyphosate-tolerant soybeans) on the immune system of rats and mice is shown. Chemical compositions were also compared with those of the non-GM soybeans. The studies failed to detect any differences in immuno-toxic activity.muno-toxic activity.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.299-307
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• 2003

The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants ($\Delta$24/83 and $\Delta$38/83) and triple mutant ($\Delta$24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.

• Genomics & Informatics
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• v.15 no.4
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• pp.142-146
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• 2017

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.206-209
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• 2001

Recombinant human GM -CSF was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, pMYN44. containing the hGM -CSF cDNA. Regulated expression and secretion of hGM -CSF from this vector achieved using the promoter, signal peptide, and terminator from a rice alfa-amylase gene Amy3D. The Amy3D gene is expressed in response to sugar deprivation. The recombinant hGM -CSF was expressed from the transgenic rice cell culture on the sugar-free medium as a yield of about 110 mg/L in the culture filtrate, which was determined by ELISA. Biological activity of hGM-CSF was confirmed by measuring the proliferation of the hGM -CSF dependent TF -1 cells.(This work was supported by a grant from the NRL program of the Korean Ministry of Science and Technology. Shin, Y.- J.. Lee. J.-H and Kwon, T.-H. have been supported by BK21 program from the Korean Ministry of Education)

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.600-605
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• 1989

For the purpose of isolation of the Zymomonas mobilis DNA fragments showing promoter activity in Escherichia coli, a promoter screening vector, PCMT215 was constructed by transferring a promoterless chloramphenicol acetyltransferase (CAT) gene of pYEJ001 into pMT21 which contains $\beta$-lactamase gene and multiple cloning sites. A library of Z, mobilis Sau3AI DNA fragments was constructed in E. coli using the newly constructed pCMT215. Fourteen clones showing resistance to chloramphenicol ranging in concentration from 30 to 750 $\mu$g/$m\ell$ were selected. From five clones of them, the Z. mobilis DNA fragments expressing CAT gene of the recombinant plasmids were sequenced and then sites of transcriptional initiation were identified. The nucleotide sequences of the cloned DNA shared AT rich regions, poly A's or T's stretches and palindromic regions. The positions of transcriptional initiation for CAT gene occurred at more than one site spaced over by 4 to 190 base pairs on the cloned fragments in E. coli.

• Journal of Life Science
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• v.18 no.7
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• pp.912-917
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• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.