• 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.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1728-1735
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• 2014

Scrub typhus, caused by infection with Orientia tsutsugamushi, is a mite-borne zoonotic disease endemic to the Asian-Pacific region. In Korea, the incidence of this disease has increased with climate changes, and over 10,000 cases of infection were reported in 2013. Although this infection is treatable with antibiotics such as doxycycline and azithromycin, an effective prophylactic vaccine against O. tsutsugamushi would be more desirable for preventing scrub typhus in endemic areas. In this study, we investigated the 56-kDa type-specific antigen (TSA56), which is a major outer membrane protein of O. tsutsugamushi, as a vaccine candidate. Intranasal immunization of recombinant TSA56 (rec56) induced a higher level of TSA56-specific IgG than that induced by intramuscular immunization of tsa56-expressing DNA (p56). Both types of immunization induced a cell-mediated immune response to TSA56, as demonstrated by the splenic cell proliferation assay. Mice immunized with p56, followed by rec56 plus heat-labile enterotoxin B subunit from E. coli, had a stronger protection from a homologous challenge with the O. tsutsugamushi Boryong strain than with other combinations. Our preliminary results suggest that an effective human vaccine for scrub typhus can include either recombinant TSA56 protein or tsa56-expressing DNA, and provide the basis for further studies to optimize vaccine performance using additional antigens or different adjuvants.

• International Journal of Industrial Entomology and Biomaterials
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• v.7 no.2
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• pp.127-131
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• 2003

Protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found to be associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. A cDNA that encodes protein disulfide isomerase was previously isolated from Bombyx mori (bPDI), in which open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal, and we report its functional characterization here. This putative bPDI cDNA is expressed in insect Sf9 cells as a recombinant proteins using baculovirus expression vector system. The bPDI recombinant proteins are successfully recognized by antirat PDI antibody, and shown to be biologically active in vitro by mediating the oxidative refolding of reduced and scrambled RNase. This suggests that bPDI may play an important role in protein folding mechanism of insects.

• Journal of Sericultural and Entomological Science
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• v.35 no.2
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• pp.129-133
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• 1993

The 44Md plasmid of Bacillus thruingiensis var. kurstaki HD-1(B. t k HD-1) was partially digested with Sau3AI and the fragments were cloned into E. coli HB101 on vector pBR322. Of 2, 950 clones with a recombinant pBR322, only one clone KC1 was determined to have the gene for crystal toxic proteins from the 44Md plasmid of B. t k HD-1 at the BamHI site of pBR322. The recombinant pBR322 was named pKC1 and its molecular size was 12kb. The KC1 produced a protein which was toxic to the silkworm and antigenically similar to the crystal toxic protein of B. t k HD-1. Also, electrophoretic mobility of the KC1 protein was apparently the same as that of the crystal toxic protein of B. t k HD-1.

• Journal of Life Science
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• v.9 no.5
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• pp.556-563
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• 1999

Presence of antibody to the capsid protein p24 is the main diagnostic criterion, since this reflects reliable antibody response to HIV infection. However, it takes about 6-8 weeks for antibody production after infection and people who are infected but antibodies are not produced yet are classified as seronegative. Therefore, there is a strong need for an improved diagnostic method for better health security. As a first step for developing such an improved diagnostic system, gag protein of human immunodificiency virus type 1 was expressed in E. coli DH5$\alpha$. The gag fragment of HIV-1 (including a portion of p17 and whole p24) was amplified by polymerase chain reaction (PCR) and BamH I/EcoR I sites were created during PCR. The amplified DNA fragment was cleaved with BamH I/EcoR I and was subcloned into the GEX-2T vector which had been digested with BamHI/EcoRI, resulting gene fusion with gst gene of pGEX-2T. The recombinant DNA was transferred into E. coli DH5$\alpha$. The transformed bacteria were grown at 37$^{\circ}C$ for 3h and protein expression was induced with 0.1mM IPTG at $25^{\circ}C$ for 3h. Recombinant gag protein or GST-gag fusion protein was purified with glutathione-sepharose 4B bead and migrated as a single band when analyzed by 10% polyacrylamide gel. These proteins were confirmed by immunoblotting with anti-GST goat sera or Korean AIDS patients sera. The results of this study establish the expression and single step pulification of HIV-1 gag protein which can specifically bind with Korean AIDS patients sera.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.215-221
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• 1991

An autonomous replication sequence (ARS) derived from Cephalosporium acremonium ATCC 20339 was cloned in Sarchuromyces cerevisiae SHY 3 using YIp5 as a cloning vector. A new recombinant plasmid, designated pCY-2, which contained a 3.7 kb BamHI fragment of C. acrenzonium DNA showed the highest stability among the 40 recombinant plasmids composed of the YIp5 2nd ARS of C. ucremoniztm. Also, Southern hybridization and transformation of E, cull with DNA purified from yeast transformants verified that pCY-2 autonomously replicates in yeasts. Transformation efficiency and plasmid stability of pCY-2 in yeast were higher than those ol YRp 7 containing ARS which originated from yeast. Detailed studies by subcloning revealed that two ARSs existed within 2.6 kb of the insert, which is a novel discovery. However, it was concluded that these two ARSs were ligated during the gene manipulation in vitro.

• Journal of Life Science
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• v.21 no.1
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• pp.31-35
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• 2011

Dual-specificity protein phosphatases (DUSPs) constitute a family of protein phosphatase characterized by the ability to dephosphorylate phospho-tyrosyl and phospho-seryl/threonyl residues. Most DUSPs are involved in regulation of cell survival and differentiation. In this study, a human dual-specificity protein phosphatase, DUSP28, was isolated from a human kidney cDNA. The recombinant protein was successfully produed in E.coli and showed sufficient phosphatase activity toward DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate). Various phosphatase inhibitors and divalent metals were tested for their effects on the DUSP28 phosphatase activity. As a result, $Zn^{2+}$ was found to strongly inhibit DUSP28 phosphatase activity, suggesting DUSP28 is involved in Zn-related signal transduction pathway. Furthermore, the DUSP28 protein preferred phospho-tyrosyl residues to phospho-threonyl residues, implying its physiological roles in the cellular process.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.57-66
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• 1987

Mutants of E. coli which showed increased sensitivity to MMS(methylmethane sulfonate)were isolated by MNNG mutagenesis and characterized by enzymatic assay, survival of simple alkylating agents and host-cell reactivation. E.coli mutant, 5-62, which showed absolute deficiency in 3-methyladenine DNA glycosylase II activity and had low capability of reactivating MMS-treated phage charon 35 was very sensitive to MMS and MNNG. NNS gene which confered resistance to the lethal effects of MMS was cloned in 5-62 strain. 5-62 mutants carrying recombinant plasmid, pMRG 1, which acquired resistance to the lethal effects of MMS had normal sensitivity to MNNG. Resistance to MMS was somewhat increased after they were treated with 0.5.$\mu$g MNNG/ml for 2 hours at $37^{\circ}C$. Although recombinant plasmid, pMRG 1, did not complement alk A mutation in 5-62 and ada mutation in 1-27 mutnat, mutnats transformed with this plasmid showed more capability of reactivating MMS treated phage than mutants.

• Journal of Microbiology
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• v.44 no.1
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.