• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.3-10
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• 1996

• International Journal of Industrial Entomology and Biomaterials
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• v.16 no.2
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• pp.87-92
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• 2008

Bombyx mori nucleopolyhedrovirus (BmNPV) orf47 gene was characterized for the first time. The coding sequence of Bm47 was amplified and subcloned into the prokaryotic expression vector pET-30a(+) in order to produce His-tagged fusion protein in the BL21 (DE3) cells. The His-Bm47 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. As the genome of BmNPV is available in GenBank and the EST database of BmNPV is expanding, identification of novel genes of BmNPV was conceivable by data-mining techniques and bioinformatics tools. Structural bioinformatics approach to analyze the properties of Bm47 encodes protein.

• International Journal of Industrial Entomology and Biomaterials
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• v.14 no.2
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• pp.69-74
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• 2007

As the genome of B. mori is available in GenBank and the EST database of B. mori is expanding, identification of novel genes of B. mori was conceivable by datamining techniques and bioinformatics tools. In this study, we used the in silico cloning method to get the 6-Phosphogluconolactonase (6PGL) gene of B. mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and prokaryotic expression. The 6PGL cDNA comtains a 702 bp ORF. The deduced protein has 233 amino acid residues, with the predicted molecular weight of 25946. 72 Da, isoelectric point of 5.41, and contains conserved NagB domains. This gene has been registered in GenBank under the accession number EF198104.

• BMB Reports
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• v.39 no.1
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• pp.22-25
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• 2006

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.827-831
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• 2007

Myostatin is a member of the transforming growth factor-${\beta}$(TGF-${\beta}$ super-family. It acts as a negative regulator for skeletal muscle growth. Myostatin mutations are characterized by a visible, generalized increase in muscle mass in double muscled cattle breeds. To understand the biochemistry and physiology of the Chinese Yellow bovine myostatin gene, we report here for the first time expression of the gene in Escherichia coli (E. coli). Primers of the myostatin gene of Chinese Yellow Cattle were designed on the basis of the reported bovine myostatin mRNA sequence (Gen-Bank Accession No. NM005259) and optimized for E. coli codon usage. XhoI and EcoRI restriction enzyme sites were incorporated in the primers, and then cloning vector and expression vector were constructed in a different host bacterium. The expressed protein had a molecule mass of about 16 kDa as determined by SDS-PAGE under reducing conditions. The expressed protein reacted specifically with myostatin monoclonal antibody on immunoblots. Our studies should lead to the investigation of the differences in myostatin genes of various cattle and could benefit human health and food animal agriculture.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.3
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• pp.77-85
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• 2020

Stable isotope enrichment in proteins is necessary for high-resolution nuclear magnetic resonance (NMR) experiments. Although methods for ¹³C, ¹⁵N and ²H-enrichment in prokaryotic cells are well established, full processing and correct folding of complex protein systems require higher organisms as the expression host. In the present study, we review recent efforts to enrich stable isotopes in mammalian cells for protein NMR studies.

• BMB Reports
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• v.44 no.4
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• pp.279-284
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• 2011

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.314-322
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• 2018

Bacillus subtilis 8 is highly efficient at degrading feather keratin. We observed integrated feather degradation over the course of 48 h in basic culture medium while studying the entire process with scanning electron microscopy. Large amounts of ammonia, sulfite, and $\text\tiny{L}$-cysteic acid were detected in the fermented liquid. In addition, four enzymes (gamma-glutamyltranspeptidase, peptidase T, serine protease, and cystathionine gamma-synthase) were identified that play an important role in this degradation pathway, all of which were verified with molecular cloning and prokaryotic expression. To the best of our knowledge, this report is the first to demonstrate that cystathionine gamma-synthase secreted by B. subtilis 8 is involved in the decomposition of feather keratin. This study provides new data characterizing the molecular mechanism of feather degradation by bacteria, as well as potential guidance for future industrial utilization of waste keratin.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Korean Journal of Biological Psychiatry
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• v.8 no.2
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• pp.203-207
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• 2001

The genome sequencing project has generated and will continue to generate enormous amounts of sequence data including 5 eukaryotic and about 60 prokaryotic genomes. Given this ever-increasing amounts of sequence information, new strategies are necessary to efficiently pursue the next phase of the genome project-the elucidation of gene expression patterns and gene product function on a whole genome scale. In order to assign functional information to the genome sequence, DNA chip(or gene microarray) technology was developed to efficiently identify the differential expression pattern of independent biological samples. DNA chip provides a new tool for genome expression analysis that may revolutionize many aspects of biotechnology including new drug discovery and disease diagnostics.