• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.29 no.4
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• pp.206-211
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• 2003

The pleomorphic adenoma is the most common neoplasm involving both the major and minor salivary glands. It is a benign, slowgrowing tumor, but local recurrences can occur. The pleomorphic adenoma gene 1 (PLAG1), which is a novel zinc finger gene, is frequently activated by reciprocal chromosomal translocations involving 8q12 in a subset of salivary gland pleomorphic adenomas. This experimental study was preformed to observe the translocation patterns between PLAG1 gene and the three translocation partner genes. We also have analyzed the presence of PLAG1 transcripts by RT-PCR. CTNNB1/PLAG1 gene fusion was observed in three of nine pleomorphic adnomas. However, LIFR/PLAG1 and SII/PLAG1 gene fusions were not detectable. All of three gene fusions was not detectable in one Warthin's tumor and three inflammatory salivary gland tissues. PLAG1 transcripts were expressed in all inflammatory salivary gland tissues and tumors except for three pleomorphic adenomas. Of particular one pleomorphic adenoma showing CTNNB1/PLAG1 gene fusion did not express PLAG1 transcipt. Our data indicate that gene fusion involving PLAG1 is a frequent event in pleomorphic adenoma, but correlation between gene fusion involving PLAG1 and PLAG1 transcription is not definite.

• KSBB Journal
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• v.23 no.3
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• pp.205-212
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• 2008

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and peri plasm region. Three fusion partners for cytoplasmic expression such as acidic tail of synuclein (ATS), thioredoxin (Trx) and lipase, and three fusion partners for periplasmic expression such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein (MBP) were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, yielding about 18.1 mg/L and three-fold higher level compared to that of insoluble non-fusion hEGF in cytoplasm. Thus, heterologous proteins containing complex disulfide bond and many hydrophobic amino acids can effectively be produced as an active form in E. coli by introducing a suitable peptide or protein.

• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Bulletin of the Korean Chemical Society
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• v.29 no.2
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• pp.381-388
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• 2008

An X-linked skeletal disorder, SEDT (spondyloepiphyseal dysplasia tarda) is a genetic disease characterized by a disproportionately short trunk and short stature caused by mutations in the SEDL gene. This gene is evolutionarily conserved from yeast to human. The yeast SEDL protein ortholog, Trs20p, has been isolated as a member of a large multi-protein complex called the transport protein particle (TRAPP), which is involved in endoplasmic reticulum (ER)-to-Golgi transport. The interaction between SEDL and partner proteins is important in order to understand the molecular mechanism of SEDL functions. We isolated several SEDL-binding proteins derived from rat cells by an immobilized GST-fusion method. Furthermore, the SEDL-homologue proteins were identified using motif based methods. Common motifs between SEDL-binding proteins and SEDL-homologue proteins were classified into seven types and 78 common motifs were revealed. Sequence similarities were contracted to seven types using phylogenetic trees. In general, types I-III and VI were classified as having the function of acetyl-CoA carboxylase, glycogen phosphorylase, isocitrate dehydrogenase, and enolase, respectively, and type IV was found to be functionally related to the GST protein. Types V and VII were found to contribute to TRAPP vesicle trafficking.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.608-609
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• 2005

• Journal of Mushroom
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• v.2 no.3
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• pp.115-126
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• 2004

Somatic hybrids of inter-compatible and inter-incompatible strains were obtained by protoplast fusion. The fusion products between compatible strains, Pleurotus ostreatus and P. florida, formed heterokaryons, while fusants between incompatible strains such as P. cornucopiae + P. florida, P. ostreatus + Ganoderma applanatum, P. florida + Ganoderma lucidum, and P. ostreatus + Flammulina velutipes formed synkaryons that retained genes from both parents. The heterokaryons showed the same level of basidioma development. In contrast, the synkaryons showed unique characteristics including clamp connection formation at mitosis, either partner basidioma development, and abnormal segregation and recombination compared with inter-compatible strains. Synkaryons can be classified into homokaryoyic and heterokaryotic type. A comparison of somatic hybrids with compatible and incompatible strains was made using random amplified polymorphic DNA (RAPD) analysis. The heterokaryons between compatible species showed the same level of variability and contained both parental RAPD bands. In contrast, most of the synkaryons between incompatible species showed similarity to those of either parental bands and non-parental RAPD bands. Synkaryons can be classified into microgenome insertion type and macrogenome insertion type. A tetrapolar mating system was found among monospore isolates in somatic hybrids and wild type P. ostreatus. Homokaryons from each somatic hybrid combination were paired with tester homokaryons of the initial wild type of P. ostreatus. The changed mating types were identified in progenies. The pattern of mating type switching in somatic hybrids depends on compatibility of fusion partner. There are several factors related to the mechanism of clamp connection formation and fruiting body development of synkaryons. Of these,the major factor may be associated with self-fertility and mating type switching such as homokaryotic fruiting of wild type P. ostreatus. This review will discuss these aspects.

• Journal of Life Science
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• v.19 no.5
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• pp.561-565
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• 2009

Human Tissue factor is an essential enzyme activator that forms a catalytic complex with factor VII/ VIIa, and catalyzes both the extrinsic and intrinsic blood coagulation cascades. The extracellular domain of human tissue factor is responsible for association with the biological partner. The efficient procedures for preparing biologically active human tissue factor are essential for the preclinical and clinical studies with coaguligands. An expression vector in Escherichia coli has been constructed to direct the production of extracellular human tissue factor without a fusion protein or a $His_6$ at the N-terminus. The recombinant human tissue factor was expressed in large amounts as a non-native state in E. coli. The recombinant protein was simply renatured during the DEAE-sephacel chromatographic purification procedure. Our expression and purification system does not require a protease treatment or an additional chromatographic step to remove a fusion contaminant, which provides a very useful alternative to conventional expression systems for the production of human tissue factor.

• KSBB Journal
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• v.15 no.3
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• pp.238-242
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• 2000

A fusion protein of human interleukin-2(hiL-2) and glucagon which was expressed in Escherichia coli. was digested with enterokinase for recovery of hIL-2 from the fused protein. To obtain hIL-2 of optimum recovery hydrolysis reaction were performed under various conditions of urea additives and reaction time. hIL-2 was finally purified by RP-HPLC(reversed phase-HPLC) to remove cleaved G3 fusion partner and residual uncleaved G3-IL-2 HIL-2 was eluted in a single peak at 100% acetonitrile at 28 min. Optimum urea concentration was found to be 0.5 M and 24 h reaction time was sufficient without any additive such as CaCl2 and Tween-20.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.179-184
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• 2007

PTK6, an intracellular protein tyrosine kinase, is significantly overexpressed in a majority of breast cancers and has a role in promoting the proliferation of the cancer cells, but not of normal cells. Here, we report high-level production of the catalytic unit of PTK6 fused with Drosophila peptidoglycan recognition protein (PGRT)-LB, in the baculovirus system. We first found that the PGRP-LB was potentially useful as a fusion partner to increase the yield of heterologous protein in the baculovirus system. The purified recombinant protein exhibited a 1.5-fold activity with much higher yield than the bacterially-expressed protein. The protein expressed in the baculovirus system will be useful for the crystallization to determine its crystal structure helping understand the molecular mechanism of PTK6 and design its inhibitors.