• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• Biomolecules & Therapeutics
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• v.21 no.2
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• pp.126-131
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• 2013

Neuropathic pain is a chronic pain disorder caused by nervous system lesions as a direct consequence of a lesion or by disease of the portions of the nervous system that normally signal pain. The spinal nerve ligation (SNL) model in rats that reflect some components of clinical pain have played a crucial role in the understanding of neuropathic pain. To investigate the direct effects of gabapentin on differential gene expression in cultured dorsal root ganglion (DRG) cells of SNL model rats, we performed a differential display reverse transcription-polymerase chain reaction analysis with random priming approach using annealing control primer. Genes encoding metallothionein 1a, transforming growth factor-${\beta}1$ and palmitoyl-protein thioesterase-2 were up-regulated in gabapentin-treated DRG cells of SNL model rats. The functional roles of these differentially expressed genes were previously suggested as neuroprotective genes. Further study of these genes is expected to reveal potential targets of gabapentin.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.776-780
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• 2004

Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation. The interferon alpha gene from human blood samples was amplified, cloned and expressed in E. coli (BL21). Leukocyte chromosomal DNA was used as a source of template DNA. Using specific primers, the gene for HulFN$\{alpha}$-2b was amplified and inserted into the E. coli vector, pET21b, by ligation of the HindIII and BamHI linkers of the vector and insert. The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E. coli strain. The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins.

• IMMUNE NETWORK
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• v.6 no.2
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• pp.59-66
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• 2006

Background: CM1 (Centrocyte/-blast Marker I) defined by a mAb developed against concanavalin-A activated PBMC, is expressed specifically on a subpopulation of centroblasts and centrocytes of human germinal center (GC) B cells. Burkitt lymphoma (BL) is a tumor consisting of tumor cells with the characteristics of GC B cell. Previously we reported that CM1 ligation with anti-CM1 mAb induced apoptosis in Ramos $(IgM^{high})$ and Raji $(IgM^{low})$ cells. Methods & Results: In the present study, we observed that CM1 ligation with anti-CM1 mAb induced Fas ligand and Fas expression in Ramos cells, but not in Raji cells. Furthermore, anti-Fas blocking antibody, ZB4, blocked CM1-mediated apoptosis effectively in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization, which was measured by $DiOC_6$, was observed only in Raji cells. In contrast to no significant change of Bax known as pro-apoptotic protein, anti-apoptotic protein Bcl-2 was significantly decreased in Raji cells. In addition, we observed that CM1 ligation increased release of mitochondrial cytochrome c and upregulated caspase-9 activity in Raji cells. Conclusion: These results suggest that apoptosis induced by CM1-ligation is mediated by Fas-Fas ligand interaction in Ramos cells, whereas apoptosis is mediated by down-regulation of Bcl-2 and subsequent decrease of mitochondrial membrane potential in Raji cells.

• Journal of Veterinary Clinics
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• v.25 no.3
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• pp.152-158
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• 2008

We investigated the changes of protein in chronic MI which was occurred with long-term ischemia, without reperfusion. Sprague Dawley (SD) rats were divided into the sham group and the experimental groups (MI groups). The sham group was treated only thoracotomy without ligation for left main descending artery (LMDA) of left coronary artery (LCA), and the experimental groups (MI7d, ligation of LMDA for 7 days and MI30d, ligation of LMDA for 30 days) were conducted an artificial chronic MI. The change of proteins according to passage of times was compared and analyzed on first and second dimension (1 and 2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Among total 46 spots expressed differentially in the sham group versus MI7d and MI30d groups on 2D gel, we selected proteins that the volume of spot was increased in the MI7d and MI30d groups compared with the sham group. After that, the proteins were identified through liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. In result, we could obtain many proteins as follows; albumin, glucose regulated protein 58 KDa, similar to tripartite motif protein 50, ubiquinol-cytochrome c reductase core protein II, sarcomeric mitochondrial creatine kinase, ATP synthetase alpha chain (mitochondrial precursor) and creatine kinase. In conclusion, we suggest many changed proteins shown at chronic ischemia after artificial MI and consider that these proteins play an important role in the function of heart after MI.

• Korean Circulation Journal
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• v.53 no.3
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• pp.151-167
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• 2023

Background and Objectives: Acute myocardial infarction (AMI) often occurs suddenly and leads to fatal consequences. Ferroptosis is closely related to the progression of AMI. However, the specific mechanism of ferroptosis in AMI remains unclear. Methods: We constructed a cell model of AMI using AC16 cells under oxygen and glucose deprivation (OGD) conditions and a mice model of AMI using the left anterior descending (LAD) ligation. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide was employed to determine cell viability. The levels of lactate dehydrogenase, creatine kinase, reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and iron were measured using corresponding kits. Dual luciferase reporter gene assay, RNA-binding protein immunoprecipitation, and RNA pull-down were performed to validate the correlations among AC005332.7, miR-331-3p, and cyclin D2 (CCND2). Hematoxylin and eosin staining was employed to evaluate myocardial damage. Results: AC005332.7 and CCND2 were lowly expressed, while miR-331-3p was highly expressed in vivo and in vitro models of AMI. AC005332.7 sufficiency reduced ROS, MDA, iron, and ACSL4 while boosting the GSH and GPX4, indicating that AC005332.7 sufficiency impeded ferroptosis to improve cardiomyocyte injury in AMI. Mechanistically, AC005332.7 interacted with miR-331-3p, and miR-331-3p targeted CCND2. Additionally, miR-331-3p overexpression or CCND2 depletion abolished the suppressive impact of AC005332.7 on ferroptosis in OGD-induced AC16 cells. Moreover, AC005332.7 overexpression suppressed ferroptosis in mice models of AMI. Conclusions: AC005332.7 suppressed ferroptosis in OGD-induced AC16 cells and LAD ligation-operated mice through modulating miR-331-3p/CCND2 axis, thereby mitigating the cardiomyocyte injury in AMI, which proposed novel targets for AMI treatment.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.2
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• pp.69-75
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• 2005

TRPM7, a cation channel protein permeable to various metal ions such as $Mg^{2+}$, is ubiquitously expressed in variety of cells including lymphocytes. The activity of TRPM7 is tightly regulated by intracellular $Mg^{2+}$, thus named $Mg^{2+}$-inhibited cation (MIC) current, and its expression is known to be critical for the viability and proliferation of B lymphocytes. In this study, the level of MIC current was compared between immature (WEHI-231) and mature (Bal-17) B lymphocytes. In both cell types, an intracellular dialysis with $Mg^{2+}$-free solution (140 mM CsCl) induced an outwardly-rectifying MIC current. The peak amplitude of MIC current and the permeability to divalent cation ($Mn^{2+}$) were several fold higher in Bal-17 than WEHI-231. Also, the level of mRNAs for TRPM7, a molecular correspondence of the MIC channel, was significantly higher in Bal-17 cells. The amplitude of MIC was further increased, and the relation between current and voltage became linear under divalent cation-free conditions, demonstrating typical properties of the TRPM7. The stimulation of B cell receptors (BCR) by ligation with antibodies did not change the amplitude of MIC current. Also, increase of extracellular $[Mg^{2+}]_c$ to enhance the $Mg^{2+}$ influx did not affect the BCR ligation-induced death of WEHI-231 cells. Although the level of TRPM7 was not directly related with the cell death of immature B cells, the remarkable difference of TRPM7 might indicate a fundamental change in the permeability to divalent cations during the development of B cells.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.1
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• pp.57-62
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• 2012

Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8), a ubiquitin-like protein, mainly functions through covalent ligation to cullin proteins. Conjugation of NEDD8 with cullins can promote ubiquitination, which plays a critical role in the degradation of many proteins. UBA3 is the subunit of NEDD8-activating enzyme which is one of the keys for NEDD8 linkage to cullin proteins. Previous research showed NEDD8 conjugation to be up-regulated in highly proliferative cell lines. In the present study, up-regulated NEDD8 conjugation was observed in melanoma cell lines by Western blot analysis. After down-regulation with a RNAi to UBA3, proliferation of M14 was suppressed in vitro and in vivo. In conclusion, up-regulated NEDD8 conjugation may be involved in the development of melanoma. Interference in this pathway might offera promising method for melanoma therapy.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.211-218
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• 2023

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Em^r), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

• Biomedical Science Letters
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• v.6 no.1
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• pp.73-82
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• 2000

Blunt-end DNA fragments can be inserted in two different orientations. Conventionally, their directions are determined by restriction enzyme digestion or by DNA sequencing, however, these methods are often limited in their use due to the lack of appropriate enzyme sites or large sample numbers, respectively. In the present study, a novel strategy and the corresponding protocol for the simple determination of insert orientation is introduced. Using conventional sequencing primers and PCR primers that have been used for amplification of the insert, single clones, which have inserted the fragment in the desired orientation, were easily identified by this PCR-based method. The fidelity of this system was confirmed by cloning of a tar urocortin cDNA, which is a recently discovered neuropeptide. Recombinant clones identified by this method were further shown to be fully functional, and using these, for the first time, urocortin was recombinantly expressed in eukaryotic cells.