• Journal of Life Science
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• v.27 no.11
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• pp.1276-1289
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• 2017

This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.140-147
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• 2010

Japanese encephalitis virus (JEV), a member of the mosquito-borne flaviviruses, causes epidemics of viral encephalitis in the Southeastern Asia. JEV is a small enveloped virus with a positive-sense RNA genome; the infectious virion consists of three structural proteins, namely capsid, membrane (M; a mature form of its prM precursor), and envelope proteins. Here, we investigated a role of the charged residues found at the N-terminus of the JEV M protein in virus production. Using an infectious JEV cDNA, we generated two mutant cDNAs, Mm1 and Mm2, by charged-to-alanine substitution for $E^9$ and $K^{15}K^{16}E^{17}$ residues of the M protein, respectively. By transfection of wild-type or each of the two mutant RNAs transcribed from the corresponding cDNAs, we found that Mm2, but not Mm1, had a ~3-log decrease in virus production, even though a comparable amount of all three structural proteins were produced in transfected cells. Interestingly, the prM protein expressed in Mm2 RNA-transfected cells was not recognized by the polyclonal antiserum raised against the N-terminal 44 amino acids of the wild type M protein, but reacted to the antiserum raised against the corresponding region of the mutant Mm2. Our results indicate that three charged residues ($K^{15}K^{16}E^{17}$) in JEV M protein play a role in virus production. Two polyclonal antisera specifically recognizing the wild-type or Mm2 version of the M protein would provide a useful reagent for the functional study of this protein in the virus life cycle.

• KSBB Journal
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• v.21 no.3
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• pp.204-211
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• 2006

For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

• Restorative Dentistry and Endodontics
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• v.41 no.2
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• pp.91-97
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• 2016

Objectives: The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human ${\beta}$-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm. Materials and Methods: C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and $300{\mu}g/mL$), CH ($100{\mu}g/mL$), and Nys ($20{\mu}g/mL$) for 7 days at $37^{\circ}C$. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05. Results: C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (${\geq}100{\mu}g/mL$). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (${\geq}100{\mu}g/mL$) showed significant fungicidal activity compared to CH group (p < 0.05). Conclusions: Synthetic HBD3-C15 peptide (${\geq}100{\mu}g/mL$) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm.

• Journal of Life Science
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• v.33 no.11
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• pp.868-875
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• 2023

Kinesin-1 is a motor protein identified as the first member of the kinesin superfamily (KIF), which plays a role in intracellular cargo transport by acting as microtubule-dependent motor proteins within cells. Kinesin-1 consists of two heavy chains (KHCs, also known as KIF5s) and two light chains (KLCs). The 93 amino acids in the carboxyl (C)-terminal tail region of KIF5A are not homologous to the C-terminal tail region of KIF5B or the C-terminal tail region of KIF5C. In this study, we used a yeast two-hybrid screen to identify the binding proteins that interacted with the C-terminal region of KIF5A. We found an association between KIF5A and CUE domain containing 2 (CUEDC2), which is proposed to function as an adaptor protein involved in ubiquitination pathways and protein trafficking. CUEDC2 bound to the C-terminal region of KIF5A and did not interact with KIF5B (the motor of kinesin-1), KIF3A (the motor of kinesin-2), or kinesin light chain 1 (KLC1). KIF5A specifically bound to the C-terminal region of CUEDC2. Furthermore, KIF5A did not interact with another isoform: CUEDC1. In addition, glutathione S-transferase (GST) pull-downs showed that KIF5A directly bound GST-CUEDC2 but did not interact with GST-CUEDC1 and GST alone. When myc-KIF5A and EGFP-CUEDC2 were co-expressed in HEK-293T cells, CUEDC2 co-immunoprecipitated with kinesin-1, and myc-KIF5A and FLAG-CUEDC2 colocalized in the cells. These results suggest that in intracellular cargo transport by kinesin-1, CUEDC2 serves as an adaptor protein connecting kinesin-1 and cargo by binding to KIF5A.