• KSBB Journal
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• 제30권6호
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• pp.291-295
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• 2015

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.

• Journal of Microbiology and Biotechnology
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• 제33권11호
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• pp.1513-1520
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• 2023

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P₁', P₂', P₄, and P₃ sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.

• BMB Reports
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• 제43권8호
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• pp.561-566
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• 2010

Though protein transduction domains (PTDs) are well known for the delivery of exogenous therapeutic proteins into living cells, the overall low efficiency of transduction is a serious obstacle. We investigated the effect of bog blueberry anthocyanins (BBA) on protein transduction efficiency and found that BBA enhanced the transduction efficiencies of Tat-SOD fusion protein into HeLa cells and mice skin. The enzymatic activities in the cells and skin tissue in the presence of BBA were markedly increased compared to controls. Further, BBA did not demonstrate any cell toxicity at various concentrations. Although the mechanism is not fully understood, we suggest that BBA might alter the conformation of the membrane, which would indicate that BBA can be used as a protein transduction enhancer for the efficient delivery of therapeutic proteins for a variety of disorders.

• 한국동물학회지
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• 제30권4호
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• pp.410-416
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• 1987

The effect of heavy metal ions on the synthesis of proteins in cultured chick embryonic muscle cells were examined by labeling the cellular proteins with 35S-methionine and the surface proteins with Nalssl and lactoperokidase. The protein pattern in the cells cultured for 48 hrs showed little or no difference whether or not the cells were treated with any of the metal ions including Cu2+, Cd2+ and Hg2+, which are known to block the fusion of mypblasts. However, a 43kd protein disappeared from the control cells cultured for 72 hrs but remained unchanged in the cells treated with the metal ions. When analyzed for the syntheiic pattern of membrane proteins, addition of the ions (particularly of Cda+ and Cr3+) caused a marked increase in the level of 66kd protein, as compared to that in the untreated cells. By contrast, the level of 29kd protein was much higher in the control cells than in the cells treated with the metal ions. These results suggest that the heavy metal ions appear to block the degradation of 43kd soluble protein and 66kd membrane protein, perhaps by inhibiting a metalloprotease, which may be essential for the myogenic process of embryonic muscle cells.

• Molecules and Cells
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• 제42권12호
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• pp.893-905
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• 2019

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion processes that closely related to their function. Disruption of mitochondrial dynamics has been demonstrated in acute kidney injury (AKI), which could eventually result in cell injury and death. Previously, we reported that augmenter of liver regeneration (ALR) alleviates renal tubular epithelial cell injury. Here, we gained further insights into whether the renoprotective roles of ALR are associated with mitochondrial dynamics. Changes in mitochondrial dynamics were examined in experimental models of renal ischemia-reperfusion (IR). In a model of hypoxia-reoxygenation (HR) injury in vitro, dynamin-related protein 1 (Drp1) and mitochondrial fission process protein 1 (MTFP1), two key proteins of mitochondrial fission, were downregulated in the Lv-ALR + HR group. ALR overexpression additionally had an impact on phosphorylation of Drp1 Ser637 during AKI. The inner membrane fusion protein, Optic Atrophy 1 (OPA1), was significantly increased whereas levels of outer membrane fusion proteins Mitofusin-1 and -2 (Mfn1, Mfn2) were not affected in the Lv-ALR + HR group, compared with the control group. Furthermore, the mTOR/4E-BP1 signaling pathway was highly activated in the Lv-ALR + HR group. ALR overexpression led to suppression of HR-induced apoptosis. Our collective findings indicate that ALR gene transfection alleviates mitochondrial injury, possibly through inhibiting fission and promoting fusion of the mitochondrial inner membrane, both of which contribute to reduction of HK-2 cell apoptosis. Additionally, fission processes are potentially mediated by promoting tubular cell survival through activating the mTOR/4E-BP1 signaling pathway.

• Bulletin of the Korean Chemical Society
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• 제30권9호
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• pp.1981-1984
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• 2009

Subcellular localization of protein kinase often plays an important role in determining its activity and specificity. Protein kinase C (PKC), a family of multi-gene protein kinases has long been known to be translocated to the particular cellular compartments in response to DAG or its analog phorbol esters. We used C-terminal green fluorescent protein (GFP) fusion proteins of PKC isoforms to visualize the subcellular distribution of individual PKC isoforms. Intracellular localization of PKC-GFP proteins was monitored by fluorescence microscopy after transient transfection of PKC-GFP expression vectors in the HeLa cells. In unstimulated HeLa cells, all PKC isoforms were found to be distributed throughout the cytoplasm with a few exceptions. PKC$\theta$ was mostly localized to the Golgi, and PKC$\gamma$, PKC$\delta$ and PKC$\eta$ showed cytoplasmic distribution with Golgi localization. DAG analog TPA induced translocation of PKC-GFP to the plasma membrane. PKC$\alpha$, PKC$\eta$ and PKC$\theta$ were also localized to the Golgi in response to TPA. Only PKC$\delta$ was found to be associated with the nuclear membrane after transient TPA treatment. These results suggest that specific PKC isoforms are translocated to different intracellular sites and exhibit distinct biological effects.

• BMB Reports
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• 제39권4호
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• pp.412-417
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• 2006

We examined the intracellular localization of NS5 protein of Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV) by expressing NS5-GFP fusion protein and proteins from deletion mutants of NS5 in baculovirus recombinant infected insect Spodoptera frugiperda (Sf-9) cells. It was found that the NS5 protein was present at the plasma membrane of the cells, and that the N-terminal portion of the protein played a key role in the localization. A transmembrane region was identified to be present in the N-terminal portion of the protein, and the detailed transmembrane domain (SQIHMVWVKSGLVFF, 57-71aa) of N-terminal portion of NS5 was further determined, which was accorded with the predicted results, these findings suggested that NS5 might have an important function in viral life cycle.

• BMB Reports
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• 제41권2호
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• pp.170-175
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• 2008

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.

• 미생물학회지
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• 제30권5호
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• pp.333-338
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• 1992

Saccharomyces cerevisiae 의 mother cell과 daughter cell 의 연결 부위의 원형질막 바로 안쪽에 존재하는 10-nm filament ring 은 세포형태 형성과정에 중요한 역할을 하리라 간주되나 그 명확한 생성기작과 기능은 밝혀지지 않았다. 본연구에서는 CDC12 유전자로부터 gene fusion technique 을 이용하여 CDC12 단백질을 만들고 이로부터 항체를 형성하였다. 이항체를 이용하여 10-nm filament ring 의 생성기작과 기능에 대하여 연구하였다. 그 결과 CDC12 단백질은 cell cycle 전주기동안 항상 정이되나 bud 가 나오기 바고 직전에 bud 가 나올 부위에 polymerization 되었다가 세포질분열 바로 직후에 unpolymerization 되며 cytoskeletal element 의 일종인 actin 과는 무관하게 행동하는 건이 밝혀졌다. 이러한 10-nm filament 는 bud 가 나올 부위의 올바른 선정과 세포질 분열에 중요한 역할을 하리라 간주된다.

• 대한바이러스학회지
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• 제27권2호
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• pp.197-207
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• 1997

To investigate the NS4 region of JEV, NS4 cDNA of K94P05 (JEV strain isolated from Korea in 1994) was amplified by RT-PCR and analyzed by sequencing PCR product. Genomic size of NS4 was 1212bp and nucleotide sequence was compared with that of other JEV strains. Nucleotide homology between JaOAr582 and K94P05 was 91.1% and that between Beijing and K94P05 was 89.8%, respectively. But the nucleotide sequence of E region of JaOAr582 and K94P05 showed 97.0% homology and that of Beijing and K94P05 did 95.8% homology. NS4 protein was expressed as a form of fusion protein by a prokaryotic expression system. The induced fusion product showed a lower molecular weight than predicted size and remained insoluble. The NS4 protein might be cleavaged by E. coli protease. Concluding above results, high hydrophobicity of the NS4 protein supported the fact that this protein played a role as a membrane component and the poor nucleotide sequence conservativity among JEV strains suggested that this region might be important to adapt each viral growth environment.