• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.13-16
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• 2000

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed in Escberichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced in E. coli cytoplasm were easily dissolved by simple alkaline pH shift $(8\rightarrow12\rightarrow8)$. Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.

• Bulletin of the Korean Chemical Society
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• v.28 no.10
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• pp.1756-1762
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• 2007

Envelope proteins of virus contain a segment of hydrophobic amino acids, called as fusion peptide, which triggers membrane fusion by insertion into membrane and perturbation of lipid bilayer structure. Potential fusion peptide sequences have been identified in the middle of L or M proteins or at the N-terminus of S protein in the envelope of human hepatitis B virus (HBV). Two 16-mer peptides representing the N-terminal fusion peptide of the S protein and the internal fusion peptide in L protein were synthesized, and their membrane disrupting activities were characterized. The internal fusion peptide in L protein showed higher activity of liposome leakage and hemolysis of human red blood cells than the N-terminal fusion peptide of S protein. Also, the membrane disrupting activity of the extracellular domain of L protein significantly increased when the internal fusion peptide region was exposed to N-terminus by the treatment of V8 protease. These results indicate that the internal fusion peptide region of L protein could activate membrane fusion when it is exposed by proteolysis.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.58-58
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• 2003

The envelope glycoprotein of HIV, gp41, mediates the membrane fusion with human cells. The extracellular domain of gp41 has two helical regions. The N-terminus helical region (N-helix) forms trimeric coiled coil, interacts with the C-terminus helical region (C-helix) of gp41 to form a stable helical bundle structure. In this study, we have shown that the N-helix of gp41 has membrane interacting and disrupting abilities. It was localized into the interface of the lipidic phase and head group of the membrane. In contrast, the N-helix region with membrane fusion defective mutations could not bind to membrane. In addition, the N-helix bound on the membrane was released from the membrane by the C-helix, and the complex of the N- and C-helix did not interact with membrane. These results suggested that the membrane binding ability of the N-helix is necessary for the fusion activity of gp41, and such property is possibly controlled by the C-helm.

• Proceedings of the Microbiological Society of Korea Conference
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• 2001.11a
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• pp.97-101
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• 2001

The fusion between viral envelope and target cell membrane is a central step of viral infection, and the fusion proteins located at viral envelope mediate such process. Gp41 of HIV is one of the fusion proteins whose structure and mechanism of membrane fusion had been extensively studied. Functionally important motives of gp41 are the N-terminus fusion peptide, the coiled-coil and the membrane proximal C-peptide regions. The role of these regions during the fusion process had been thoroughly examined. Specially, insertion of the fusion peptide into membrane and conformational change of the coiled-coil and C-peptide regions are assumed to be critical for the fusion mechanism. In addition, the coiled-coil region has been shown to interact with membrane, and the C-peptide region regulates the interaction in a dose dependent manner. Furthermore, fusion defective mutations of the coiled-coil region dramatically changed its binding affinity to membrane. These results suggested that the membrane binding property of the coiled-coil region is important for the fusion activity of gp41, and such property could be modulated by the interaction with the C-peptide region.

• Journal of Life Science
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• v.28 no.3
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• pp.275-283
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• 2018

Human fibroblast growth factor (FGF) has the potential to be a commercially important therapeutic or cosmeceutical agent due to its ability to generate tissue and heal wounds. Granting permeability into skin tissues increases the therapeutic effects of FGF. Thus, several researchers have attempted the fusion of FGF conjugates with protein transduction domains (PTDs) to investigate the transduction ability and therapeutic effects of FGF. Less is known, however, about whether the location of PTD fused to the N- or C-terminus of FGF proteins has a significant impact on the folding and stability in Escherichia coli, and eventually, on transduction. Here, we report cloning of human basic fibroblast growth factor (FGF2) as a control and FGF2 with PTD fused to the N- or C-terminal ends of FGF proteins by an overlap extension PCR. We performed expression, verified expression properties of recombinant FGF2 without or with PTD fused to the N-terminus and the C-terminus, and investigated transduction ability into tissue by treating the dorsal skin of mice subjects. As a result, FGF2 and FGF2-PTD (fused to C-terminus) fusion protein were expressed as soluble forms suitable for straight-forward purification, unlike insoluble PTD-FGF2 (fused to N-terminus), but only FGF2-PTD fusion protein could transduce into the dorsal skin tissue of the mice subjects. Our results suggest that FGF2 with PTD fused to the C-terminus is more efficient than other options in terms of expression, purification, and delivery into skin tissue, as it does not require labor-intensive, costly, and time-consuming methods.

• Molecules and Cells
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• v.26 no.1
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• pp.34-40
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• 2008

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (${\geq}10.55$). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP ($OmpASP_{tr}$) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with $OmpASP_{tr}$ peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an $OmpASP_{tr}$ Xa leader sequence that contains the recognition site for Xa protease.

• BMB Reports
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• v.46 no.12
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• pp.606-610
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• 2013

Glucagon like peptide-1 (GLP-1) regulates glucose mediated-insulin secretion, nutrient accumulation, and ${\beta}$-cell growth. Despite the potential therapeutic usage for type 2 diabetes (T2D), GLP-1 has a short half-life in vivo ($t_{1/2}$ <2 min). In an attempt to prolong half-life, GLP-1 fusion proteins were genetically engineered: GLP-1 human serum albumin fusion (GLP-1/HSA), AGLP-1/HSA which has an additional alanine at the N-terminus of GLP-1, and AGLP-1-L/HSA, in which a peptide linker is inserted between AGLP-1 and HSA. Recombinant fusion proteins secreted from the Chinese Hamster Ovary-K1 (CHO-K1) cell line were purified with high purity (>96%). AGLP-1 fusion protein was resistant against the dipeptidyl peptidase-IV (DPP-IV). The fusion proteins activated cAMP-mediated signaling in rat insulinoma INS-1 cells. Furthermore, a C57BL/6N mice pharmacodynamics study exhibited that AGLP-1-L/HSA effectively reduced blood glucose level compared to AGLP-1/HSA.

• Biomedical Science Letters
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• v.13 no.4
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• pp.263-272
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• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.

• Journal of Life Science
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• v.14 no.5
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• pp.770-777
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• 2004

It has been previously reported that unacetylated a-tropomyosin(TM) produced in E. coli failed to bind to actin while acetylated muscle TM and Ala-Ser dipeptide fusion TM (AS-TM) bound well to actin. In order to determine the structural requirement of the amino terminus for high actin affinity, a recombinant tropomyosin (Ala-TM) that a single Ala residue was added to the amino terminus of Ala-TM was constructed, overexpressed, and purified from E. coli. Actin affinity of Ala-TM was 2.3$\times$$10^{6}$$M^{-1}$, whereas that of unacetylated TM was considerably lower than 0.1$\times$$10^{-6}$$M^{-1}$ indicating that addition of a single Ala residue to the amino terminus drastically increased, at least twenty times, actin affinity of TM. Ala-TM, however, bound to actin about three times weaker than acetylated TM and AS- TM, implying that the addition of an Ala residue was insufficient for complete restoration of high actin affinity. While Ala-TM, AS-TM, and muscle TM showed inhibition and activation of actomyosin Sl ATPase activity depending on myosin Sl concentration, the degree of inhibition and activation was different from each other. AS-TM exhibited the greatest inhibition of the ATPase at low Sl concentration, whereas the greatest activation of the ATPase was observed with muscle TM. These results, together with previous findings, strongly suggested that local structure of the amino terminus is the crucial functional determinant of TM.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.169-177
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• 2013

Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus ${\rightarrow}$ tobacco E2 signal peptide ${\rightarrow}$ B-peptide (1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-peptide (1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.