• 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.

• Bulletin of the Korean Chemical Society
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• v.25 no.6
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• pp.838-842
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• 2004

Amyloid peptide (A${\beta}$) is the major component of senile plaques found in the brain of patient of Alzheimer's disease. ${\beta}$-amyloid peptide (25-35) (A${\beta}$25-35) is biologically active fragment of A${\beta}$. The three-dimensional structure of A${\beta}$25-35 in aqueous solution with 50% (vol/vol) TFE determined by NMR spectroscopy previously adopts an ${\alpha}$-helical conformation from $Ala^{30}$ to $Met^{35}$. It has been proposed that A${\beta}$(25-35) exhibits pH- and concentration-dependent ${\alpha}-helix{\leftrightarrow}{\beta}$sheet transition. This conformational transition with concomitant peptide aggregation is a possible mechanism of plaque formation. Here, in order to gain more insight into the mechanism of ${\alpha}$-helix formation of A${\beta}$25-35 peptide by TFE, which particularly stabilizes ${\alpha}$-helical conformation, we studied the secondary-structural elements of A${\beta}$25-35 peptide by molecular dynamics simulations. Secondary structural elements determined from NMR spectroscopy in aqueous TFE solution are preserved during the MD simulation. TFE/water mixed solvent has reduced capacity for forming hydrogen bond to the peptide compared to pure water solvent. TFE allows A${\beta}$25-35 to form bifurcated hydrogen bonds to TFE as well as to residues in peptide itself. MD simulation in this study supports the notion that TFE can act as an ${\alpha}$-helical structure forming solvent.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1429-1433
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• 2006

Application of recombinant protein production and particularly their isotopic enrichment has stimulated development of a range of novel multidimensional heteronuclear NMR techniques. Peptides in most cases are amenable to assignment and structure determination without the need for isotopic labeling. However, there are many cases where the availability of $^{15}N$ and/or $^{13}C$ labeled peptides is useful to study the structure of peptides with more than 30 residues and the interaction between peptides and membrane. CRAMP (Cathelicidin-Related AntiMicrobial Peptide) was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. CRAMP was successfully expressed as a GST-fused form in E. coli and purified using affinity chromatography and reverse-phase chromatography. The yield of the CRAMP was 1.5 mg/l 1. According to CD spectra, CRAMP adopted ${\alpha}$-helical conformation in membrane-mimetic environments. Isotope labeling of CRAMP is expected to make it possible to study the structure and dynamic properties of CRAMP in various membrane systems.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.280-287
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• 2015

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein ($2{\mu}g/mouse$) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.669-671
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• 2000

Antimicrobial cationic peptides have attracted increasing research and clinical interest as a natural antibiotics due to their broad spectrum of antimicrobial activites and the rapid development of multidrug-resistant pathogenic microorganisms. In this study, first, we synthesized artificial fusion partner and cationic peptide genes (lactoferricin, magainin, protegrin-1, and indolicidin). Second, we constructed recombinant expression vectors and then transformed Pichia pastoris. Finally, expressed cationic peptides were purified and tested for their antimicrobial activites. Antimicrobial activity has been tested upon the appearance of clearing zone on the plate with the lawn of gram negative E.coli XL- I blue and garm positive Staphylococcus aureus. Protegrin-1 and Indolicidin have apparant activity of cationic peotides. This fusion technique may lead to a general and suitable tool for production of pure antimicrobial cationic peptides in Pichia pastoris.

• BMB Reports
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• v.32 no.5
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• pp.497-501
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• 1999

Biotinylation of recombinant proteins is a powerful tool for the detection and analysis of proteins of interest in a large variety of assay systems. The recent development of in vivo biotinylation techniques in E. coli has opened new possibilities for the production of site-specifically biotinylated proteins without the need for further manipulation after the isolation of the recombinantly expressed proteins. In the present study, a novel vector set was generated which allows the convenient cloning and expression of proteins of interest fused with an N-terminal in vivo biotinylated thioredoxin (TRX) protein. These vectors were derived from the previously reported pBIOTRX vector into which was incorporated part of the pBluescript II+phagemid multiple cloning site (MCS), amplified by PCR using a pair of sophisticated oligonucleotide primers. The functionality of these novel vectors was examined in this system by recombinant expression of rat transforming growth factor-$\beta$. Western-blot analysis using TRX-specific antibodies or peroxidase-conjugated streptavidin confirmed the successful induction of the fusion protein and the in vivo conjugation of biotin molecules, respectively. The convenience of molecular subcloning provided by the MCS and the effective in vivo biotinylation of proteins of interest makes this novel vector set an interesting alternative for the production of biotinylated proteins.

• Molecules and Cells
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• v.21 no.2
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• pp.244-250
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• 2006

Gangliosides are receptors for various peptides and proteins including neuropeptides, ${\beta}$-amyloid proteins, and prions. Recently, the role of gangliosides in mucosal immunization has attracted attention due to the emerging interest in oral vaccination. Ganglioside GM1 exists in abundance on the surface of the M cells of Peyer's patch, a well-known mucosal immunity induction site. In the present study we identified a peptide ligand for GM1 and tested whether it played a role in immune induction. GM1-binding peptides were selected from a phage-displayed dodecapeptide library and one peptide motif, GWKERLSSWNRF, was fused to the C-terminus of enhanced green fluorescent protein (EGFP). The fusion protein, but not EGFP fused with a control peptide, was concentrated around Peyer's patch after incubation in the lumen of the intestine ex vivo. Furthermore, oral feeding of the fusion protein but not control EGFP induced mucosal and systemic immune responses against EGFP resembling Th2-type immune responses.

• Journal of Microbiology
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• v.37 no.2
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• pp.97-101
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• 1999

Random hexapeptide library on the surface of filamentous bacteriophage was constructed using the SurfZAP vector. The size of the library was approximately 105. The peptide insert was flanked by two cysteines to constrain the peptide structure with a disulfide bond. This library was screened for the topoisomerase II binding peptide. Dramatic enrichment of the fusion phage over the VCS M13 helper phage was demonstrated by bio-panning affinity selection.

• Journal of Life Science
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• v.31 no.1
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• pp.83-89
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• 2021

Uu-ilys, an i-type lysozyme from spoon worm (Urechis unicinctus), is an innate immune factor that plays an important role in the defense against pathogens. It also possesses non-enzymatic antibacterial activity. Thus, there is a possibility to develop an antimicrobial model peptide from Uu-ilys. In this study, we report the design, production, and antibacterial activity of an Uu-ilys analog that exhibits antibacterial activity. The Uu-ilys structure was fragmented according to its secondary structures to predict the regions with antimicrobial activity using antimicrobial peptide (AMP) prediction tools from different AMP databases. A peptide containing the C-terminal fragment was predicted to exert antimicrobial activity. The chosen fragment was designated as an Uu-ilys analog containing the C-terminal fragment, Uu-ilys-CF. To examine the possibility of developing an AMP using the sequence of Uu-ilys-CF, recombinant fusion protein (TrxA-Uu-ilys-CF) was produced in an expression system that was heterologous. The produced fusion protein was cleaved after methionine leaving Uu-ilys-CF free from the fusion protein. This was then isolated through high performance liquid chromatography and reverse phase column, CapCell-Pak C18. The antibacterial activity of Uu-ilys-CF against different microbial strains (four gram-positive, six gram-negative, and one fungal strain) were assessed through the ultrasensitive radial diffusion assay (URDA). Among the bacterial strains tested, Salmonella enterica was the most susceptible. While the fungal strain tested was not susceptible to Uu-ilys-CF, broad spectrum antibacterial activity was observed.

• Journal of the Korean Magnetic Resonance Society
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• v.13 no.2
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• pp.96-107
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• 2009

Lactophoricin (LPcin-I) is a cationic amphipathic peptide with 23-mer peptide, and corresponds to the carboxy terminal 113-135 region of Component-3 of proteose-peptone. LPcin-I is a good candidate as a peptide antibiotic, because it has an antibacterial activity, but no hemolytic activity. On the other hand, its shorter analog (LPcin-II), which corresponds to the 119-135 region of PP3, has no antibacterial activity. In order to understand the structure-activity relationship under the membrane environments, we succeed to produce large amounts of LPcin-I and LPcin-II peptides. Peptides were over expressed in the form of fusion protein in Escherichia coli, and purified with several chromatography techniques. In this paper, we introduce the optimizing processes of purification and NMR measurement.