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

• Journal of Microbiology and Biotechnology
• /
• v.9 no.3
• /
• pp.303-310
• /
• 1999

A cost-effective mass production method for a strong antimicrobial peptide, buforin II, which was isolated from the stomach of Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charge of buforin II by fusion with a cysteine-rich acidic peptide (CAP) to avoid any lethal effect on the host. The neutralized fusion peptide was multimerized and expressed in Escherichia coli as tandem repeats to increase the production yield. Multimers of the CAP-buforin II fusion peptide were successfully expressed at high levels in E. coli as inclusion bodies. More than 100mg of pure buforin II was obtained per 11 of E. coli culture after cleaving the multimeric polypeptide with CNBr. The buforin II obtained from the recombinant E. coli had antimicrobial activity identical to that of natural buforin II. The proposed expression system can provide a cost-effective mass production method for both antimicrobial peptides and other host-lethal basic proteins.

• Molecules and Cells
• /
• v.42 no.3
• /
• pp.262-269
• /
• 2019

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration ($128{\mu}M$) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.

• BMB Reports
• /
• v.41 no.2
• /
• pp.170-175
• /
• 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.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.26 no.2
• /
• pp.119-123
• /
• 2013

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.1
• /
• pp.34-41
• /
• 1998

A method was developed for the controlled expression of an antimicrobial peptide magainin in Escherichia coli. A series of concatemeric magainin genes was constructed with a gene amplification vector, and fused to the 3'end of malE gene encoding the affinity ligand, E. coli maltose-binding protein (MBP). The construct directed the synthesis of the fusion protein with the magainin polypeptide fused to the C-terminus of MBP. The fusion protein was expressed in a tightly regulatable expression system which was under the control of an invertible promoter. The MBP-fused magainin monomer was expressed efficiently. However, the expression level of the MBP-fused magainin in E. coli decreased with the increasing size of multimers possibly because of the transcription and translation inhibition by the multimeric peptides. After purification using an amylose affinity column, the fusion protein was digested by factor Xa at a specific cleavage site between the monomers. The recombinant magainin had an antimicrobial activity identical to that of synthetic magainin. This experiment shows that a biologically active, antimicrobial peptide magainin can be produced by fusing to MBP, along with a promoter inversion vector system.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1994.11a
• /
• pp.143-145
• /
• 1994

Chimeric fusion proteins involving IgG have proven valuable in studying protein-protein interactions and may possess therapeutic applications as well. For example, three receptor subtypes for the natriuretic peptides, when fused to the Fc portion of human IgG ${\gamma}$ chain, were quantitatively and qualitatively indistinguishable from the native receptor, thus allowing detailed structure-function studies of the receptor. In an attempt to block human immunodeficiency virus infectivity with soluble derivatives of CD4, a CD4/IgG Fc chimeric molecule was shown to increase the plasma half life of soluble CD4 and possessed the added advantage of IgG Fc-mediated placental transfer. In the case of the KGFR, this approach provided a framework for dissection of its ligand binding domains and made it possible to demonstrate that high affinity binding sites for two ligands, aFGF and KGF, reside within different receptor Ig-like domains. Chimeric molecules fused to immunoglobulins would have the advantages of secretion from transfected cells as well as detection and purification from medium utilizing Staphylococcus aureus Protein A. In addition, where highly related receptors make their discrimination very hard due to the difficulties in generating specific immunochemical probes, IgG fusion protein with tailor-made specificities confers particular advantages to elucidate patterns of receptor distribution and expression. The approach described here may have general applications in defining ligand-receptor interactions as well as searching for specific agonists and antagonists of receptor function.

• Journal of the Korean Magnetic Resonance Society
• /
• v.15 no.2
• /
• pp.175-186
• /
• 2011

Amyloid fibrils have long been known to be the well known ${\alpha}$-helix to ${\beta}$-sheet transition characterizing the conversion of cellular to scrapie forms of the prion protein. A very short sequence of Yeast prion-like protein, GNNQQNY (SupN), is responsible for aggregation that induces diseases. KSI-fused tandem repeats of SupN vector are constructed and used to express SupN peptide in Escherichia coli (E.Coli). A method for a production, purification, and cleavage of tandem repeats of recombinant isotopically enriched SupN in E. coli is described. This method yields as much as 20 mg/L of isotope-enriched fusion proteins in minimal media. Synthetic SupN peptides and $^{13}C$ Gly labeled SupN peptides are studied by Congo Red staining, Birefringence and transmission electron microscopy to characterize amyloid fibril formation. To get a better understanding of aggregation-structure relationship of 7 residues of Yeast prion-like protein, the change of a conformational structure will be studied by $^{13}C$ solid-state nmr spectroscopy as powder of both amorphous and fibrillar forms.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.1
• /
• pp.59-64
• /
• 2002

An angiotensin I-converting enzyme (EC 3.4.15.1) (ACE), which can convert inactive angiotensin I into angiotensin II, a vasoconstrictor, is one of the key enzymes in controlling hypertension. It is suggested that the inhibition of ACE prevents hypertension, and many inhibitory peptides have already been reported. In the current study, oligonucleotides encoding ACE inhibitory peptides (IY, VKY) were chemically synthesized and designed to be multimerised due to isoschizomer sites (BamHI, BglII). The cloned gene named AP3 was multimerised up to 6 times in pBluescript and expressed in BL2l containing pGEX-KG. The fusion protein (GST-AP3) was easily purified with a high recovery by an affinity resin, yielding 38 mg of synthetic AP3 from a 1-1 culture. The digestion of AP3 by chymotrypsin exhibited an $IC_50$ value of $18.53{\mu}M$. In conclusion, the present experiment indicated that AP3 could be used as a dietary antihypertensive drug, since the potent ACE inhibitory activity of AP3 could be activated by chymotrypsin in human intestine.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.2
• /
• pp.350-355
• /
• 2010

Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.