• 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 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.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

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

• Biomolecules & Therapeutics
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• v.29 no.3
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• pp.282-289
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• 2021

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

• BMB Reports
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• v.31 no.5
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• pp.427-431
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• 1998

In the previous report (Choi et al., 1997), the angiogenin binding peptides identified from a phage-peptide library were analyzed by using the fusion proteins composed of the Escherichia coli maltose binding protein and its corresponding peptides. However, it was difficult to obtain a sufficient amount of the fusion proteins required for further analysis because of the low expression level. We now report a high level expression of the fusion protein and analysis of its anti-angiogenin activity. The use of strong T7 promoter and removal of signal sequence allowed about a 20-fold increase in the expression efficiency of the fusion protein. We were able to obtain about 10 mg of purified fusion protein from one liter of culture. The purified fusion protein showed angiogenin-specific affinity and inhibited the binding of biotinylated actin to human angiogenin at $IC_{50}$ of 0.6 mM. Its anti-angiogenin activity was also revealed by the chorioallantoic membrane assay.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.4
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• pp.576-584
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• 2017

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ${\beta}-defensin-2$ (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and grampositive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

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

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.82-86
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• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.

• 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.