• Bulletin of the Korean Chemical Society
• /
• 제31권9호
• /
• pp.2509-2513
• /
• 2010

In order to increase protease stability of a novel Trp-rich model antimicrobial peptide, $K_6L_2W_3$ (KLWKKWKKWLK-$NH_2$)and investigate the effect of L-amino acid to peptoid residue conversion on biological functions, we synthesized its antimicrobial peptoid, $k_6l_2w_3$. Peptoid $k_6l_2w_3$ had similar bacterial selectivity compared to peptide $k_66L_2W_3$. The bactericidal rate of $k_6l_2w_3$ was somewhat slower than that of $K_6L_2W_3$. Peptoid $k_6l_2w_3$ exhibited very little dye leakage from bacterial outer-membrane mimicking PE/PG liposomes, as observed in $K_6L_2W_3$, indicating that the major target site of $K_6L_2W_3$ and $k_6l_2w_3$ may be not the cell membrane but the cytoplasm of bacteria. Trypsin treatment of $K_6L_2W_3$ completely abolished antimicrobial activities against Escherichia coli and Staphylococcus aureus. In contrast, the antimicrobial activity of $k_6l_2w_3$ was completely preserved after trypsin treatment. Taken together, our results suggested that antimicrobial peptoid $k_6l_2w_3$ can potentially serves as a promising therapeutic agent for the treatment of microbial infection.

• BMB Reports
• /
• 제40권6호
• /
• pp.1090-1094
• /
• 2007

Melittin (ME), a linear 26-residue non-cell-selective antimicrobial peptide, displays strong lytic activity against bacterial and human red blood cells. To design ME analogue with improved cell selectivity, we synthesized a melittin diastereomer (ME-D) with D-amino acid in the leucine zipper sequence (Leu-6, Lue-13 and Ile-20). Compared to ME, ME-D exhibited the same or 2-fold higher antibacterial activity but 8-fold less hemolytic activity. Circular dichroism analysis revealed that ME-D has much less $\alpha$-helical content in $\alpha$-helical content in the presence of zwitterionic EYPC/cholesterol (10 : 1, w/w) liposomes compared to negatively charged EYPE/EYPG (7 : 3, w/w) liposomes. The blue shift of the fluorescence emission maximum of ME-D in zwitterionic EYPC/cholesterol (10 : 1, w/w) liposomes was much smaller than in negatively charged EYPE/EYPG (7 : 3, w/w) liposomes. These results suggested that the improvement in therapeutic index/cell selectivity of ME-D is correlated with its less permeability to zwitterionic membranes.

• 산업식품공학
• /
• 제21권4호
• /
• pp.360-366
• /
• 2017

본 연구에서는 lab scale 용량으로 100 mL, 1 L 단위로 리포좀을 제조하였으며, prototype scale로서 10 L 단위로 blank 리포좀의 제조한 뒤 입자 크기 및 포집 효율을 측정하여 최적 제조 조건 선정하였다. 선정한 최적조건으로 어피 펩타이드를 리포좀으로 포집하여 그에 따른 저장 안정성을 평가하였다. 1차 균질 조건은 초고속균질기를 사용하여 3분간 각각 4,000 rpm, 8,000 rpm, 12,000 rpm으로 균질하였으며, 2차 균질 조건은 초음파균질기를 이용하여 각각 40 W, 60 W, 80 W로 3분간 균질하여 최적 균질 조건을 확립한 뒤 어피 펩타이드 리포좀을 농도에 따라 제조하여 $4^{\circ}C$에서 냉장 저장하였다. 최적 제조 조건 실험 결과를 two-way ANOVA로 분석한 결과 1, 2차 균질에서는 제조 용량이 입자크기와 제타 전위에 유의적으로 가장 큰 영향을 미쳤으며(p<0.001), pdI는 2차 균질 조건에서 제조용량을 제외하고는 어떤 요인도 유의적으로 영향을 미치지 못하였다(p>0.05). 1차 균질 실험 결과 lab scale에서 prototype scale로 제조 용량이 증가하였을 때, 유의적으로 입자크기가 증가하였으며(p<0.05), 가장 입자크기가 작고 제타전위의 절대값이 높은 4,000 rpm을 최적조건으로 선정하였다. 2차 균질 실험결과 40 W에서 제조 용량이 증가하였을 때 유의적으로 제타전위가 감소하였으며, 60 W 이상에서는 안정적인 결과가 나타났다. 리포좀의 산업적 적용을 고려할 때 공정비용 감소 측면에서 80 W보다 60 W가 적절하다고 사료된다. 선정된 리포좀 최적 조건으로 농도(3, 6, 12, 24%)별로 어피 펩타이드를 포집하였을 때, 24%에서 입자크기가 1 mm 이상으로 크게 나타났다. 이후 저장실험에서는 24%를 제외한 3가지 조건으로 진행하였다. 1달간 어피 펩타이드 리포좀을 냉장 저장한 결과를 two-way ANOVA로 분석한 결과, 펩타이드 농도에 따라 제조하였을 때는 입자크기와 제타 전위가 제조용량보다 펩타이드 농도에 더 큰 영향을 받았다. 또한 저장기간은 pdI에 유의적으로 영향을 미치는 것으로 나타났다(p<0.001). 입자크기는 제조용량과 펩타이드 농도가 증가함에 따라서 유의적으로 증가하였으며, 저장기간에 따라서 감소하였다. 제타전위는 10 L 용량에서 저장기간에 따라 증가하는 경향을 보였다. 이는 리포좀이 풀리면서 비표면적이 증가하여 제타전위가 증가한 것으로 사료된다. 또한 12%에서 3주차부터 침전물층이 형성되는 것이 관찰되어 6%가 가장 산업적용으로 적합한 농도로 사료된다.

• Bulletin of the Korean Chemical Society
• /
• 제33권9호
• /
• pp.2883-2889
• /
• 2012

LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric antimicrobial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and ${\alpha}$-helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis.

• Bulletin of the Korean Chemical Society
• /
• 제32권12호
• /
• pp.4337-4340
• /
• 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
• /
• 제27권4호
• /
• pp.759-767
• /
• 2017

Lactophoricin (LPcin), which is a part of proteose peptone isolated from bovine milk, is a cationic amphipathic ${\alpha}-helical$ antimicrobial peptide. Its truncated variants and mutated analogs were designed and their antimicrobial activities were evaluated by using various assays, like broth dilution methods and disk diffusion methods as well as hemolysis assay. Three analogs, LPcin-C8 (LPcin-YK1), LPcin-T2&6W (LPcin-YK2), and LPcin-T2&6W-C8 (LPcin-YK3), which showed better antibiotic activities than LPcin, were selected. Their secondary structures were also characterized by using CD spectropolarimetry. These three analogs of LPcin could be used as an alternative source of powerful antibacterial agents.

• Mass Spectrometry Letters
• /
• 제6권3호
• /
• pp.80-84
• /
• 2015

Here, we demonstrate the use of MALDI-TOF as a fast and simple analytical approach to evaluate the DNA-binding capability of various peptides. Specifically, by varying the amino acid sequence of the peptides consisting of lysine (K) and tryptophan (W), we identified peptides with strong DNA-binding capabilities using MALDI-TOF. Mass spectrometric analysis reveals an interesting novel finding that lysine residues show sequence selective preference, which used to be considered as mediator of electrostatic interactions with DNA phosphate backbones. Moreover, tryptophan residues show higher affinity to DNA than lysine residues. Since there are numerous possible combinations to make peptide oligomers, it is valuable to introduce a simple and reliable analytical approach in order to quickly identify DNA-binding peptides.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2001년도 Proceedings of the Pharmaceutical Society of Korea
• /
• pp.195.3-196
• /
• 2001

• 한국수산과학회지
• /
• 제30권3호
• /
• pp.466-472
• /
• 1997

명태 (Alask pollack, Theragia chalcogramma) 고기풀 수세액 중의 단백질 활용방안으로 기능성 peptide생산을 위해 고정화 protease를 이용한 생물반응기에서 가수분해하여 gel 여과후 정제 peptide의 특성 및 아미노산 존재 여부를 파악하기 위해 실험한 결과는 다음과 같다. 1. $0.5\%$ 농도의 고기를 수세액을 반응온도 $50^{\circ}C$에서 24시간 가수분해 하였을 때 분해율은 $50\%$ (OPA법)였다. 2. Sephadex G-50 column chromatography에 의한 고기풀 수세 액과 조peptide의 분자량은 각각 $10,000\~50,000Da$과, $600\~12,000Da$의 광범위한 분포를 나타내었다. 3. SP-Sephadex C-25 $(H^+)$ column chromatography에서 식염농도 $0\~3\%$의 농도구배에서 $0.6\~0.9\%\;NaCl$, pH 2 범위에서 염기성이 약한 아미노산을 함유하였고, $1.2\~2.0\%$에서는 염기성이 강한 아미노산을 함유하는 peptide가 용출되었다. 4. SP-Sephadex C-25 ($(H^+)$ column chromatography에서 분획한 SP-분획을 Sephadex G-50 colum chromatography에서 peptide의 분자량 측정 결과는 대게 저분자로 분자량 1,000Da에서 3,000Da 범위였고, SP-4>SP-3>SP-2>SP-1의 순으로 분자량 분포를 나타내었다. 5. 정제 peptide의 아미노산 조성은 수세 peptide에서 glycine, arginine이 가장 많이 함유하였으며 그외 glutamic acid, alanine, aspartic aicd 등이였고, SP-분획 peptide는 수세 peptide와 대동소이하였으나 SP-2에서 glycine이 $33\%$로 가장 높았다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제16권10호
• /
• pp.1460-1468
• /
• 2003

An experiment was conducted to quantify the flow of soluble non-ammonia nitrogen (SNAN) in the liquid phase of ruminal (RD) and omasal digesta (OD), and to investigate diurnal pattern in SNAN flow in OD. Five ruminally cannulated Finnish-Ayrshire dairy cows in a $5{\times}5$ Latin square design consumed a basal diet of grass silage and barley grain, and that supplemented with four protein feeds (kg/d DM basis) as follows: skimmed milk powder (2.1), wet distiller' solubles (3.0), untreated rapeseed meal (2.1) and treated rapeseed meal (2.1). Ruminal digesta was sampled using a vacuum pump, whereas OD was collected using an omasal sampling system at 1.0 h interval during a 12 h feeding cycle. Both RD and OD were acidified, centrifuged to remove microbes and precipitated with trichloroacetic acid followed by centrifugation. The SNAN fractions (free amino acid (AA), peptide and soluble protein) in RD and OD were assessed using ninhydrin assay. Free AA, peptide and soluble protein averaged 60.0, 89.4 and 2.1 g/d, respectively, for RD, and 81.8, 121.5 and 2.5 g/d, respectively, for OD. Although free AA flow was relatively high, mean peptide flow was quantitatively the most important fraction of SNAN, indicating that degradation of peptide to AA rather than hydrolysis of soluble protein to peptide or deamination may be the most limiting step in rumen proteolysis. Diurnal pattern in flow of peptide including free AA in OD during a 12 h feeding cycle peaked 1 h post-feeding, decreased by 3 h post-feeding and was relatively constant thereafter. Protein supplementation showed higher flow of peptide including free AA immediately after feeding compared with no supplemented diet. There were no differences among protein supplements in diurnal pattern in flow of peptide including free AA in OD.