A novel cecropin‐like peptide from black soldier fly, Hermetia illucens: Isolation, structural and functional characterization

박순익,여성문 한국곤충학회 2017 Entomological Research Vol.47 No.2

Cecropins are basic antibacterial peptides that have potent antimicrobial activities. We induced and purified a novel antimicrobial peptide exhibiting activity against Gram‐negative bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel cecropin‐like peptide 1 (CLP1) was purified using solid‐phase extraction and reverse‐phase chromatography. The purified CLP1 demonstrated a molecular weight of 4,840 Da, as determined by matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF). From analysis of CLP1 by N‐terminal amino acid sequencing using Edman degradation, combined with MALDI‐TOF and rapid amplification of cDNA ends‐polymerase chain reaction (RACE‐PCR), the amino acid sequence of the mature peptide was determined to be GWRKRVFKPVEKFGQRVRDAGVQGIAIAQQGANVLATARGGP PQQG. In NCBI BLAST, the amino acid sequence of CLP1 was found to be 60 % identical to the Drosophila melanogaster cecropin C. In silico analysis revealed that CLP1 was suggested to be part of the cecropin superfamily of AMPs characterized as cationic, linear, α‐helical, and amphipathic polypeptides. Analysis of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) showed that CLP1 exerted antibacterial effects against Gram‐negative bacteria. The expression of CLP1 transcripts in several tissues after bacterial challenge was measured by quantitative real‐time PCR. CLP1 expression was negligible throughout the body before immunization, and was mostly evident in the fat body after immunization.

비수임형 양대혈관 우심실기시증에서의 양심실 교정술

박순익,박정준,윤태진,서동만 대한흉부외과학회 2004 Journal of Chest Surgery (J Chest Surg) Vol.37 No.1

The remote location of VSD relative to the aortic valve imposes considerable surgical difficulties in the repair of DORV with noncommitted VSD. We report a successful biventricular repair of the anomaly with VSD rerouting to pulmonary artery followed by arterial switching operation. 비수임형 양대혈관 우심실기시증은 심실중격결손(DORV with remote VSD)이 대동맥과 상당히 떨어져 있어 양심실 교정에 상당한 어려움이 따른다. 본 교실에서는 심실중격결손과 폐동맥을 배플(baffle)을 이용하여 연결한 후 대혈관 전환술(arterial switching operation)을 시행하여 양심실 교정을 성공적으로 치험하였기에 보고하는 바이다.

Overexpression and purification of recombinant lysozyme from Agrius convolvuli expressed as inclusion body in Escherichia coli

박순익,여성문 한국통합생물학회 2012 Animal cells and systems Vol.16 No.6

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector,and the fusion protein was induced by ioporpyl-b-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

Expression, cDNA cloning, and characterization of the antibacterial peptide cecropin D from Agrius convolvuli

박순익,안홍선,여성문,장병수 한국통합생물학회 2013 Animal cells and systems Vol.17 No.1

Cecropins are basic antibacterial peptides that have potent activities against microorganisms. We have cloned and characterized a cecropin-like peptide of the lepidopteran insect Agrius convolvuli and analyzed its expression in Escherichia coli. The full-length cDNA of A. convolvuli cecropin D3 (AcCec) was 318 bp, containing a 5? untranslated region (UTR) of 47 bp, a 3? UTRof 82 bp with a poly (A) tail, and an open reading frame (ORF) of 189 bp encoding a polypeptide of 63 amino acids, including a 24 amino acid signal sequence and a 38 amino acid mature peptide 15 (GenBank accession no. GQ888768). The mature peptide is highly similar to D-type cecropin. To understand the effect of C-terminal amidation, while overcoming the disadvantage of its lack in the prokaryote system, we added a lysine residue to AcCec (AcCec-K) and compared its antibacterial activity to the purified AcCec. The recombinant AcCec and AcCec-K were expressed, respectively, in E. coli Rosetta cells using a pGEX-4T-1 expression vector, which contained the glutathione S-transferase (GST) gene for fusion partner, and the fusion proteins were induced by 20 isopropyl-b-d-thiogalactopyranoside (IPTG). The recombinant proteins were purified by FPLC using GSTrap FF and Resource RPC column. The result of the inhibition zone suggests that C-terminal lysine residue could increase the activity due to activated phosphorylation.

1140 gm의 미숙아에 대한 동맥치환술

박순익,이승현,박정준,서동만,김영휘,고재곤,박인숙 대한흉부외과학회 2005 Journal of Chest Surgery (J Chest Surg) Vol.38 No.11

Cardiac surgery in the neonate with congenital heart disease has progressed dramatically in the past three decades. However, low-birth-weight premie with congenital heart disease continue to challenge the intellectual and technical skills of those who care for them. We report a case of successful arterial switch operation in 1140 g premie with TGA, IVS after 4 week care 1317 gm. 최근 30년 동안 선천성 심기형의 수술적 교정은 많은 발전을 거듭해, 어리고 적은 체중에서도 큰 문제 없이 시행되고 있지만, 아직 저체중(low birth weight), 극저체중(very low birth weight) 출산아의 개심술에는 많은 어려움을 겪고 있다. 본원에서는 대혈관전위증(TGA)을 동반한 태내주수 35주, 출생체중 1140 g의 미숙아를 생후 27일까지 키워 체중 1317 gm일때 동맥치환술을 성공적으로 시행하였으며, 추적관찰한 결과 양호하였기에 보고하는 바이다.

비디오 흉강경을 이용한 종격동 종양 절제술

박순익,김동관,유양기,김용희,박기성,박창률,박승일 대한흉부외과학회 2002 Journal of Chest Surgery (J Chest Surg) Vol.35 No.11

배경 : 종격동 종양의 경우 그 분포가 다양하나 양성질환이 더 많고, 흉강경 수술시 시야가 좋으며, 수술에 필요한 공간을 쉽게 확보할 수 있다는 점에서 비디오 흉강경 절제술이 기존의 개흉술에 못지 않은 수술방법이라고 생각되어 본원에서 양성 종격동 종양으로 진단 받은 환자들을 대상으로 비디오 흉강경을 이용한 종격동 종양 절제술을 시행하고 고찰하였다. 대상 및 방법 : 1995년 1월부터 2001년 8월까지 근무력증을 제외한 양성 종격동 종양 43례의 환자에서 비디오 흉강경술을 시도하여 이중 개흉술로 전환된 5례를 제외한 38례를 분석하였다. 결과 : 성별은 남자가 13명(34.2%), 여자가 25명(65.8%)이었으며, 평균 연령은 39.2±35.4세였다. 진단은 신경초종(neurilemmoma) 8례(21.1%), 흉선 낭종(thymic cyst) 6례(15.8%), 기형종(teratoma) 5례(13.2%), 신경절신경종(ganglioneuroma) 5례(13.2%), 기관지원성 낭종(bronchogenic cyst) 4례(10.5%), 심막 낭종(pericardial cyst) 3례(7.9%), 흉선종(thymoma) 3례(7.9%), 림프관종(lymphangioma) 2례(5.3%) 등이었다. 평균 수술 시간은 110.6±7.0분, 평균 흉관 거치기간은 4.2±0.4일, 수술후 평균 재원기간은 5.2±0.4일, 평균 술후 진통제 근주 횟수는 1.9 ± 0.4회이었으며, 이상의 측정치들은 개흉술로 전환된 5례와 비교하여 평균값이 적었으나, 통계적으로 유의하지는 않았다. 수술후 합병증으로는 유미흉, 장기간 공기유출 및 일측성 횡격막 마비가 각 1례가 있었으나 퇴원시 소실된 일과성이었으며, 편측안검하수 1례가 있었다. 결론 : 비디오 흉강경을 이용한 종격동 종양 절제술은 안전하게 시행될 수 있으며, 수술 후 통증경감과 빠른 회복 및 미용상의 장점이 있어 계속 발전되고 적극적으로 시도되어야 한다고 사료된다.13. Roviaro G, Rebuffat C, Varoli F, Vergani C, Maciocco M, Scalambra SM. Videothoracoscopic excision of media stinal masses:indications and technique. Ann Thorac surg 1994;58:1679-84.14. Demmy TL, Krasna MJ, Detterbeck FC, et al. Multicenter VATS experience with mediastinal tumors. Ann Thorac surg 1998;66:187-92.15. Kern JA, Daniel TM, Tribble CG, Silen ML, Rodgers BM. Thoracoscopic diagnosis and treatment of mediastinal masses. Ann Thorac Surg 1993;56:92-6.16. Hazelrigg SR, Boley TM, Krasna MJ, Landreneau RJ, Yim AP. Thoracoscopic resection of posterior neurogenic tumors. American surgeon 1999;65:1129-1133.17. Landreneau RJ, Dowling RD, Castillo WM, Ferson PF. Thoracoscopic resection of an anterior mediastinal tumor. Ann Thorac Surg 1992;54:142-4.18. Kelemen JJ III, Naunheim KS. Minimally invasive approaches to medias tneoplasms. Seminars in Thoracic and Cardiovascular surgery 2000;12:301-306. Video-assisted Thoracoscopic Excision of Mediastinal Masses

Detection of antimicrobial substances from larvae of the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae)

박순익,장병수,여성문 한국곤충학회 2014 Entomological Research Vol.44 No.2

Maggots have become highly successful in the treatment of non‐healing wounds and multidrug‐resistant pathogen infections. The main objective of this study was to extract antibacterial substances from larvae of the black soldier fly, Hermetia illucens. To induce immune responses, we septically injured the larvae with a contaminated needle. Lyophilized H. illucens larvae were homogenized and extracted with acidic methanol. We examined the antifungal and antibacterial effects of the low molecular weight antimicrobial factors within the larval extract on the growth of a broad range of microorganisms, including Gram‐positive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), and Gram‐negative Pseudomonas aeruginosa. Furthermore, we isolated the anti‐MRSA substances from the larval extract using high performance liquid chromatography. These investigations revealed that the larval extract possessed a broad‐spectrum of antibacterial activity, demonstrating that secretions of H. illucens larvae prove useful in the fight against MRSA and can potentially be a source of novel antibiotic‐like compounds for infection control.

Defensin‐like peptide3 from black solder fly: Identification, characterization, and key amino acids for anti‐Gram‐negative bacteria

박순익,여성문 한국곤충학회 2017 Entomological Research Vol.47 No.1

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.

기관 절제 및 단단문합술의 성적 고찰

유양기,박승일,박순익,김용희,박기성,김동관,최인철 대한흉부외과학회 2003 Journal of Chest Surgery (J Chest Surg) Vol.36 No.4

배경: 기관 협착의 치료에는 주기적인 확장, 기관내시경을 통한 협착 조직의 제거, T-tube 삽입 등의 보존적 방법과 수술적 방법이 있다. 그러나, 최근 수술을 통한 기관 절제 및 단단문합술이 기능적으로나 해부학적으로 정상적인 상기도 확보에 효과적인 것으로 보고되고 있어 그 결과를 알아보고자 한다. 대상 및 방법: 1990년 3월부터 2002년 7월까지 울산대학교 의과대학 서울아산병원에서 기관 절제 및 단단문합술을 시행한 환자 41예를 대상으로 의무기록을 참조하여 후향적으로 분석하였다. 결과: 원인 질환으로는 postintubation stenosis 26예, 기관에 발생된 원발성 기관종양이 10예(양성 3예, 악성 7예), 기관내 결핵 1예, 외상 2예, 그리고 갑상선암이 2예였다. 기관 절제 및 단단문합술을 시행한 환자 41예 중 29예는 기관 절제 및 단단 문합술을 시행하였고, 12예는 갑상연골 또는 윤상연골 절제가 동반된 후두 기관 문합술을 시행하였다. 후두 기관 문합술을 시행한 11예 중 4예에서 후두분리술(supralaryngeal release)을 시행하였다. 절제된 기관의 길이는 평균 3.6±1.0 cm였다. 기관 절제 및 단단문합술을 시행한 41예의 환자 중 수술 후 상태가 양호한 환자는 30명(73.2%)이었고, 수술 직후 일부 문합 부위에 육아종 증식 또는 감염이 있었으나 보존적 치료 후 호전된 환자는 8명(19.5%)으로 전체 환자 중 92.7%에서 수술 후 양호한 기도의 확보가 가능하였다. 합병증으로는 육아종 증식을 보여 보존적 치료를 시행한 환자가 7예, 창상감염 2예, 문합부 dehiscence 2예, 운동 시만 호흡곤란을 보이면서 특별한 치료없이 지내는 재협착이 1예, 수술 후 반복적인 기도 흡인으로 재기관절개술을 시행한 환자가 1예 있었다. 기관 절제 및 단단문합술과 직접 관련된 30일 내의 조기사망은 없었고, 원내사망이 3예 있었다. 결론: 기관 절제 및 단단문합술은 높은 성공률과 낮은 유병률 및 사망률을 보여 병변의 길이가 적절한 경우, 기관 수술의 표준이 될 수 있을 것으로 생각한다. 그러나, 수술부위 육아조직 증식이 가장 심각한 합병증으로 이를 방지하기 위한 지속적인 연구와 노력이 필요할 것으로 생각한다.

Cloning and overexpression of lysozyme from Spodoptera litura in prokaryotic system

김종완,박순익,여지현,여성문 한국통합생물학회 2011 Animal cells and systems Vol.15 No.1

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5? UTR and a 559 bp long 3? UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.