천잠 cecropin-A 유전자 클로닝 및 재조합 발현

김성렬 ( Seong Ryul Kim ),최광호 ( Kwang Ho Choi ),김성완 ( Sung Wan Kim ),구태원 ( Tae Won Goo ),황재삼 ( Jae Sam Hwang ) 한국잠사학회 2014 한국잠사곤충학회지 Vol.52 No.1

면역 유도된 천잠(Antheraea yamamai) 유충에서 cecropin- A 유전자를 분리하였고 이 유전자를 Ay-CecA로 명명하였다. 전체 Ay-CecA cDNA 크기는 419 bp로 64개의 아미노산 잔기를 인코딩하는 195 bp ORF로 구성되어 있다. 천잠 CecA 유전자는 22개 잔기의 signal peptide, 4개 잔기의 propeptide 및 항균활성을 갖는 37개 잔기로 구성된 성숙 단백질(mature protein) 영역으로 구성되고 예상 분자량은 4046.81 Da으로 산출되었다. 천잠 CecA의 아미노산 서열은 다른 나비목 곤충에서 분리된 cecropin와 매우 높은 상동성(62 ~ 78%)을 나타냈다. Ay-CecA 유전자의 C 말단에 기존에 보고된 곤충의 cecropin에서와 동일하게 C 말단 아미드화를 위한 glycine 잔기가 존재하고 있다. 이펩타이드의 항균활성을 검정하기 위해서 대장균 발현 시스템을 이용하여 활성이 있는 재조합 Ay-CecA 발현에 성공하였다. 발현 기주인 대장균에 대한 재조합 CecA 독성 중화를 위해서 불용성 단백질인 ketosteroid isomerase(KSI) 유전자를 CecA 유전자와 융합하였다. 융합 CecA-KSI 단백질은 대부분 불용성 단백질로 발현되었다. 발현된 융합 단백질은 Ni-NTA immobilized metal affinity chromatography (IMAC)에 의해서 정제하였으며 CNBr 반응을 통하여 재조합 CecA를 절단하여 용출하였다. 최종적으로 양이온 교환 chromatography 과정을 통하여 CecA를 순수 정제하였다. 정제된 재조합 Ay-CecA는 그람음성균인 E. cori ML 35, Klebsiella pneumonia 및 Pseudomonas aeruginosa에 대해 매우 높은 항균활성을 나타냈었다. 따라서 본 연구 결과, 높은 항균활성 지닌 CecA는 천잠의 면역 반응에서 중요한 역할을 담당할 것으로 사료된다. A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.

Structure-activity relationships of cecropin-like peptides and their interactions with phospholipid membrane

( Eun Jung Lee ),( Ki Woong Jeong ),( Ju Ho Lee ),( Areum Shin ),( Jin Kyoung Kim ),( June Young Lee ),( Dong Gun Lee ),( Yang Mee Kim ) 생화학분자생물학회 2013 BMB Reports Vol.46 No.5

Cecropin A and papiliocin are novel 37-residue cecropin-like antimicrobial peptides isolated from insect. We have confirmed that papiliocin possess high bacterial cell selectivity and has an α-helical structure from Lys3 to Lys21 and from Ala25 to Val35, linked by a hinge region. In this study, we demonstrated that both peptides showed high antimicrobial activities against multi-drug resistant Gram negative bacteria as well as fungi. Interactions between these cecropin-like peptides and phospholipid membrane were studied using CD, dye leakage experiments, and NMR experiments, showing that both peptides have strong permeabilizing activities against bacterial cell membranes and fungal membranes as well as Trp2 and Phe5 at the N-terminal helix play an important role in attracting cecropin-like peptides to the negatively charged bacterial cell membrane. Cecropin-like peptides can be potent peptide antibiotics against multi-drug resistant Gram negative bacteria and fungi.

Purification and cDNA Cloning of a Cecropin-like Peptide from the Great Wax Moth, Galleria mellonella

김정한,이인희,Joon Ha Lee,김익수,서숙재,손석민,Ki Young Lee 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a pu-tative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calcu-lated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.

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.

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.

천잠 세크로핀 항균펩타이드 분리 및 정제

김성렬 ( Seong Ryul Kim ),구태원 ( Tae Won Goo ),최광호 ( Kwang Ho Choi ),박승원 ( Seung Won Park ),김성완 ( Sung Wan Kim ),황재삼 ( Jae Sam Hwang ),강석우 ( Seok Woo Kang ) 한국잠사학회 2012 한국잠사곤충학회지 Vol.50 No.2

Cecropin is a well-studied antimicrobial peptide that play important role as key factor in insect humoral immunity. In this study, cecropin-like antimicrobial peptide was isolated and purified from the larval haemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. To isolate antimicrobial peptide, we separated and compared acidic extracted hemolymph protein bends between control and immune-challenged larvae using SDS-PAGE analysis. In the immune hemolymph extract, but not of non-immune hemolymph, we detected differential expressed peptide band with molecular mass 4,223.01 Da. To understand this peptide better, we successfully purified this peptide using cation exchange chromatography and gel permeation chromatography. Its N-terminal amino acid sequence obtained by Edman degradation evidenced a significant degree of identity with other lepidopteran cecropins. The purified A. yamamai cecropin-like peptide showed a broad spectrum of activity against fungi, Gram-negative and Gram-positive bacteria.

Molecular Cloning and Characterization of Papiliocin, Cecropin-like Antimicrobial Peptide from the Swallowtail Butterfly, Papilio xuthus

Seong-Ryul Kim,Mee-Yeon Hong,Kwang-Ho Choi,Min-Uk Kang,Tae-Won Goo,Seok-Woo Kang,Kwang-Gill Lee,Iksoo Kim,Jae Sam Hwang 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

Cecropin is an antimicrobial peptide that is synthesized in fat body cells and hemocytes of insect in response to a hypodermic injury or bacterial infection. A 503 bp cDNA encoding a cecropin-like antimicrobial peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE from immunized Papilio xuthus larvae. The open reading frame (ORF) of isolated cDNA encoded a 63 amino acid prepropeptide with a putative 22-residue signal peptide, a 3-residue propeptide and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of peptide showed significant identities with other Lepidopteran cecropins. This peptide was named as papiliocin. RT-PCR revealed that the papiliocin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). Based on the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized by Fmoc method, and analyzed antimicrobial activity. The synthetic papiliocin peptide had a broad spectrum of activity against fungi, Gram-positive and negative bacteria, and also showed no hemolytic activity against human red blood cell.

Characterization and cDNA Cloning of a Cecropin-Like Antimicrobial Peptide, Papiliocin, from the Swallowtail Butterfly, Papilio xuthus

Seong Ryul Kim,Mee Yeon Hong,박승원,최광호,Eun Young Yun,구태원,강석우,Hwa Jin Suh,김익수 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.4

Cecropin is a well-studied antimicrobial peptide that is synthesized in fat body cells and hemocytes of insects in response to hypodermic injury or bacterial infection. A 503 bp cDNA encoding for a cecropin-like peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5′-RACE with immunized Papilio xuthus larvae. The open reading frame of the isolated cDNA encoded for a 62-amino acid prepropeptide with a putative 22-residue signal peptide, a 2-residue propeptide, and a 38-residue mature peptide with a theoretical mass of 4060.89Da. The deduced amino acid sequence of the peptide evidenced a significant degree of identity with other lepidopteran cecropins. This peptide was named papiliocin. RTPCR results revealed that the papiliocin transcript was detected at significant levels after injection with bacterial lipopolysaccharide (LPS). On the basis of the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized via the Fmoc method, and its antimicrobial activity was analyzed. The synthetic papiliocin peptide evidenced a broad spectrum of activity against fungi,Gram-positive and Gram-negative bacteria, and also evidenced no hemolytic activity against human red blood cells.

Characterization and cDNA Cloning of a Cecropin-Like Antimicrobial Peptide, Papiliocin, from the Swallowtail Butterfly, Papilio xuthus

Kim, Seong-Ryul,Hong, Mee-Yeon,Park, Seung-Won,Choi, Kwang-Ho,Yun, Eun-Young,Goo, Tae-Won,Kang, Seok-Woo,Suh, Hwa-Jin,Kim, Ik-Soo,Hwang, Jae-Sam Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.29 No.4

Cecropin is a well-studied antimicrobial peptide that is synthesized in fat body cells and hemocytes of insects in response to hypodermic injury or bacterial infection. A 503 bp cDNA encoding for a cecropin-like peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE with immunized Papilio xuthus larvae. The open reading frame of the isolated cDNA encoded for a 62-amino acid prepropeptide with a putative 22-residue signal peptide, a 2-residue propeptide, and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of the peptide evidenced a significant degree of identity with other lepidopteran cecropins. This peptide was named papiliocin. RTPCR results revealed that the papiliocin transcript was detected at significant levels after injection with bacterial lipopolysaccharide (LPS). On the basis of the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized via the Fmoc method, and its antimicrobial activity was analyzed. The synthetic papiliocin peptide evidenced a broad spectrum of activity against fungi, Gram-positive and Gram-negative bacteria, and also evidenced no hemolytic activity against human red blood cells.

Identification, in silico characterization, and expression analysis of Tenebrio molitor Cecropin‐2

Ali Mohammadie Kojour Maryam,Jang Ho Am,Edosa Tariku Tesfaye,Keshavarz Maryam,Kim Bo Bae,Bae Young Min,Patnaik Bharat Bhusan,Han Yeon Soo,Jo Yong Hun 한국곤충학회 2021 Entomological Research Vol.51 No.2

Antimicrobial peptides (AMPs) are considered to be candidate effectors for eliciting humoral immune responses against infectious pathogens in the host. Cecropins are α‐ helical peptides of 30–40 amino acids, which are known to permeabilize bacterial membranes, and play authoritative roles in the innate immune system of insects. In the present study, we identified the full‐length open reading frame (ORF) encoding the Tenebrio molitor cecropin‐2 (TmCec2) gene using the Tribolium castaneum cecropin‐2 (TcCec2) gene to query a T. molitor Rnaseq database. Phylogenetic analysis revealed close identity of TmCec2 with TcCec2. TmCec2 was ubiquitously expressed in the insect during all developmental stages, with the highest expression observed in the adult. Tissue‐specific TmCec2 expression was highest in larval hemocytes and in the adult integument and hemocytes. Microbial challenge experiments revealed that TmCec2 was highly induced in response to gram‐positive and gram‐negative bacteria, and fungi. These data provide credible evidence for a putative role of TmCec2 in insect innate immunity against a plethora of pathogens.