Design,Characterization and Expression of a Novel Hybrid Peptide Melittin(1-13)-LL37(17-30)

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• 英文论文题名：Design,Characterization and Expression of a Novel Hybrid Peptide Melittin(1-13)-LL37(17-30)
• 论文作者：Xubiao Wei ; Dayong Si ; Zhaojun Zheng ; Lulu Zhang ; Rujuan Wu ; Rijun Zhang
• 英文论文作者：Xubiao Wei ; Dayong Si ; Zhaojun Zheng ; Lulu Zhang ; Rujuan Wu ; Rijun Zhang ; Laboratory of Feed Biotechnology ; State Key Laboratory of Animal Nutrition ; College of Animal Science and Technology ; China Agricultural University
• 年：2016
• 作者机构：Laboratory of Feed Biotechnology,State Key Laboratory of Animal Nutrition,College of Animal Science and Technology,China Agricultural University;
• 英文论文关键词：Hybrid peptide ; Melittin(1-13)-LL-37(17-30) ; Antibacterial activity ; Hemolytic activity ; Escherichia coli ; Fusion expression
• 会议召开时间：2016-11-04
• 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
• 语种：英文
• 分类号：Q51
• 学会代码：ZGXJ
• 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
• 会议地点：中国山东青岛
• 主办单位：中国畜牧兽医学会动物微生态学分会
• 学会名称：中国畜牧兽医学会
• 页数：1
• 文件大小：71k
• 原文格式：O
• 会议级别：全国

摘要

Both Melittin(ME) and LL37(L) are small linear helical peptides with multiply effects including antibacterial,anti-inflammation,anti-cancer and so on[1-3].However,they also have the undesirable property of cytotoxicity to red blood cells at a lower concentration(2 μM and 25-30 mM) which largely limit application widely in clinic[1-3].Recently,hybridizing of different antimicrobial peptides(AMPs) has been a common practice for obtaining novel hybrid AMPs with elevated antibacterial activity but minimized cytotoxicity[4,5].The hybrid peptide ME(1-13)-L(17-30)(M-L)combining the hydrophobic N-terminal fragment of melittin(M) with the core antibacterial fragment of LL37(L),was designed for the first time to explore its antibacterial activity and hemolytic activity against bacteria and sheep erythrocyte respectively.Results showed that M-L had an even more potent antibacterial activity against all indicator strains(especially gram-positive bacteria) than M and L,whereas did not exhibit hemolytic activity to sheep erythrocytes,implying M-L can be served as a potential therapeutic drug to substitute traditional antibiotics.However,the high expense of biosynthesis limited its further research,therefore fusion expression of M-L was carried out in Escherichia coli(E.coli)for overproducing the hybrid peptide so as to solve the problem.The DNA sequence encoding M-L with preferred codons was cloned into the pET-SUMO vector for protein expression in E.coli BL21(DE3).After IPTG induction,approximately 165 mg soluble fusion protein SUMO-M-L was recovered per liter supernatant of the fermentation ultrasonic lysate using Ni-NTA sepharose column(92%purity).And 23 mg recombinant M-L was obtained per liter culture after cleavage of SUMO protease and purification of Ni-NTA sepharose column.In sum,this research not only supplied an effective approach for overproducing hybrid peptide M-L,but paved the way for its further exploration on pharmaceutical potential and medical importance.
Both Melittin(ME) and LL37(L) are small linear helical peptides with multiply effects including antibacterial,anti-inflammation,anti-cancer and so on[1-3].However,they also have the undesirable property of cytotoxicity to red blood cells at a lower concentration(2 μM and 25-30 mM) which largely limit application widely in clinic[1-3].Recently,hybridizing of different antimicrobial peptides(AMPs) has been a common practice for obtaining novel hybrid AMPs with elevated antibacterial activity but minimized cytotoxicity[4,5].The hybrid peptide ME(1-13)-L(17-30)(M-L)combining the hydrophobic N-terminal fragment of melittin(M) with the core antibacterial fragment of LL37(L),was designed for the first time to explore its antibacterial activity and hemolytic activity against bacteria and sheep erythrocyte respectively.Results showed that M-L had an even more potent antibacterial activity against all indicator strains(especially gram-positive bacteria) than M and L,whereas did not exhibit hemolytic activity to sheep erythrocytes,implying M-L can be served as a potential therapeutic drug to substitute traditional antibiotics.However,the high expense of biosynthesis limited its further research,therefore fusion expression of M-L was carried out in Escherichia coli(E.coli)for overproducing the hybrid peptide so as to solve the problem.The DNA sequence encoding M-L with preferred codons was cloned into the pET-SUMO vector for protein expression in E.coli BL21(DE3).After IPTG induction,approximately 165 mg soluble fusion protein SUMO-M-L was recovered per liter supernatant of the fermentation ultrasonic lysate using Ni-NTA sepharose column(92%purity).And 23 mg recombinant M-L was obtained per liter culture after cleavage of SUMO protease and purification of Ni-NTA sepharose column.In sum,this research not only supplied an effective approach for overproducing hybrid peptide M-L,but paved the way for its further exploration on pharmaceutical potential and medical importance.

引文

[1]G.Kreil,H.Bachmayer,Biosynthesis of melittin,a toxic peptide from bee venom.Detection of a possible precursor.Eur J Biochem 20(1971)344-350.
    [2]S.Y.Shin,M.K.Lee,K.L.Kim,K.S.Hahm,Structure-antitumor and hemolytic activity relationships of synthetic peptides derived from cecropin A-magainin 2 and cecropin A-melittin hybrid peptides.J Pept Res 50(1997)279-285.
    [3]D.W.Hoskin,A.Ramamoorthy,Studies on anticancer activities of antimicrobial peptides.Biochim Biophys Acta1778(2008)357-375.
    [4]M.Bastos,G.Bai,P.Gomes,D.Andreu,E.Goormaghtigh,M.Prieto,Energetics and partition of two cecropin-melittin hybrid peptides to model membranes of different composition.BIOPHYS J 94(2008)2128-2141.
    [5]S.Y.Shin,J.H.Kang,K.S.Hahm,Structure-antibacterial,antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides.J Pept Res 53(1999)82-90