抗菌肽异源表达的研究进展

刘　辉,张兰威,易华西,韩　雪

(哈尔滨工业大学化学工程与技术学院,黑龙江哈尔滨 150090)



抗菌肽异源表达的研究进展

刘辉,张兰威*,易华西,韩雪

(哈尔滨工业大学化学工程与技术学院,黑龙江哈尔滨 150090)

抗菌肽是一种小肽类物质,可以抑制多种细菌、真菌、病毒以及抗生素抗性细菌。但是抗菌肽产量低提取工艺繁琐,制约其工业化生产和科学研究。异源表达技术为提高抗菌肽产量提供了十分有效的途径。目前抗菌肽异源表达系统,主要包括大肠杆菌和酵母菌表达系统。本文主要综述了大肠杆菌和酵母菌表达系统的优缺点以及发展方向,为利用生物技术方法提高抗菌肽产量提供理论依据和技术支持。

抗菌肽,异源表达,大肠杆菌,酵母菌

抗菌肽是由微生物核糖体合成的一种小肽类抑菌物质。抗菌肽可以有效的抑制细菌、真菌和病毒,被认为是一种天然无毒的食品防腐剂[1]。近几年,研究发现抗菌肽可以调节免疫系统,抑制炎性因子以及肿瘤细菌和癌细胞的作用[2-4]。抗菌肽的广谱作用,使得越来越多的新型抗菌肽被发现。抗菌肽性质、机理、结构以及功能研究,需要大量的高纯度的抗菌肽。目前,获得高纯度的抗菌肽主要有三个途径:源料提取、化学合成以及异源表达。异源表达抗菌肽的优势在于生产成本低,提取工艺简单,产量高,更好的满足生产和研究所需的抗菌肽[5]。虽然异源表达技术为提高抗菌肽产量提供了有效途径,但基因表达的效果往往依赖于表达载体、宿主菌、分泌蛋白所需要的信号序列以及外部因素,如:温度、诱导剂等[6-7]。在异源表达抗菌肽表达方面大肠杆菌和酵母菌是最为常用的表达宿主。虽然大肠杆菌作为宿主的占80%以上,但是酵母菌的表达也有其独特的优势,如肽类分子折叠、二硫键的形成和基团修饰方面[8-9]。本文综述了目前在抗菌肽异源表达方面的研究和表达系统的最新进展。

1　大肠杆菌表达抗菌肽

在大肠杆菌表达系统中质粒主要应用是Novagen公司的pET系列,表达宿主最为常用的是Novagen公司的BL21(DE3)、pLys S Origami、Rosetta和C41(DE3)大肠杆菌宿主[10]。利用大肠杆菌异源表达抗菌肽主要存在两个问题:如何减少表达后抗菌肽对宿主的毒性[11]，如何正确表达抗菌肽[12]。研究人员最为常用的两种方法是:将抗菌肽基因与融合蛋白基因共同表达，密码子优化。

1.1融合蛋白辅助表达抗菌肽

表1　大肠杆菌表达抗菌肽的主要融合蛋白及产量

注:ND:表示没有测定；表2同。

利用大肠杆菌表达系统的主要问题是如何减少抗菌肽表达后对宿主的毒性。在抗菌肽基因附加一个阴离子特性的融合蛋白基因(这种融合蛋白并无抑菌性),可以有效减少抗菌肽对宿主的毒性[13]。融合蛋白表达后,往往包含着特殊的理化性质和结构,可以中和抗菌肽的阳离子,阻止表达后的抗菌肽对宿主的毒性。含融合蛋白的抗菌肽可以通过化学试剂特异性切除融合蛋白,恢复抗菌肽的抑菌活力[14-15]。目前有很多融合蛋白在抗菌肽的表达中得到应用,最为常用的硫氧还原蛋白、His标签以及谷胱甘肽转移酶(表1)。Pediocin PA-1融合了ABC转运子和辅助蛋白,不仅正确表达PA-1中二硫键而且对宿主并无毒害作用[16]。Divergicin A在融合了碱性磷酸酶后,在大肠杆菌发酵液中融合体抗菌肽对宿主无伤害[17]。融合蛋白PurF已经使得抗菌肽Magainin和Pleurocidin达到毫克的产量,buforin IIb达到克的产量[18-20]。Wang等人利用小分子融合蛋白(SUMO)在大肠杆菌中表达Bacterocin E50-52,经纯化后达到16 mg[21]。Yu等人利用SUMO融合蛋白表达出抗菌肽Lacticin Q,经纯化后达到2.5 mg[22]。Chen等人利用绿色荧光蛋白(GFP)成功在大肠杆菌中表达出新型抗菌肽NB-C1表达量为2.2 mg/mL[23]。虽然融合蛋白帮助抗菌肽成功表达,但是融合蛋白也存在着不足,它很容易在细胞内部形成包涵体而导致表达产物不溶,因此必需加入特定的蛋白酶或化学试剂(Factor Xa、Hydroxylamine、CNBr、Enterokinase)切出融合蛋白才能让抗菌肽溶于提取液中,但这样就会导致抗菌肽产量造成2～8倍的损失[10,24]。

Morin等人在融合蛋白的基础上设计出一种新的方法,将一个融合蛋白与多个抗菌肽indolicidin基因连接,表达出三聚体或六聚体的融合蛋白与抗菌肽复合物,经纯化后用溴化氢切除融合蛋白释放抗菌肽单体[41]。既减少抗菌肽对宿主的毒性同时又提高抗菌肽的产量,Rao和Peng等人分别使得His-Tagged融合蛋白和GST融合蛋白与多个抗菌肽β-defensin 2串连基因表达,提高了抗菌肽β-defensin 2的产量[6,42]。这种技术应用性不强,因为重组重复抗菌肽基因,并不能保证一定会形成聚集体,部份抗菌肽形成聚集体也会对宿主有毒性。

内含肽融合蛋白的提出解决抗菌肽去除融合蛋白的问题。这种技术的优势是通过在一定条件下纯化抗菌肽,诱导抗菌肽自身切除融合体而释放出有活性的抗菌肽。这个系统要求有一个恰当的内含肽与抗菌肽的融合体,它不能与其它融合蛋白酶切位相同。抗菌肽OG2和乳酸菌Class IIa抗菌肽bacterocins BacR1、divercin V41、enterocin P、pediocin PA-1和piscicolin 126利用这种技术成功的表达[43-45]。这种表达系统缺点在于只能表达小分子量的蛋白,而且对于质粒的构建是要求十分严格,内含肽融合蛋白使得抗菌肽失去活力,因此在抗菌肽的表达方面,内含肽的应用还有待进一步的研究。

1.2密码子优化

在提高抗菌肽遗传稳定性方面,密码子优化是最为有效的方法。通过对不同来源抗菌肽的遗传密码子优化,达到宿主本身的密码子偏好,可以使抗菌肽mRNA稳定转录和翻译,提高抗菌肽的稳定性[46]。优化后的抗菌肽密码子不仅可以有效的合成抗菌肽,而且还可以减少抗菌肽对宿主细胞的毒性[47-48]。Richard等人优化抗菌肽divercin V41编码基因并与组氨酸标签以及硫氧还原蛋白融合后成功表达,divercin V41产量达到23 mg/L。抗菌肽Hepcidin的基因通过密码子优化后成功在大肠杆菌中表达[49]。Ingham等人通过优化多种乳酸菌Class IIa抗菌肽基因,并通过与内含肽融合蛋白融合,成功表达多种Class IIa抗菌肽[24]。研究表明在一定区域内重复插入优化后抗菌肽的基因单元,可以有效提高抗菌肽的表达率。利用这种方法抗菌肽β-defensin-2产量提高了6倍[50]。该方法在乳酸菌抗菌肽没有得到很好的应用,但是在其它类型的抗菌肽已经得到成功应用[50-51]。

2　酵母表达抗菌肽

利用大肠杆菌表达抗菌肽是一种有效的方法,但是却无法表达一些修饰型抗菌肽[10]。酵母菌表达宿主是一种可以表达修饰蛋白的表达宿主。目前酵母菌宿主主要有酿酒酵母和毕赤酵母。近些年来酵母表达宿主也逐渐丰富,包括安格斯毕赤酵母(Pichia angusta)、解脂耶氏酵母(Yarrowia lipolytica)[52-53]。酵母表达系统与大肠杆菌表达系统相比,主要体现在翻译后对蛋白的修饰作用,比如糖基化的修饰[54]。尽管酵母菌表达抗菌肽的时间比大肠杆菌表达的时间长,但是酵母菌表达的可修饰的抗菌肽可以分泌到胞外,更重要的是酵母表达并不需要辅助蛋白(大肠杆菌表达必需)[55]。

表2　抗菌肽在毕赤酵母中表达

2.1酿酒酵母异源表达抗菌肽

酿酒酵母是酵母异源表达重组蛋白的主要宿主。因为对于酿酒酵母的理化性质和相关基因都十分明确,在表达蛋白方面也取得了丰硕的研究成果[56]。在一些条件下,酿酒酵母比大肠杆菌能更好的表达可溶性蛋白,并且表达量可以满足结构分析[57]。酿酒酵母表达抗菌肽存在一些限制因素阻碍了其在抗菌肽表达方面的应用,例如抗菌肽PA-1虽然在酿酒酵母中成功表达,但是其产量水平却没有报道[58];但是在毕赤酵母中抗菌肽PA-1的产量达到74 μg/mL[59]。Van等人利用酿酒酵母成功表达出抗菌肽Plantaricin 423,但也无法定量[60]。此外,酿酒酵母是一种发酵型酵母,可以发酵糖类物质产生乙醇,乙醇又会影响其菌群数量,导致蛋白或肽类的产量低[55]。这些不利的因素,使得酿酒酵母并不适合用于大规模发酵产生异源抗菌肽。

2.2毕赤酵母表达异源抗菌肽

近几年,毕赤酵母作为异源表达抗菌肽宿主的研究报道明显增加。十年前,大约只有400多种蛋白利用毕赤酵母表达[61]。如今毕赤酵母表达多达上千种,在2009年美国食品药品管理局第一次通过了利用毕赤酵母表达的重组药品上市,更是促进了毕赤酵母在表达方面的应用[55]。与酿酒酵母不同,毕赤酵母不是发酵型微生物,菌体浓度可以达到100～200 g/L,而且其代谢产物并无毒性,也不会产生乙醇。毕赤酵母的另外一个优势是,易扩大培养、培养基成本低、易纯化、可以表达具有修饰作用的蛋白[61]。相比与酿酒酵母和大肠杆菌中限制性表达因素,毕赤酵母的表达启动子相对简单(采用AOX启动子,利用甲醇启动表达系统)。毕赤酵母表达抗菌肽主要分为两步:高密度培养菌体;加入甲醇诱导重组蛋白的表达。这一过程的优势在于在表达抗菌肽之前,可以得到大量的菌体,避免了重组抗菌肽对宿主的毒性。

毕赤酵母表达不同的抗菌肽,表达量也不相同(表2)。在大肠杆菌或酿酒酵母表达的抗菌肽,在毕赤酵母中表达产量有所提高[62-63]。部分抗菌肽在大肠杆菌表达失败后,在毕赤酵母中可以很好的表达,例如抗菌肽defensin SPE10[64]。从表达产物的结构来说,抗菌肽defensin PDC1分别在大肠杆菌和毕赤酵母菌表达,两者表达的抗菌肽都有抗真菌的活性,但是毕赤酵母表达量是大肠杆菌的2倍。比较两种表达产物的结构,发现在毕赤酵母表达的抗菌肽defensin PDC1结构中具有较多的β-折叠,而无规则卷曲较少[65]。另外毕赤酵母要可以更好的表达抗菌肽的活性,Basanta等人利用酿酒酵母和毕赤酵母表达了无前导肽序列的enterocinL50A和50B,成功表达了双肽链抗菌肽enterocinL50A和L50B,但是毕赤酵母表达的两种抗菌肽活力分别是酿酒酵母的6～60倍[66-67]。目前大多数利用毕赤酵母表达异源抗菌肽依然处于实验室阶段。主要是在毕赤酵母表达中需要一个适合的启动子伴随表达,从而达到产业化生产所需的产量。

3　结论与展望

抗菌肽在食品、药品、化妆品、农业方面的应用越来越广泛,吸引大量的研究人员开发和利用抗菌肽。异源表达技术很好的解决抗菌肽产量不足的问题。通过对启动子、信号肽、融合蛋白以及相关的辅助因子的估化,成功表达了抗菌肽并且抗菌肽的产量已经有大范围的提升，为研究新的表达系统和发酵工艺提供理论依据,为建立大规模表达抗菌肽的方法提供思路。抗菌肽的种类繁多,不同的抗菌肽在大规模生产技术上依然存在困难,如抗菌肽稳定性、提取工艺、抗菌肽的聚集、抗菌肽表达后的修饰、降低成本等。随着基因工程技术的不断的发展和改良,这些困难有望逐一得到解决。希望研究人员利用异源系统,生产出用途更广、种类更多、效果更好的抗菌肽产品。

[1]Gálvez A,Abriouel H,López R L,et al. Bacteriocin-Based Strategies for Food Biopreservation[J]. International Journal of Food Microbiology,2007,120(1):51-70.

[2]Kamarajan P,Hayami T,Matte B,et al. Nisin Zp,a Bacteriocin and Food Preservative,Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival[J]. PloS one,2015,10(7):1-20.

[3]Choi K-Y,Chow L N,Mookherjee N. Cationic Host Defence Peptides:Multifaceted Role in Immune Modulation and Inflammation[J]. Journal of Innate Immunity,2012,4(4):361-370.

[4]Hilchie A L,Wuerth K,Hancock R E. Immune Modulation by Multifaceted Cationic Host Defense(Antimicrobial)Peptides[J]. Nature Chemical Biology,2013,9(12):761-768.

[5]Klint J K,Senff S,Saez N J,et al. Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis Via Expression in the Periplasm ofE.Coli[J]. Periplasmic Expression of Disulfide-Rich Peptides，2013,8(5):e63865.

[6]Rao X,Hu J,Li S,et al. Design and Expression of Peptide Antibiotic Hpab-Β as Tandem Multimers in Escherichia Coli[J]. Peptides,2005,26(5):721-729.

[7]Xu X,Jin F,Yu X,et al. Expression and Purification of a Recombinant Antibacterial Peptide,Cecropin,from Escherichia Coli[J]. Protein Expression and Purification,2007,53(2):293-301.

[8]Li Y,Chen Z. Rapd:A Database of Recombinantly-Produced Antimicrobial Peptides[J]. FEMS Microbiology Letters,2008,289(2):126-129.

[9]Guo C,Huang Y,Zheng H,et al. Secretion and Activity of Antimicrobial Peptide Cecropin D Expressed in Pichia Pastoris[J]. Experimental and Therapeutic Medicine,2012,4(6):1063-1068.

[10]Parachin N S,Mulder K C,Viana A A B,et al. Expression Systems for Heterologous Production of Antimicrobial Peptides[J]. Peptides,2012,38(2):446-456.

[11]Choi J H,Lee S Y. Secretory and Extracellular Production of Recombinant Proteins Using Escherichia Coli[J]. Applied Microbiology and Biotechnology,2004,64(5):625-635.

[12]Morello E,Bermudez-Humaran L G,Llull D,et al. Lactococcus Lactis,an Efficient Cell Factory for Recombinant Protein Production and Secretion[J]. Journal of Molecular Microbiology and Biotechnology,2008,14(1-3):48-58.

[13]Metlitskaia L,Cabralda J E,Suleman D,et al. Recombinant Antimicrobial Peptides Efficiently Produced Using Novel Cloning and Purification Processes[J]. Biotechnology and Applied Biochemistry,2004,39(3):339-345.

[14]Li Y. Recombinant Production of Antimicrobial Peptides in Escherichia Coli:A Review[J]. Protein Expression and Purification,2011,80(2):260-267.

[15]Sørensen H P,Mortensen K K. Soluble Expression of Recombinant Proteins in the Cytoplasm of Escherichia Coli[J]. Microbial Cell Factories,2005,4(1):1.

[16]Oppegård C,Fimland G,Haug Anonsen J,et al. The Pediocin Pa-1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin Pa-1[J]. Biochemistry,2015(54):2967-2974.

[17]Worobo R W,Van Belkum M J,Sailer M,et al. A Signal Peptide Secretion-Dependent Bacteriocin from Carnobacterium Divergens[J]. Journal of Bacteriology,1995,177(11):3143-3149.

[18]Lee J,Kim J,Hwang S,et al. High-Level Expression of Antimicrobial Peptide Mediated by a Fusion Partner Reinforcing Formation of Inclusion Bodies[J]. Biochemical and Biophysical Research Communications,2000,277(3):575-580.

[19]Bryksa B C,MacDonald L D,Patrzykat A,et al. A C-Terminal Glycine Suppresses Production of Pleurocidin as a Fusion Peptide in Escherichia Coli[J]. Protein Expression and Purification,2006,45(1):88-98.

[20]Pyo S-H,Lee J-H,Park H-B,et al. Expression and Purification of a Recombinant Buforin Derivative from Escherichia Coli[J]. Process Biochemistry,2004,39(11):1731-1736.

[21]Wang Q,Fu W,Ma Q,et al. Production of Bacteriocin E50-52 by Small Ubiquitin-Related Modifier Fusion in Escherichia Coli[J]. Polish Journal of Microbiology,2013,62(4):345-350.

[22]Yu Z,Wang Q,Ma Q,et al. Secretory Expression of Lacticin Q Fused with Sumo in Bacillus Subtilis[J]. Protein Expression and Purification,2013,89(1):51-55.

[23]Chen H,Yan X,Tian F,et al. Cloning,Expression,and Identification of a Novel Class Iia Bacteriocin in the Escherichia Coli Cell-Free Protein Expression System[J]. Biotechnology Letters,2012,34(2):359-364.

[24]Ingham A B,Moore R J. Recombinant Production of Antimicrobial Peptides in Heterologous Microbial Systems[J]. Biotechnology and Applied Biochemistry,2007,47(1):1-9.

[25]Liu S-n,Han Y,Zhou Z-j. Fusion Expression of Peda Gene to Obtain Biologically Active Pediocin Pa-1 in Escherichia Coli[J]. Journal of Zhejiang University Science B,2011,12(1):65-71.

[26]Meng F,Zhao H,Zhang C,et al. Expression of a Novel Bacteriocin—the Plantaricin Pln1—in Escherichia Coli and Its Functional Analysis[J]. Protein Expression and Purification,2016,119:85-93.

[27]Wang Q,Zhu F,Xin Y,et al. Expression and Purification of Antimicrobial Peptide Buforin Iib in Escherichia Coli[J]. Biotechnology Letters,2011,33(11):2121-2126.

[28]Xie Y-G,Luan C,Zhang H-W,et al. Effects of Thioredoxin:Sumo and Intein on Soluble Fusion Expression of an Antimicrobial Peptide Og2 in Escherichia Coli[J]. Protein and Peptide Letters,2013,20(1):54-60.

[29]Huang L,Wang J,Zhong Z,et al. Production of Bioactive Human Β-Defensin-3 in Escherichia Coli by Soluble Fusion Expression[J]. Biotechnology Letters,2006,28(9):627-632.

[30]Xu Z,Zhong Z,Huang L,et al. High-Level Production of Bioactive Human Beta-Defensin-4 in Escherichia Coli by Soluble Fusion Expression[J]. Applied Microbiology and Biotechnology,2006,72(3):471-479.

[31]Huang L,Ching C B,Jiang R,et al. Production of Bioactive Human Beta-Defensin 5 and 6 in Escherichia Coli by Soluble Fusion Expression[J]. Protein Expression and Purification,2008,61(2):168-174.

[32]Hernandez-Garcia C M,Bouchard R A,Rushton P J,et al. High Level Transgenic Expression of Soybean(Glycine Max)Gmerf and Gmubi Gene Promoters Isolated by a Novel Promoter Analysis Pipeline[J]. BMC Plant Biology,2010,10(1):237.

[33]Huang L,Leong S S J,Jiang R. Soluble Fusion Expression and Characterization of Bioactive Human Beta-Defensin 26 and 27[J]. Applied Microbiology and Biotechnology,2009,84(2):301-308.

[34]Song D,Chen Y,Li X,et al. Heterologous Expression and Purification of Dermaseptin S4 Fusion in Escherichia coli and Recovery of Biological Activity[J]. Preparative Biochemistry and Biotechnology,2014,44(6):598-607.

[35]Song D,Li X,Zhang Y,et al. Mutational Analysis of Positively Charged Residues in the N‐Terminal Region of the Class Iia Bacteriocin Pediocin Pa‐1[J]. Letters in Applied Microbiology,2014,58(4):356-361.

[36]Feng X-J,Xing L-W,Liu D,et al. Design and High-Level Expression of a Hybrid Antimicrobial Peptide Lf15-Ca8 in Escherichia Coli[J]. Journal of Industrial Microbiology & Biotechnology,2014,41(3):527-534.

[37]Zhang T,Pan Y,Li B,et al. Molecular Cloning and Antimicrobial Activity of Enterolysin a and Helveticin J of Bacteriolysins from Metagenome of Chinese Traditional Fermented Foods[J]. Food Control,2013,31(2):499-507.

[38]Richard C,Drider D,Elmorjani K,et al. Heterologous Expression and Purification of Active Divercin V41,a Class Iia Bacteriocin Encoded by a Synthetic Gene in Escherichia Coli[J]. J Bacteriol,2004,186(13):4276-4284.

[39]Yildirim S,Konrad D,Calvez S,et al. Production of Recombinant Bacteriocin Divercin V41 by High Cell Density Escherichia Coli Batch and Fed-Batch Cultures[J]. Applied Microbiology and Biotechnology,2007,77(3):525-531.

[40]Pal G,Srivastava S. Cloning and Heterologous Expression of Plne,-F,-J and-K Genes Derived from Soil Metagenome and Purification of Active Plantaricin Peptides[J]. Applied Microbiology and Biotechnology,2014,98(3):1441-1447.

[41]Morin K,Arcidiacono S,Beckwitt R,et al. Recombinant Expression of Indolicidin Concatamers in Escherichia Coli[J]. Applied Microbiology and Biotechnology,2006,70(6):698-704.

[42]Peng L,Xu Z,Fang X,et al. High-Level Expression of Soluble Human Β-Defensin-2 in Escherichia Coli[J]. Process Biochemistry,2004,39(12):2199-2205.

[43]Ingham A,Sproat K,Tizard M,et al. A Versatile System for the Expression of Nonmodified Bacteriocins in Escherichia Coli[J]. Journal of Applied Microbiology,2005,98(3):676-683.

[44]Le T N,Do T H,Nguyen T N,et al. Expression and Simple Purification Strategy for the Generation of Anti-Microbial Active Enterocin P from Enterococcus Faecium Expressed in Escherichia coli Er2566[J]. Iranian Journal of Biotechnology,2015,12(4):17-25.

[45]Xie Y-G,Han F-F,Luan C,et al. High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide Og2 Using the Intein System in Escherichia Coli[J]. BioMed Research International,2013,2013:1-6.

[46]Mathiesen G,Namløs H,Risøen P,et al. Use of Bacteriocin Promoters for Gene Expression in Lactobacillus Plantarum C11[J]. Journal of Applied Microbiology,2004,96(4):819-827.

[47]Richard C,Drider D,Elmorjani K,et al. Heterologous Expression and Purification of Active Divercin V41,a Class Iia Bacteriocin Encoded by a Synthetic Gene in Escherichia Coli[J]. Journal of Bacteriology,2004,186(13):4276-4284.

[48]Pazgier M,Lubkowski J. Expression and Purification of Recombinant Human Α-Defensins in Escherichia Coli[J]. Protein Expression and Purification,2006,49(1):1-8.

[49]Zhang H,Yuan Q,Zhu Y,et al. Expression and Preparation of Recombinant Hepcidin in Escherichia coli[J]. Protein Expression and Purification,2005,41(2):409-416.

[50]Zhong Z,Xu Z,Peng L,et al. Tandem Repeat Mhbd2 Gene Enhance the Soluble Fusion Expression of Hbd2 in Escherichia Coli[J]. Applied Microbiology and Biotechnology,2006,71(5):661-667.

[51]Xu Z,Peng L,Zhong Z,et al. High‐Level Expression of a Soluble Functional Antimicrobial Peptide,Human Β‐Defensin 2,in Escherichia coli[J]. Biotechnology Progress,2006,22(2):382-386.

[52]Gellissen G,Kunze G,Gaillardin C,et al. New Yeast Expression Platforms Based on Methylotrophic Hansenula Polymorpha and Pichia Pastoris and on Dimorphic Arxula Adeninivorans and Yarrowia Lipolytica-a Comparison[J]. Fems Yeast Research,2005,5(11):1079-1096.

[53]Boer E,Steinborn G,Kunze G,et al. Yeast Expression Platforms[J]. Applied Microbiology and Biotechnology,2007,77(3):513-523.

[54]Cregg J M,Tolstorukov I,Kusari A,et al. Expression of Recombinant Genes in the Yeast Pichia Pastoris[J]. Current Protocols Essential Laboratory Techniques,2009(463):169-189.

[55]Mattanovich D,Branduardi P,Dato L,et al. Recombinant Gene Expression[J]. Springer,2012(824):329-358.

[56]Romanos M A,Scorer C A,Clare J J. Foreign Gene Expression in Yeast:A Review[J]. Yeast,1992,8(6):423-488.

[57]Quartley E,Alexandrov A,Mikucki M,et al. Heterologous Expression of L. Major Proteins in S. Cerevisiae:A Test of Solubility,Purity,and Gene Recoding[J]. Journal of Structural and Functional Genomics,2009,10(3):233-247.

[58]Schoeman H,Vivier M A,Du Toit M,et al. The Development of Bactericidal Yeast Strains by Expressing the Pediococcus Acidilactici Pediocin Gene(Peda)in Saccharomyces Cerevisiae[J]. Yeast,1999,15(8):647-656.

[59]Beaulieu L,Groleau D,Miguez C B,et al. Production of Pediocin Pa-1 in the Methylotrophic Yeast Pichia Pastoris Reveals Unexpected Inhibition of Its Biological Activity Due to the Presence of Collagen-Like Material[J]. Protein Expression and Purification,2005,43(2):111-125.

[60]Van Reenen C,Chikindas M,Van Zyl W,et al. Characterization and Heterologous Expression of a Class Iia Bacteriocin,Plantaricin 423 from Lactobacillus Plantarum 423,in Saccharomyces Cerevisiae[J]. International Journal of Food Microbiology,2003,81(1):29-40.

[61]Cereghino G P L,Cereghino J L,Ilgen C,et al. Production of Recombinant Proteins in Fermenter Cultures of the Yeast Pichia Pastoris[J]. Current Opinion in Biotechnology,2002,13(4):329-332.

[62]Li L,Wang J-X,Zhao X-F,et al. High Level Expression,Purification,and Characterization of the Shrimp Antimicrobial Peptide,Ch-Penaeidin,in Pichia Pastoris[J]. Protein Expression and Purification,2005,39(2):144-151.

[63]Peng H,Liu H-P,Chen B,et al. Optimized Production of Scygonadin in Pichia Pastoris and Analysis of Its Antimicrobial and Antiviral Activities[J]. Protein Expression and Purification,2012,82(1):37-44.

[64]Song X,Wang J,Wu F,et al. Cdna Cloning,Functional Expression and Antifungal Activities of a Dimeric Plant Defensin Spe10 from Pachyrrhizus Erosus Seeds[J]. Plant Molecular Biology,2005,57(1):13-20.

[65]Kant P,Liu W-Z,Pauls K P. Pdc1,a Corn Defensin Peptide Expressed in Escherichia Coli and Pichia Pastoris Inhibits Growth of Fusarium Graminearum[J]. Peptides,2009,30(9):1593-1599.

[66]Basanta A,Herranz C,Gutierrez J,et al. Development of Bacteriocinogenic Strains of Saccharomyces Cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50a and L50b from Enterococcus Faecium L50[J]. Applied and Environmental Microbiology,2009,75(8):2382-2392.

[67]Basanta A,Gomez-Sala B,Sanchez J,et al. Use of the Yeast Pichia Pastoris as an Expression Host for Secretion of Enterocin L50,a Leaderless Two-Peptide(L50a and L50b)Bacteriocin from Enterococcus Faecium L50[J]. Applied and Environmental Microbiology,2010,76(10):3314-3324.

[68]Niu M,Chai S,You X,et al. Expression of Porcine Protegrin-1 in Pichia Pastoris and Its Anticancer ActivityinVitro[J]. Experimental and Therapeutic Medicine,2015,9(3):1075-1079.

[69]Wang X,Wang X,Teng D,et al. Recombinant Production of the Antimicrobial Peptide Nz17074 in Pichia Pastoris Using Sumo3 as a Fusion Partner[J]. Letters in Applied Microbiology,2014,59(1):71-78.

[70]Basanta A,Gómez-Sala B,Sánchez J,et al. Use of the Yeast Pichia Pastoris as an Expression Host for Secretion of Enterocin L50,a Leaderless Two-Peptide(L50a and L50b)Bacteriocin from Enterococcus Faecium L50[J]. Applied and Environmental Microbiology,2010,76(10):3314-3324.

[71]Peng Z,Wang A,Feng Q,et al. High-Level Expression,Purification and Characterisation of Porcine Β-Defensin 2 in Pichia Pastoris and Its Potential as a Cost-Efficient Growth Promoter in Porcine Feed[J]. Applied Microbiology and Biotechnology,2014,98(12):5487-5497.

[72]López-García B,Moreno A B,San Segundo B,et al. Production of the Biotechnologically Relevant Afp from Aspergillus Giganteus in the Yeast Pichia Pastoris[J]. Protein Expression and Purification,2010,70(2):206-210.

Heterologous expression systems for recombinant production of antimicrobial peptides

LIU Hui,ZHANG Lan-wei*,YI Hua-xi,HAN Xue

(School of Chemcial Engneering and Technology,Harbin Institute of Technology,Harbin 150090,China)

Antimicrobial peptide(AMP)is a kind of small peptides. AMP has the inhibition of various bacteria,fungi and viruses. However,the low quantity of peptides and complex purification process are still a remarkable bottleneck for scientific and industrial research development. Heterologous expression technology provides a effective way to improve the production of AMP. The main heterologous systems currently used for AMP production,including bacteria,yeast. This review described the heterologous expression system of antimicrobial peptide,compared the advantages and disadvantages in each expression system. It was designed to provide the basis of theoretical and technical support for the mass production of antimicrobial peptides by biotechnological tools.

antimicrobial peptides;heterologous expression;E.coli;yeast

2015-11-30

刘辉(1982-)，男，博士研究生，研究方向：食品防腐剂，E-mail:liuhui_hit@foxmail.com。

张兰威(1961-)，男，博士，教授，研究方向：乳品科学、食品发酵，E-mail:zhanglw@hit.edu.cn。

国家自然科学学基金(31271906/C2002204，31571850/C200502)；高等学校博士学科点专项科研基金(20112302110051)。

TS201.3

A

1002-0306(2016)12-0380-06

10.13386/j.issn1002-0306.2016.12.064