劉　輝,張?zhí)m威,易華西,韓　雪

(哈爾濱工業(yè)大學化學工程與技術(shù)學院,黑龍江哈爾濱 150090)

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抗菌肽異源表達的研究進展

劉輝,張?zhí)m威*,易華西,韓雪

(哈爾濱工業(yè)大學化學工程與技術(shù)學院,黑龍江哈爾濱 150090)

抗菌肽是一種小肽類物質(zhì),可以抑制多種細菌、真菌、病毒以及抗生素抗性細菌。但是抗菌肽產(chǎn)量低提取工藝繁瑣,制約其工業(yè)化生產(chǎn)和科學研究。異源表達技術(shù)為提高抗菌肽產(chǎn)量提供了十分有效的途徑。目前抗菌肽異源表達系統(tǒng),主要包括大腸桿菌和酵母菌表達系統(tǒng)。本文主要綜述了大腸桿菌和酵母菌表達系統(tǒng)的優(yōu)缺點以及發(fā)展方向,為利用生物技術(shù)方法提高抗菌肽產(chǎn)量提供理論依據(jù)和技術(shù)支持。

抗菌肽,異源表達,大腸桿菌,酵母菌

抗菌肽是由微生物核糖體合成的一種小肽類抑菌物質(zhì)?？咕目梢杂行У囊种萍毦?、真菌和病毒,被認為是一種天然無毒的食品防腐劑[1]。近幾年,研究發(fā)現(xiàn)抗菌肽可以調(diào)節(jié)免疫系統(tǒng),抑制炎性因子以及腫瘤細菌和癌細胞的作用[2-4]?？咕牡膹V譜作用,使得越來越多的新型抗菌肽被發(fā)現(xiàn)?？咕男再|(zhì)、機理、結(jié)構(gòu)以及功能研究,需要大量的高純度的抗菌肽。目前,獲得高純度的抗菌肽主要有三個途徑:源料提取、化學合成以及異源表達。異源表達抗菌肽的優(yōu)勢在于生產(chǎn)成本低,提取工藝簡單,產(chǎn)量高,更好的滿足生產(chǎn)和研究所需的抗菌肽[5]。雖然異源表達技術(shù)為提高抗菌肽產(chǎn)量提供了有效途徑,但基因表達的效果往往依賴于表達載體、宿主菌、分泌蛋白所需要的信號序列以及外部因素,如:溫度、誘導劑等[6-7]。在異源表達抗菌肽表達方面大腸桿菌和酵母菌是最為常用的表達宿主。雖然大腸桿菌作為宿主的占80%以上,但是酵母菌的表達也有其獨特的優(yōu)勢,如肽類分子折疊、二硫鍵的形成和基團修飾方面[8-9]。本文綜述了目前在抗菌肽異源表達方面的研究和表達系統(tǒng)的最新進展。

1　大腸桿菌表達抗菌肽

在大腸桿菌表達系統(tǒng)中質(zhì)粒主要應用是Novagen公司的pET系列,表達宿主最為常用的是Novagen公司的BL21(DE3)、pLys S Origami、Rosetta和C41(DE3)大腸桿菌宿主[10]。利用大腸桿菌異源表達抗菌肽主要存在兩個問題:如何減少表達后抗菌肽對宿主的毒性[11]，如何正確表達抗菌肽[12]。研究人員最為常用的兩種方法是:將抗菌肽基因與融合蛋白基因共同表達，密碼子優(yōu)化。

1.1融合蛋白輔助表達抗菌肽

表1　大腸桿菌表達抗菌肽的主要融合蛋白及產(chǎn)量

注:ND:表示沒有測定；表2同。

利用大腸桿菌表達系統(tǒng)的主要問題是如何減少抗菌肽表達后對宿主的毒性。在抗菌肽基因附加一個陰離子特性的融合蛋白基因(這種融合蛋白并無抑菌性),可以有效減少抗菌肽對宿主的毒性[13]。融合蛋白表達后,往往包含著特殊的理化性質(zhì)和結(jié)構(gòu),可以中和抗菌肽的陽離子,阻止表達后的抗菌肽對宿主的毒性。含融合蛋白的抗菌肽可以通過化學試劑特異性切除融合蛋白,恢復抗菌肽的抑菌活力[14-15]。目前有很多融合蛋白在抗菌肽的表達中得到應用,最為常用的硫氧還原蛋白、His標簽以及谷胱甘肽轉(zhuǎn)移酶(表1)。Pediocin PA-1融合了ABC轉(zhuǎn)運子和輔助蛋白,不僅正確表達PA-1中二硫鍵而且對宿主并無毒害作用[16]。Divergicin A在融合了堿性磷酸酶后,在大腸桿菌發(fā)酵液中融合體抗菌肽對宿主無傷害[17]。融合蛋白PurF已經(jīng)使得抗菌肽Magainin和Pleurocidin達到毫克的產(chǎn)量,buforin IIb達到克的產(chǎn)量[18-20]。Wang等人利用小分子融合蛋白(SUMO)在大腸桿菌中表達Bacterocin E50-52,經(jīng)純化后達到16 mg[21]。Yu等人利用SUMO融合蛋白表達出抗菌肽Lacticin Q,經(jīng)純化后達到2.5 mg[22]。Chen等人利用綠色熒光蛋白(GFP)成功在大腸桿菌中表達出新型抗菌肽NB-C1表達量為2.2 mg/mL[23]。雖然融合蛋白幫助抗菌肽成功表達,但是融合蛋白也存在著不足,它很容易在細胞內(nèi)部形成包涵體而導致表達產(chǎn)物不溶,因此必需加入特定的蛋白酶或化學試劑(Factor Xa、Hydroxylamine、CNBr、Enterokinase)切出融合蛋白才能讓抗菌肽溶于提取液中,但這樣就會導致抗菌肽產(chǎn)量造成2～8倍的損失[10,24]。

Morin等人在融合蛋白的基礎(chǔ)上設(shè)計出一種新的方法,將一個融合蛋白與多個抗菌肽indolicidin基因連接,表達出三聚體或六聚體的融合蛋白與抗菌肽復合物,經(jīng)純化后用溴化氫切除融合蛋白釋放抗菌肽單體[41]。既減少抗菌肽對宿主的毒性同時又提高抗菌肽的產(chǎn)量,Rao和Peng等人分別使得His-Tagged融合蛋白和GST融合蛋白與多個抗菌肽β-defensin 2串連基因表達,提高了抗菌肽β-defensin 2的產(chǎn)量[6,42]。這種技術(shù)應用性不強,因為重組重復抗菌肽基因,并不能保證一定會形成聚集體,部份抗菌肽形成聚集體也會對宿主有毒性。

內(nèi)含肽融合蛋白的提出解決抗菌肽去除融合蛋白的問題。這種技術(shù)的優(yōu)勢是通過在一定條件下純化抗菌肽,誘導抗菌肽自身切除融合體而釋放出有活性的抗菌肽。這個系統(tǒng)要求有一個恰當?shù)膬?nèi)含肽與抗菌肽的融合體,它不能與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利用這種技術(shù)成功的表達[43-45]。這種表達系統(tǒng)缺點在于只能表達小分子量的蛋白,而且對于質(zhì)粒的構(gòu)建是要求十分嚴格,內(nèi)含肽融合蛋白使得抗菌肽失去活力,因此在抗菌肽的表達方面,內(nèi)含肽的應用還有待進一步的研究。

1.2密碼子優(yōu)化

在提高抗菌肽遺傳穩(wěn)定性方面,密碼子優(yōu)化是最為有效的方法。通過對不同來源抗菌肽的遺傳密碼子優(yōu)化,達到宿主本身的密碼子偏好,可以使抗菌肽mRNA穩(wěn)定轉(zhuǎn)錄和翻譯,提高抗菌肽的穩(wěn)定性[46]。優(yōu)化后的抗菌肽密碼子不僅可以有效的合成抗菌肽,而且還可以減少抗菌肽對宿主細胞的毒性[47-48]。Richard等人優(yōu)化抗菌肽divercin V41編碼基因并與組氨酸標簽以及硫氧還原蛋白融合后成功表達,divercin V41產(chǎn)量達到23 mg/L?？咕腍epcidin的基因通過密碼子優(yōu)化后成功在大腸桿菌中表達[49]。Ingham等人通過優(yōu)化多種乳酸菌Class IIa抗菌肽基因,并通過與內(nèi)含肽融合蛋白融合,成功表達多種Class IIa抗菌肽[24]。研究表明在一定區(qū)域內(nèi)重復插入優(yōu)化后抗菌肽的基因單元,可以有效提高抗菌肽的表達率。利用這種方法抗菌肽β-defensin-2產(chǎn)量提高了6倍[50]。該方法在乳酸菌抗菌肽沒有得到很好的應用,但是在其它類型的抗菌肽已經(jīng)得到成功應用[50-51]。

2　酵母表達抗菌肽

利用大腸桿菌表達抗菌肽是一種有效的方法,但是卻無法表達一些修飾型抗菌肽[10]。酵母菌表達宿主是一種可以表達修飾蛋白的表達宿主。目前酵母菌宿主主要有釀酒酵母和畢赤酵母。近些年來酵母表達宿主也逐漸豐富,包括安格斯畢赤酵母(Pichia angusta)、解脂耶氏酵母(Yarrowia lipolytica)[52-53]。酵母表達系統(tǒng)與大腸桿菌表達系統(tǒng)相比,主要體現(xiàn)在翻譯后對蛋白的修飾作用,比如糖基化的修飾[54]。盡管酵母菌表達抗菌肽的時間比大腸桿菌表達的時間長,但是酵母菌表達的可修飾的抗菌肽可以分泌到胞外,更重要的是酵母表達并不需要輔助蛋白(大腸桿菌表達必需)[55]。

表2　抗菌肽在畢赤酵母中表達

2.1釀酒酵母異源表達抗菌肽

釀酒酵母是酵母異源表達重組蛋白的主要宿主。因為對于釀酒酵母的理化性質(zhì)和相關(guān)基因都十分明確,在表達蛋白方面也取得了豐碩的研究成果[56]。在一些條件下,釀酒酵母比大腸桿菌能更好的表達可溶性蛋白,并且表達量可以滿足結(jié)構(gòu)分析[57]。釀酒酵母表達抗菌肽存在一些限制因素阻礙了其在抗菌肽表達方面的應用,例如抗菌肽PA-1雖然在釀酒酵母中成功表達,但是其產(chǎn)量水平卻沒有報道[58];但是在畢赤酵母中抗菌肽PA-1的產(chǎn)量達到74 μg/mL[59]。Van等人利用釀酒酵母成功表達出抗菌肽Plantaricin 423,但也無法定量[60]。此外,釀酒酵母是一種發(fā)酵型酵母,可以發(fā)酵糖類物質(zhì)產(chǎn)生乙醇,乙醇又會影響其菌群數(shù)量,導致蛋白或肽類的產(chǎn)量低[55]。這些不利的因素,使得釀酒酵母并不適合用于大規(guī)模發(fā)酵產(chǎn)生異源抗菌肽。

2.2畢赤酵母表達異源抗菌肽

近幾年,畢赤酵母作為異源表達抗菌肽宿主的研究報道明顯增加。十年前,大約只有400多種蛋白利用畢赤酵母表達[61]。如今畢赤酵母表達多達上千種,在2009年美國食品藥品管理局第一次通過了利用畢赤酵母表達的重組藥品上市,更是促進了畢赤酵母在表達方面的應用[55]。與釀酒酵母不同,畢赤酵母不是發(fā)酵型微生物,菌體濃度可以達到100～200 g/L,而且其代謝產(chǎn)物并無毒性,也不會產(chǎn)生乙醇。畢赤酵母的另外一個優(yōu)勢是,易擴大培養(yǎng)、培養(yǎng)基成本低、易純化、可以表達具有修飾作用的蛋白[61]。相比與釀酒酵母和大腸桿菌中限制性表達因素,畢赤酵母的表達啟動子相對簡單(采用AOX啟動子,利用甲醇啟動表達系統(tǒng))。畢赤酵母表達抗菌肽主要分為兩步:高密度培養(yǎng)菌體;加入甲醇誘導重組蛋白的表達。這一過程的優(yōu)勢在于在表達抗菌肽之前,可以得到大量的菌體,避免了重組抗菌肽對宿主的毒性。

畢赤酵母表達不同的抗菌肽,表達量也不相同(表2)。在大腸桿菌或釀酒酵母表達的抗菌肽,在畢赤酵母中表達產(chǎn)量有所提高[62-63]。部分抗菌肽在大腸桿菌表達失敗后,在畢赤酵母中可以很好的表達,例如抗菌肽defensin SPE10[64]。從表達產(chǎn)物的結(jié)構(gòu)來說,抗菌肽defensin PDC1分別在大腸桿菌和畢赤酵母菌表達,兩者表達的抗菌肽都有抗真菌的活性,但是畢赤酵母表達量是大腸桿菌的2倍。比較兩種表達產(chǎn)物的結(jié)構(gòu),發(fā)現(xiàn)在畢赤酵母表達的抗菌肽defensin PDC1結(jié)構(gòu)中具有較多的β-折疊,而無規(guī)則卷曲較少[65]。另外畢赤酵母要可以更好的表達抗菌肽的活性,Basanta等人利用釀酒酵母和畢赤酵母表達了無前導肽序列的enterocinL50A和50B,成功表達了雙肽鏈抗菌肽enterocinL50A和L50B,但是畢赤酵母表達的兩種抗菌肽活力分別是釀酒酵母的6～60倍[66-67]。目前大多數(shù)利用畢赤酵母表達異源抗菌肽依然處于實驗室階段。主要是在畢赤酵母表達中需要一個適合的啟動子伴隨表達,從而達到產(chǎn)業(yè)化生產(chǎn)所需的產(chǎn)量。

3　結(jié)論與展望

抗菌肽在食品、藥品、化妝品、農(nóng)業(yè)方面的應用越來越廣泛,吸引大量的研究人員開發(fā)和利用抗菌肽。異源表達技術(shù)很好的解決抗菌肽產(chǎn)量不足的問題。通過對啟動子、信號肽、融合蛋白以及相關(guān)的輔助因子的估化,成功表達了抗菌肽并且抗菌肽的產(chǎn)量已經(jīng)有大范圍的提升，為研究新的表達系統(tǒng)和發(fā)酵工藝提供理論依據(jù),為建立大規(guī)模表達抗菌肽的方法提供思路?？咕牡姆N類繁多,不同的抗菌肽在大規(guī)模生產(chǎn)技術(shù)上依然存在困難,如抗菌肽穩(wěn)定性、提取工藝、抗菌肽的聚集、抗菌肽表達后的修飾、降低成本等。隨著基因工程技術(shù)的不斷的發(fā)展和改良,這些困難有望逐一得到解決。希望研究人員利用異源系統(tǒng),生產(chǎn)出用途更廣、種類更多、效果更好的抗菌肽產(chǎn)品。

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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

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

張?zhí)m威(1961-)，男，博士，教授，研究方向：乳品科學、食品發(fā)酵，E-mail:zhanglw@hit.edu.cn。

國家自然科學學基金(31271906/C2002204，31571850/C200502)；高等學校博士學科點專項科研基金(20112302110051)。

TS201.3

A

1002-0306(2016)12-0380-06

10.13386/j.issn1002-0306.2016.12.064