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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (4): 69-77    DOI: 10.13523/j.cb.20190409
技术与方法     
大肠杆菌细胞裂解系统的构建及其在真菌毒素降解酶表达中的应用 *
赵程程,孙长坡,常晓娇,伍松陵,林振泉()
国家粮食和物资储备局科学研究院 粮油加工研究所 北京 100037
Construction and Application of Cell Lysis Systems in the Expression of Mycotoxin Degrading Enzyme in Escherichia coli
Cheng-cheng ZHAO,Chang-po SUN,Xiao-jiao CHANG,Song-ling WU,Zhen-quan LIN()
Academy of National Food and Strategic Reserves Administration, Institute of Cereal Processing Science and Technology, Beijing 100037,China
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摘要：

目的:大肠杆菌中分泌表达重组蛋白受限于其分泌效率,为此设计构建大肠杆菌诱导裂解系统以实现胞内重组蛋白的快速高效分泌。方法:利用大肠菌素E7对细胞的裂解能力,构建共表达目标重组蛋白和E7的大肠杆菌细胞裂解系统,使目标重组蛋白在E7表达后得以释放到培养基中。结果:首先以红色荧光蛋白(red fluorescent protein, RFP)为报告基因,在pET28a(+)载体上构建大肠杆菌素E7和红色荧光蛋白两个表达盒,通过对比分析IPTG一步诱导和IPTG-阿拉伯糖分步诱导系统蛋白质的表达效果,发现分步诱导系统能够更高效地表达并释放目标蛋白到培养基。在IPTG-阿拉伯糖分步诱导裂解系统中表达玉米赤霉烯酮降解酶基因,培养基上清液中检测到玉米赤霉烯酮降解酶有较好的表达量和较高的活性,能够在37℃反应30min的条件下降解约5.8μg玉米赤霉烯酮毒素。结论:利用大肠菌素E7成功构建大肠杆菌细胞裂解系统,并且此系统在快速释放胞内表达外源蛋白方面有适用性。
关键词： 大肠杆菌;  细胞裂解系统;  红色荧光蛋白;  玉米赤霉烯酮降解酶    
Abstract:

Objective: Extracellular production of recombinant proteins in Escherichia coli is limited by the inefficiency of inherent secretion system. An inducible cell lysis system was designed and constructed to enhance secretion of intracellular recombinant protein in E.coli.Methods: Considering colicin E7 could promote cell lysis, E.coli cell lysis systems were constructed by co-expressing target protein and colicin E7 lysis to release the recombinant proteins to culture medium.Results: A reporter protein (red fluorescent protein, RFP), as a recombinant protein, was co-expressed with E7 in E.coli to evaluate the cell lysis systems. Expression of recombinant protein was controlled by T7 promoter. While E7 cassette was controlled by two promoters (T7 promoter and araBAD promoter) which determined the expression timing of E7 and therefore determined the timing of cell lysis. Compared to one-step induction by IPTG, the two-step induction by IPTG and L-arabinose was better for the production and secretion of recombinant proteins. The two-step inducible lysis system was also used to express zearalenone (ZEN) degrading enzyme, and high enzyme activity was detectable in the culture supernatant samples. The secreted enzyme could degrade about 5.8μg ZEN in 30minutes at 37℃.Conclusion: The colicin E7 assisted two-step inducible cell lysis system could be potential for expression of recombinant proteins and their secretion to extracellular in E. coli.
Key words: E. coli    Cell lysis system    Red fluorescent protein    Zearalenone degrading enzyme
收稿日期: 2018-08-21 出版日期: 2019-05-08
ZTFLH:  Q819  
基金资助: * 公益性行业(粮食)科研专项(201513006);国家自然科学基金资助项目(U1604234)
通讯作者: 林振泉     E-mail: zhenquanlin09@gmail.com
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赵程程
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引用本文:

赵程程,孙长坡,常晓娇,伍松陵,林振泉. 大肠杆菌细胞裂解系统的构建及其在真菌毒素降解酶表达中的应用 *[J]. 中国生物工程杂志, 2019, 39(4): 69-77.

Cheng-cheng ZHAO,Chang-po SUN,Xiao-jiao CHANG,Song-ling WU,Zhen-quan LIN. Construction and Application of Cell Lysis Systems in the Expression of Mycotoxin Degrading Enzyme in Escherichia coli. China Biotechnology, 2019, 39(4): 69-77.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190409    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I4/69

Strains and plasmids Characteristics Source
E.coli DH5α F-; φ80lacZΔM15; Δ(lacZYA-argF)U169; deoR; recA1; endA1; hsdR17(rk-, mk+); phoA; supE44; λ-; thi-1; gyrA96; relA1 Lab collection
E.coli BL21(DE3) F-; ompT; hsdSB(rB-, mB-); gal; dcm(DE3) Lab collection
pKD46 Ampr, λ-red recombinase under araBAD promoter, temperature-conditional replicon Lab collection
pET28a(+) Kanr, T7lac promoter, His-Tag (N, C) and T7-Tag (I) Lab collection
pET28a(+)-E7 pET28a(+) containing E7 gene This study
pET28a(+)-AraE7 colicin E7 under the control of araBAD promoter in pET28a(+)-E7 This study
pET28a(+)-T7E7 pET28a(+) containing a cassette of colicin E7 controlled by T7 promoter This study
pET28a(+)-T7E7-T7rfp pET28a(+)-T7E7 containing a cassette of rfp controlled by T7 promoter This study
pET28a(+)-AraE7 pET28a(+) containing a cassette of colicin E7 controlled by araBAD promoter This study
pET28a(+)-AraE7-T7rfp pET28a(+)-AraE7 containing a cassette of rfp controlled by T7 promoter This study
pET28a(+)-AraE7-T7ZENd pET28a(+)-AraE7 containing a cassette of ZENd controlled by T7 promoter This study
表1  本研究中所用菌株和质粒
Primers Sequences(5'→3') Restriction sites Target genes
E7-F 5'-CATGCCATGGATGAAAAAAATAACAGG-3' Nco I Colicin E7
E7-R 5'-CCGCTCGAGTTACTGCGTTTCCACTCC-3' Xho I Colicin E7
PBAD-F 5'- CACTGATCCGCATGCTTATGAC -3' Sph I ParaBAD
PBAD-R 5'-TCTAGATCTAGATTCCCAAAAAAACGGGTATGGA-3' Xba I ParaBAD
rfp-F 5'-CCGGAATTCATGGCTTCCTCCGAAGACGTTATC-3' EcoR I rfp
rfp-R 5'-ACGCGTCGACTTAAGCACCGGTGGAGTGACGACC-3' Sal I rfp
ZENd-F 5'-CCGGAATTCATGCGTATCCGTAGCACCATTAG-3' EcoR I ZENd
ZENd-R 5'-ACGCGTCGACTTACAGATATTTCTGGGTAAATTC-3' Sal I ZENd
表2  本研究中使用的引物
图1  大肠杆菌诱导裂解系统中细胞裂解示意图
图2  大肠杆菌细胞裂解系统的质粒构建过程
图3  大肠杆菌诱导裂解菌株生长曲线
图4  大肠杆菌裂解系统中红色荧光蛋白表达菌株培养基上清液的SDS-PAGE
图5  红色荧光蛋白荧光强度随诱导时间的变化
图6  大肠杆菌RFP表达菌株显微镜下的菌体形态观察
图7  大肠杆菌裂解系统中玉米赤霉烯酮降解酶表达菌株培养基上清液浓缩10倍后的SDS-PAGE
图8  玉米赤霉烯酮降解酶表达菌株诱导过程培养基上清液对ZEN的降解
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