Please wait a minute...

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  2018, Vol. 38 Issue (2): 54-60    DOI: 10.13523/j.cb.20180208
技术与方法     
ELP30-tag蛋白纯化能力的原核表达研究
陈远侨,龙定沛,豆晓雪,祁润,赵爱春()
家蚕基因组生物学国家重点实验室 西南大学生物技术学院 重庆 400716
Studies on the Protein Purification Ability of an ELP30-Tag in Prokaryotic Expression System
Yuan-qiao CHEN,Ding-pei LONG,Xiao-xue DOU,Run QI,Ai-chun ZHAO()
State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University,Chongqing 400716, China
 全文: PDF(813 KB)   HTML
摘要：

目的：探究一种小分子量类弹性蛋白标签(elastin-like protein tag,ELP tag)-ELP30-tag在原核表达系统中的蛋白纯化能力。方法：人工合成ELP30-tag基因并将其构建于pET-28a(+)载体,结合2种内含肽(intein1和intein2)基因和增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)基因,构建4个含有不同元件序列的原核表达载体:pET-ELP30、pET-ELP30-eGFP、pET-ELP30-intein1-eGFP和pET-eGFP-intein2-ELP30;将表达载体转化大肠杆菌BL21(DE3),经IPTG诱导重组蛋白表达,并通过可逆相变循环(inverse transition cycling,ITC)纯化重组蛋白ELP30、ELP30-eGFP、ELP30-intein1-eGFP和eGFP-intein2-ELP30,随后通过调节溶液pH值或添加二硫苏糖醇(DL-Dithiothreitol,DTT)分别诱导intein1和intein2断裂,最后再经ITC分离获得纯eGFP。结果：利用设计的ELP30-tag成功纯化获得了重组蛋白ELP30、ELP30-eGFP和eGFP-intein2-ELP30;重组蛋白ELP30-intein1-eGFP和eGFP-intein2-ELP30中的内含肽可经诱导发生断裂而释放eGFP,但未能分离获得纯eGFP。由此为小分子量ELP-tag的运用和优化设计奠定了一定基础。
关键词： 类弹性蛋白;  内含肽;  增强绿色荧光蛋白;  原核表达;  蛋白质纯化    
Abstract:

Objective： Exploration of the purification efficiency of proteins in prokaryotic expression system(Escherichia coli) by using an elastin-like protein tag(ELP30-tag) with small molecular weight.Methods: The ELP30-tag gene was synthesized and inserted into the pET-28a(+) vector,two intein genes (intein1 & intein2) and an enhanced green fluorescent protein (eGFP) gene were cloned and applied to construct four prokaryotic expression vectors: pET-ELP30, pET-ELP30-eGFP, pET-ELP30-intein1-eGFP and pET-eGFP-intein2-ELP30. All the recombinant plasmids were transferred into E. coli BL21(DE3)and induced by IPTG, respectively. Recombinant proteins ELP30, ELP30-eGFP,ELP30-intein1-eGFP and eGFP-intein2-ELP30 were purified by inverse transition cycling (ITC), and then the cleavage reaction of intein1 and intein2 were induced by adjusting the pH value of the solution or adding DL-Dithiothreitol (DTT), respectively, and last the pure eGFPs were separated by one more ITC reaction.Results: The recombinant proteins ELP30, ELP30-eGFP and eGFP-intein2-ELP30 were purified by using the designed ELP30-tag; the cleavage reaction of inteins from the recombinant proteins ELP30-intein1-eGFP and eGFP-intein2-ELP30, which could be successfully induced,and then the eGFPs were released into the solution but not separated. This lays some foundations for the application and optimization of the ELP-tags with small molecular weight.
Key words: Elastin-like protein(ELP)    Intein    Enhanced green fluorescent protein (eGFP)    Prokaryotic expression    Protein purification
收稿日期: 2017-07-05 出版日期: 2018-03-21
ZTFLH:  Q819  
基金资助: * 国家蚕桑产业技术体系资助项目(CARS-18-ZJ0201)
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
陈远侨
龙定沛
豆晓雪
祁润
赵爱春

引用本文:

陈远侨,龙定沛,豆晓雪,祁润,赵爱春. ELP30-tag蛋白纯化能力的原核表达研究[J]. 中国生物工程杂志, 2018, 38(2): 54-60.

Yuan-qiao CHEN,Ding-pei LONG,Xiao-xue DOU,Run QI,Ai-chun ZHAO. Studies on the Protein Purification Ability of an ELP30-Tag in Prokaryotic Expression System. China Biotechnology, 2018, 38(2): 54-60.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180208    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I2/54

引物名称 序列(5’-3’)
F1 ACATgcatgcAAGGAGATGGCGCC
R1 CCGctcgagggatccgcatgcggtaccgtcgacCATTATATCTCCTTCTTAAA
eGFP-F1 GGggtaccAACAACAACAACAACAACAACAACAACAACATGGTGAGCAAGGGCGAGGA
eGFP-R1 CGggatccCTTGTACAGCTCGTCCATGC
eGFP-F2 GCtctagaAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGTGAGCAAGGGCGAGGA
eGFP-R2 GCATGGACGAGCTGTACAAGTGCATCACGGGAGATGCACT
intein2-F AGTGCATCTCCCGTGATGCACTTGTACAGCTCGTCCATGC
intein2-R CCGctcgagGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTAGCGTGGCTGACGAACCCGT
表1  目的片段扩增所用的引物
图1  表达载体的构建
图2  ELP30和ELP30-eGFP纯化检测
图3  重组蛋白纯化产物荧光活性检测
图4  ELP30-intein1-eGFP纯化及切割产物检测
图5  eGFP-intein2-ELP30纯化及切割产物检测
重组蛋白 重组蛋白回收率 蛋白浓度
(mg/ml,
ITC-3)
ITC-1 ITC-2 ITC-3
ELP30 93% 70% 61% 0.18
ELP30-eGFP 96% 84% 65% 0.44
eGFP-intein2-ELP30 95% 88% 72% 0.29
表2  重组蛋白回收率分析
[1] Urry D W, Trapane T L, Prasad K U . Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies. Biopolymers, 1985,24(12):2345-2356.
doi: 10.1002/bip.360241212 pmid: 4092092
[2] Mcpherson D T, Morrow C, Minehan D S , et al. Production and purification of a recombinant elastomeric polypeptide, G-(VPGVG) 19-VPGV, from Escherichia coli. Biotechnology Progress, 1992,8(4):347-352.
[3] Meyer D E, Chilkoti A . Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nature Biotechnology, 1999,17(11):1112-1115.
doi: 10.1038/15100 pmid: 10545920
[4] Mcpherson D T, Xu J, Dan W U . Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP. Protein Expression & Purification, 1996,7(1):51-57.
doi: 10.1006/prep.1996.0008 pmid: 9172783
[5] Banki M R, Feng L, Wood D W . Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nature Methods, 2005,2(9):659-661.
doi: 10.1038/nmeth787 pmid: 16074986
[6] Trabbiccarlson K, Liu L, Kim B , et al. Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Science, 2004,13(12):3274-3284.
doi: 10.1110/ps.04931604 pmid: 2287301
[7] Banki M R, Wood D W . Inteins and affinity resin substitutes for protein purification and scale up. Microbial Cell Factories, 2005,4(1):32-37.
doi: 10.1186/1475-2859-4-32 pmid: 11876
[8] Ge X, Trabbic-Carlson K, Chilkoti A , et al. Purification of an elastin-like fusion protein by microfiltration. Biotechnology & Bioengineering, 2006,95(3):424-432.
doi: 10.1002/bit.21046 pmid: 16767781
[9] Schipperus R, Teeuwen R L, Werten M W , et al. Secreted production of an elastin-like polypeptide by Pichia pastoris. Applied Microbiology & Biotechnology, 2009,85(2):293-301.
[10] Lin M, Rose-John S, Gr?tzinger J , et al. Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: purification via inverse transition cycling. Biochemical Journal, 2006,398(3):577-583.
doi: 10.1042/BJ20060544 pmid: 16716147
[11] Li Tian, Samuel S M . A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform. Plos One, 2011,6(8):e24183.
doi: 10.1371/journal.pone.0024183 pmid: 3168869
[12] 李敏, 杨谦 . 一种高效构建同源重组DNA片段的方法--融合PCR. 中国生物工程杂志, 2007,27(8):53-58.
Li M, Yang Q . China Biotechnology, 2007,27(8):53-58.
[13] Meyer D E, Chilkoti A . Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules, 2002,3(2):357-367.
doi: 10.1021/bm015630n
[14] Evans T C J, Benner J, Xu M Q . The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins. Journal of Biological Chemistry, 1999,274(26):18359-18363.
doi: 10.1074/jbc.274.26.18359 pmid: 10373440
[15] Cho Y, Zhang Y, Christensen T , et al. Effects of hofmeister anions on the phase transition temperature of elastin-like polypeptides. Journal of Physical Chemistry B, 2008,112(44):13765-13771.
doi: 10.1021/jp8062977 pmid: 3475179
[16] Chong S, Mersha F B, Comb D G , et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene, 1997,192(2):271-281.
doi: 10.1109/84.825779 pmid: 9224900
[17] Shi C, Han T C, Wood D W . Purification of microbially expressed recombinant proteins via a dual ELP split intein system. Methods in Molecular Biology, 2017,1495:13-25.
doi: 10.1007/978-1-4939-6451-2
[1] 乔圣泰,王曼琦,徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析*[J]. 中国生物工程杂志, 2021, 41(8): 25-32.
[2] 张磊,唐永凯,李红霞,李建林,徐逾鑫,李迎宾,俞菊华. 促进原核表达蛋白可溶性的研究进展 *[J]. 中国生物工程杂志, 2021, 41(2/3): 138-149.
[3] 张潇航,李媛媛,贾敏晅,顾奇. 弹性蛋白样生物材料的制备及性质鉴定 *[J]. 中国生物工程杂志, 2020, 40(8): 33-40.
[4] 吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. LbCpf1基因的原核表达、纯化与体外切割检测 *[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[5] 李彤彤,宋彩玲,杨凯越,王文静,陈慧宇,刘明. 抗犬细小病毒VP2蛋白单链抗体的制备与中和活性研究 *[J]. 中国生物工程杂志, 2020, 40(4): 10-16.
[6] 陈秋利,杨丽超,李辉,温莎,李刚,何敏. 人Nek2蛋白原核表达纯化及其多克隆抗体制备 *[J]. 中国生物工程杂志, 2020, 40(3): 31-37.
[7] 朱彤彤,杨磊,刘应保,孙文秀,张修国. 辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 *[J]. 中国生物工程杂志, 2020, 40(1-2): 116-123.
[8] 杨隆兵,国果,马慧玲,李妍,赵欣宇,苏佩佩,张勇. 家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测 *[J]. 中国生物工程杂志, 2019, 39(4): 24-31.
[9] 李明英,王仁军,张帆,迟彦. β2糖蛋白Ⅰ第五结构域及其突变体、短肽片段的原核表达及活性分析 *[J]. 中国生物工程杂志, 2018, 38(8): 1-9.
[10] 陈军军,娄颖,张元兴,刘琴,刘晓红. 增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化 *[J]. 中国生物工程杂志, 2018, 38(7): 14-20.
[11] 何亚南,孙钰椋,任雅坤,梁盛英,杨芬,刘彦礼,林俊堂. 金黄色葡萄球菌类肠毒素K与GFP融合蛋白工程菌的构建及其表达蛋白生物学活性分析 *[J]. 中国生物工程杂志, 2018, 38(12): 14-20.
[12] 任建委,李军,李尚泽. 人源CT55蛋白原核表达及单克隆抗体的制备 *[J]. 中国生物工程杂志, 2018, 38(11): 1-8.
[13] 王明轩, 陈海琴, 顾震南, 陈卫, 陈永泉. 高山被孢霉中Δ9脂肪酸脱饱和酶的表达、纯化和其细胞色素b5功能域的鉴定[J]. 中国生物工程杂志, 2017, 37(3): 43-50.
[14] 孙文佳, 姚宇峰, 杨旭, 黄惟巍, 刘存宝, 龙琼, 褚晓杰, 马雁冰. 乙肝核心抗原病毒样颗粒呈现HPV 16L1抗原表位及特异抗体诱导[J]. 中国生物工程杂志, 2017, 37(3): 58-64.
[15] 王路路, 权春善, 许永斌, 陈金利, 吴懿, 王冠天, 董悦生. 金黄色葡萄球菌arl双组分信号转导系统受体蛋白ArlSCA的表达、纯化及活性研究[J]. 中国生物工程杂志, 2017, 37(11): 52-58.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会