牛肠激酶轻链在大肠杆菌中的非融合表达及生物学活性分析
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摘要
该研究尝试利用大肠杆菌表达体系以非融合蛋白形式表达牛肠激酶轻链(EK_(LC))。利用基因重组技术构建重组表达载体p6×His-D_4K-EK_(LC)并转化至E. coli DH5α,37℃过夜培养,超声破壁法释放菌体蛋白并收集包涵体,考察包涵体在不同浓度下的复性效率。复性后的目的蛋白经金属离子亲和层析纯化,并以EK_(LC)自切方式移除重组EK_(LC)的N-端6×His-D_4K序列得到具有天然N-端氨基酸序列的EK_(LC),酶活性测定结果显示该研究所制备的重组EK_(LC)高于商品化牛肠激酶。重组EK_(LC)在大肠杆菌中非融合表达成功,为实现EK_(LC)的经济、高效制备研究奠定了实验基础。
In this study,the author attempt to express bovine enterokinase light chain( EK_(LC)) without fusion partner in E. coli system. The DNA sequence of EK_(LC)containing 6 × His-D_4K DNA at its 5' end,was synthesized using a chemical method,and then sub-cloned into the Bam HI/Hind Ⅲ site of p Green-S plasmid by employing gene recombination technology,yielding the vector p6 × His-D_4K-EK_(LC)for producing the recombinant 6 × His-D_4K-EK_(LC). The cells E. coli DH5α harboring p6 × His-D_4K-EK_(LC) were cultured at 37 ℃ for overnight,then the cells were harvested by centrifugation,and the bacterial protein was released by ultrasonic wall breaking method and inclusion bodies were collected and analyzed by SDS-PAGE. Then,the inclusion bodies were endured protein renaturation procedure. The renaturation efficiency of inclusion bodies at different concentrations was also investigated. After renaturation,the target protein was purified by IMAC,and the natural N-terminal amino acid sequence of EK_(LC)was exposed via autocatalytic cleavage reaction. The enzyme activity was determined by using Gly-( Asp) 4-Lys-β-naphthylamide as a substrate. The result of double restriction enzyme digestion showed that the recombinant vector was constructed correctly. SDS-PAGE assay showed that the product was mainly inclusion body,and in the author' s renaturation procedure,the inclusion bodies were successfully refold at high concentration. The target protein was successfully purified by IMAC. After autocatalytic cleavage reaction,the 6 × His-D_4K sequence was removed and exposed the natural N-terminal amino acid sequence of EK_(LC). And the results of enzyme bioactivity analysis showed that the EK_(LC)prepared in this study was 1. 5 fold of the commercial one at A_(580 nm). The successful expression of EK_(LC)in E. coli provided an experimental foundation for the economic and efficient preparation of EK_(LC)in future.
In this study,the author attempt to express bovine enterokinase light chain( EK_(LC)) without fusion partner in E. coli system. The DNA sequence of EK_(LC)containing 6 × His-D_4K DNA at its 5' end,was synthesized using a chemical method,and then sub-cloned into the Bam HI/Hind Ⅲ site of p Green-S plasmid by employing gene recombination technology,yielding the vector p6 × His-D_4K-EK_(LC)for producing the recombinant 6 × His-D_4K-EK_(LC). The cells E. coli DH5α harboring p6 × His-D_4K-EK_(LC) were cultured at 37 ℃ for overnight,then the cells were harvested by centrifugation,and the bacterial protein was released by ultrasonic wall breaking method and inclusion bodies were collected and analyzed by SDS-PAGE. Then,the inclusion bodies were endured protein renaturation procedure. The renaturation efficiency of inclusion bodies at different concentrations was also investigated. After renaturation,the target protein was purified by IMAC,and the natural N-terminal amino acid sequence of EK_(LC)was exposed via autocatalytic cleavage reaction. The enzyme activity was determined by using Gly-( Asp) 4-Lys-β-naphthylamide as a substrate. The result of double restriction enzyme digestion showed that the recombinant vector was constructed correctly. SDS-PAGE assay showed that the product was mainly inclusion body,and in the author' s renaturation procedure,the inclusion bodies were successfully refold at high concentration. The target protein was successfully purified by IMAC. After autocatalytic cleavage reaction,the 6 × His-D_4K sequence was removed and exposed the natural N-terminal amino acid sequence of EK_(LC). And the results of enzyme bioactivity analysis showed that the EK_(LC)prepared in this study was 1. 5 fold of the commercial one at A_(580 nm). The successful expression of EK_(LC)in E. coli provided an experimental foundation for the economic and efficient preparation of EK_(LC)in future.
引文
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[5]刘国强,林杨勇,赵林,等.肠激酶轻链在毕赤酵母中的组成型表达[J].药物生物技术,2017,27(05):446-451.
[6]Collins-Racie LA,Mc Colgan JM,Grant KL,et al.Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner Dsb A[J].Biotechnol(NY),1995,13(9):982-987.
[7]Yuan LD,Hua ZC.Expression,purification,and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli[J].Protein Expr Purif,2002,25(2):300-304.
[8]Tan H,Wang J,Zhao ZK.Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli[J].Protein Expr Purif,2007,56(1):40-47.
[9]Tang J,Liang S,Zhang J,et al.p Green-S:a clone vector bearing absence of enhanced green fluorescent protein for screening recombinants[J].Anal Biochem,2009,388(1):173-174.
[10]Chang Woo Suha,Sin Hye ParkbSeung,GookPark,et al.Covalent immobilization and solid-phase refolding of enterokinase for fusion protein cleavage[J].Process Biochem,2005,40(5):1755-1762.
[11]Terpe K.Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,2003,60(5):523-533.
[12]Lu D,Fütterer K,Korolev S,et al.Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide[J].J Mol Biol,1999,292(2):361-373.
[13]Song HW,Choi SI,Seong BL.Engineered recombinant enteropeptidase catalytic subunit:effect of N-terminal modification[J].Arch Biochem Biophys,2002,400(1):1-6.
[14]张笑岩,张会勇,范豪,等.重组猪羧肽酶原B在大肠杆菌中的表达与复性研究[J].药物生物技术,2010,17(1):39-44.
[15]李国柱,杜敏,陆源,等.干扰素α-2a的复性DOE研究[J].药物生物技术,2017,24(5):386-391.
[2]Tan H,Wang J,Zhao ZK.Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli[J].Protein Expr Purif,2007,56(1):40-47.
[3]La Vallie ER,Rehemtulla A,Racie LA,et al.Cloning and functional expression of a c DNA encoding the catalytic subunit of bovine enterokinase[J].J Biol Chem,1993,268(31):23311-23317.
[4]Peng L,Zhong X,Ou J,et al.High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris[J].J Biotechnol,2004,08(2):185-192.
[5]刘国强,林杨勇,赵林,等.肠激酶轻链在毕赤酵母中的组成型表达[J].药物生物技术,2017,27(05):446-451.
[6]Collins-Racie LA,Mc Colgan JM,Grant KL,et al.Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner Dsb A[J].Biotechnol(NY),1995,13(9):982-987.
[7]Yuan LD,Hua ZC.Expression,purification,and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli[J].Protein Expr Purif,2002,25(2):300-304.
[8]Tan H,Wang J,Zhao ZK.Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli[J].Protein Expr Purif,2007,56(1):40-47.
[9]Tang J,Liang S,Zhang J,et al.p Green-S:a clone vector bearing absence of enhanced green fluorescent protein for screening recombinants[J].Anal Biochem,2009,388(1):173-174.
[10]Chang Woo Suha,Sin Hye ParkbSeung,GookPark,et al.Covalent immobilization and solid-phase refolding of enterokinase for fusion protein cleavage[J].Process Biochem,2005,40(5):1755-1762.
[11]Terpe K.Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,2003,60(5):523-533.
[12]Lu D,Fütterer K,Korolev S,et al.Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide[J].J Mol Biol,1999,292(2):361-373.
[13]Song HW,Choi SI,Seong BL.Engineered recombinant enteropeptidase catalytic subunit:effect of N-terminal modification[J].Arch Biochem Biophys,2002,400(1):1-6.
[14]张笑岩,张会勇,范豪,等.重组猪羧肽酶原B在大肠杆菌中的表达与复性研究[J].药物生物技术,2010,17(1):39-44.
[15]李国柱,杜敏,陆源,等.干扰素α-2a的复性DOE研究[J].药物生物技术,2017,24(5):386-391.