肠激酶轻链在毕赤酵母中的组成型表达
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摘要
[目的]构建牛肠激酶轻链(EK_L)毕赤酵母组成型高效分泌表达系统。[方法]人工合成MF-α信号肽与EK_L融合基因,克隆于pGAPzα-A和野生毕赤酵母X-33。镍柱纯化EK_L,内切糖苷酶H(Endo H)消化法鉴定糖基化,软件分析糖基化位点,以含EK_L位点的融合蛋白作底物验证酶学活性。[结果]获得了8株EK_L的高抗性毕赤酵母转化子,其中7株可组成型分泌表达目的蛋白,在摇瓶中表达水平约为20 mg/L。分析表明,EK_L表达产物为分子量约43 kDa的N型糖基化蛋白,可有效切割含识别位点的融合蛋白。[结论]成功构建了EK_L毕赤酵母组成型高效分泌表达系统。
[Objective]To establish a high efficient production system of enterokinase light chain( EK_L) based on the Pichia pastoris constitutive expression system. [Methods]The fusion gene of MF-α signal peptide and EK_Lwas synthesized and cloned into pGAPzα-A and Pichia pastoris X-33. The nickel column was used to purify EK_L. The glycosylation of EK_Lwas identified by Endoglycosidase H( Endo H) and the glycosylation sites were analyzed by software. The activity of the product was verified by digesting the fusion protein contains the enterokinase cleavage site. [Results]Eight strains of highly resistant Pichia pastoris were obtained,of which seven strains can be constitutively secretory expression of the target protein,and the expression levels were about 20 mg/L in shake-flask cultures. The product was about 43 kDa and N-glycosylated protein. The glycosylated modified EK_Lhas good enzymatic activity on the fusion protein contains the enterokinase cleavage site. [Conclusion]An efficient constitutive expression system for EK_Lwas established base on Pichia pastoris.
[Objective]To establish a high efficient production system of enterokinase light chain( EK_L) based on the Pichia pastoris constitutive expression system. [Methods]The fusion gene of MF-α signal peptide and EK_Lwas synthesized and cloned into pGAPzα-A and Pichia pastoris X-33. The nickel column was used to purify EK_L. The glycosylation of EK_Lwas identified by Endoglycosidase H( Endo H) and the glycosylation sites were analyzed by software. The activity of the product was verified by digesting the fusion protein contains the enterokinase cleavage site. [Results]Eight strains of highly resistant Pichia pastoris were obtained,of which seven strains can be constitutively secretory expression of the target protein,and the expression levels were about 20 mg/L in shake-flask cultures. The product was about 43 kDa and N-glycosylated protein. The glycosylated modified EK_Lhas good enzymatic activity on the fusion protein contains the enterokinase cleavage site. [Conclusion]An efficient constitutive expression system for EK_Lwas established base on Pichia pastoris.
引文
[1]Savithri HS,Light A.Retention of enzymatic activity of bovine enterokinase after a limited reduction of disulfide bonds[J].Biochemical&Biophysical Research Communications,1980,94(1):360-365.
[2]Liepnieks JJ,Light A.The preparation and properties of bovine enterokinase[J].Journal of Biological Chemistry,1979,254(5):1677-1683.
[3]Braud S,Ciufolini MA,Harosh I.Enteropeptidase:a gene associated with a starvation human phenotype and a novel target for obesity treatment[J].Obe Therapy Biotechnology,2012,7(11):e49612.
[4]Hosfield T,Lu Q.Influence of the amino acid residue downstream of(Asp)4Lys on enterokinase cleavage of a fusion protein[J].Analytical Biochemistry,1999,269(1):10-16.
[5]卢雪梅,黄演婷,金小宝,等.重组融合蛋白Trx-IFN-CSP的肠激酶酶切及纯化[J].生物技术,2015,25(5):491-494.
[6]Wolfgang Skala,Peter Goetting,Hans Brandstetter.Do-it-yourself histidine-tagged bovine enterokinase:A handy member of the protein engineer's toolbox[J].Journal of Biotechnology,2013,168(4):421-425.
[7]林辉煌,刘春凤,尹凤红,等.重组人白细胞介素-13在大肠杆菌中的表达纯化及鉴定[J].厦门大学学报:自然科学版,2016,55(6):836-841.
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[9]朱文赫,郭志刚,刘红建,等.具有自激活切割活性肠激酶轻链基因设计、表达及活性研究[J].中国生化药物杂志,2011,32(1):6-10.
[10]陈展歌,陈锡贤,杨梦琳,等.昆明鼠肠激酶轻链的原核表达、复性与纯化[J].生物技术,2014,24(1):48-51.
[11]郝思怡,姚远航,郭土敬,等.金环蛇抗菌肽在毕赤酵母中的组成型表达[J].生物技术,2015,25(2):123-127.
[12]郑国军,倪玲,郭德军,等.人胰岛素前体在毕赤酵母中的组成型表达及其活性[J].中国生物制品学杂志,2013,26(12):1753-1763.
[13]孙自勇,陈均勇,陈新圆.牛肠激酶轻链在毕赤酵母中的分泌表达_纯化和活性鉴定[J].南京大学学报,2004,40(1):42-48.
[14]郭超,王志彦,甘一如,等.理性设计改造牛肠激酶的热稳定性[J].中国生物工程杂志,2016,36(8):46-54.
[15]刘龙飞.猪肠激酶催化亚基基因的克隆及其在毕赤酵母和大肠杆菌中的表达[D].保定:河北农业大学,2011.
[16]Eliot T.Smith,David A.Johnson.Human enteropeptidase light chain:Bioengineering of recombinants and kinetic investigations of structure and function[J].Protein Science,2013,22(5):577-585.
[17]曹东艳,柳倩,贺晓云,等.源于GAP启动子的毕赤酵母组成型表达系统的研究进展[J].食品安全质量检测学报,2013,4(04):1217-1221.
[18]Yang M,Teymorian S,Olivares P,et al.Extracellular expression of alkaline phytase in Pichia pastoris:Influence of signal peptides,promoters and growth medium[J]Biotechnology Reports,2015,6(1):112-118.
[19]Mate DM,David GP,Roman K,et al.Functional expression of a blood tolerant laccase in Pichia pastoris[J].Bmc Biotechnology,2013,13(1):1-12.
[20]毛根平.毕赤酵母表达系统糖基化修饰改造研究[D].合肥:安徽大学,2015.
[21]CHOi BK,Warburton S,Lin H,et al.Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris[J].Applied Microbiology&Biotechnology,2012,95(3):671-682.
[22]Pepeliaev S,Krahulec J,ˇCerny Z,et al.High level expression of human enteropeptidase light chain in Pichia pastoris[J].Journal of Biotechnology,2011,156(1):67-75.
[2]Liepnieks JJ,Light A.The preparation and properties of bovine enterokinase[J].Journal of Biological Chemistry,1979,254(5):1677-1683.
[3]Braud S,Ciufolini MA,Harosh I.Enteropeptidase:a gene associated with a starvation human phenotype and a novel target for obesity treatment[J].Obe Therapy Biotechnology,2012,7(11):e49612.
[4]Hosfield T,Lu Q.Influence of the amino acid residue downstream of(Asp)4Lys on enterokinase cleavage of a fusion protein[J].Analytical Biochemistry,1999,269(1):10-16.
[5]卢雪梅,黄演婷,金小宝,等.重组融合蛋白Trx-IFN-CSP的肠激酶酶切及纯化[J].生物技术,2015,25(5):491-494.
[6]Wolfgang Skala,Peter Goetting,Hans Brandstetter.Do-it-yourself histidine-tagged bovine enterokinase:A handy member of the protein engineer's toolbox[J].Journal of Biotechnology,2013,168(4):421-425.
[7]林辉煌,刘春凤,尹凤红,等.重组人白细胞介素-13在大肠杆菌中的表达纯化及鉴定[J].厦门大学学报:自然科学版,2016,55(6):836-841.
[8]吕永通,李文燕,杨帆,等.牛肠激酶轻链基因的构建与原核表达[J].辽宁大学学报:自然科学版,2012,39(1):12-14.
[9]朱文赫,郭志刚,刘红建,等.具有自激活切割活性肠激酶轻链基因设计、表达及活性研究[J].中国生化药物杂志,2011,32(1):6-10.
[10]陈展歌,陈锡贤,杨梦琳,等.昆明鼠肠激酶轻链的原核表达、复性与纯化[J].生物技术,2014,24(1):48-51.
[11]郝思怡,姚远航,郭土敬,等.金环蛇抗菌肽在毕赤酵母中的组成型表达[J].生物技术,2015,25(2):123-127.
[12]郑国军,倪玲,郭德军,等.人胰岛素前体在毕赤酵母中的组成型表达及其活性[J].中国生物制品学杂志,2013,26(12):1753-1763.
[13]孙自勇,陈均勇,陈新圆.牛肠激酶轻链在毕赤酵母中的分泌表达_纯化和活性鉴定[J].南京大学学报,2004,40(1):42-48.
[14]郭超,王志彦,甘一如,等.理性设计改造牛肠激酶的热稳定性[J].中国生物工程杂志,2016,36(8):46-54.
[15]刘龙飞.猪肠激酶催化亚基基因的克隆及其在毕赤酵母和大肠杆菌中的表达[D].保定:河北农业大学,2011.
[16]Eliot T.Smith,David A.Johnson.Human enteropeptidase light chain:Bioengineering of recombinants and kinetic investigations of structure and function[J].Protein Science,2013,22(5):577-585.
[17]曹东艳,柳倩,贺晓云,等.源于GAP启动子的毕赤酵母组成型表达系统的研究进展[J].食品安全质量检测学报,2013,4(04):1217-1221.
[18]Yang M,Teymorian S,Olivares P,et al.Extracellular expression of alkaline phytase in Pichia pastoris:Influence of signal peptides,promoters and growth medium[J]Biotechnology Reports,2015,6(1):112-118.
[19]Mate DM,David GP,Roman K,et al.Functional expression of a blood tolerant laccase in Pichia pastoris[J].Bmc Biotechnology,2013,13(1):1-12.
[20]毛根平.毕赤酵母表达系统糖基化修饰改造研究[D].合肥:安徽大学,2015.
[21]CHOi BK,Warburton S,Lin H,et al.Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris[J].Applied Microbiology&Biotechnology,2012,95(3):671-682.
[22]Pepeliaev S,Krahulec J,ˇCerny Z,et al.High level expression of human enteropeptidase light chain in Pichia pastoris[J].Journal of Biotechnology,2011,156(1):67-75.