多形拟杆菌肝素酶Ⅰ的SUMO融合表达及酶学特性分析
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摘要
【目的】克隆多形拟杆菌(Bacteroides thetaiotaomicron) HeparinaseⅠ基因,在大肠杆菌(Escherichia coli)中进行基因工程表达获得重组酶SUMO-Bt-HepI和Bt-HepI,并研究其酶学特性。【方法】对B.thetaiotaomicron肝素酶I (Bt-HepI)的基因序列进行密码子优化,PCR扩增得到目的基因,构建表达载体pET-28a-Bt-HepⅠ和pE-SUMO-Bt-HepⅠ,并转化至E.coliRosetta(DE3)进行表达,分别得到重组产物Bt-HepⅠ和SUMO-Bt-HepⅠ,以肝素钠为底物研究两者的酶学性质。【结果】SDS-PAGE检测显示Bt-HepⅠ和SUMO-Bt-HepⅠ的分子量大小分别约为42.5 kDa和55 kDa。与Bt-HepI相比,融合SUMO-Tag后的肝素酶I比酶活提高了48.9%。酶学性质表明:Bt-HepⅠ和SUMO-Bt-HepⅠ的最适pH和温度均为pH 9、45°C,二者在pH 5–9都具有很好的稳定性,但pH<5时,SUMO-Bt-HepI的耐酸性明显高于Bt-HepI。同时,在温度低于50°C时,SUMO-Bt-HepⅠ的比酶活高于Bt-HepⅠ。此外,Ca~(2+)和Mg~(2+)对重组肝素酶I具有明显的促进作用,而Cu~(2+)、Mn~(2+)、Zn~(2+)则表现出一定的抑制作用,提示在多形拟杆菌肝素酶I的结构中除了存在已知的Ca~(2+)结合位点外,可能还存在Mg~(2+)的结合位点。【结论】本研究首次将多形拟杆菌来源的肝素酶I和SUMO-Tag进行了融合表达,使其比酶活得到了显著的提高,为其生产应用奠定了基础。
[Objective] To clone and recombinant express the gene Heparinase I from Bacteroides thetaiotaomicron,and then characterize the recombinant SUMO-Bt-HepⅠ and Bt-HepⅠ. [Methods] Codon optimization was done on the gene sequence of B. thetaiotaomicron heparinase I. The target gene was obtained by PCR amplification, inserted into the expression vectors pET-28 a and pE-SUMO, and then transformed into E. coli Rosetta(DE3) to obtain recombinant products Bt-HepⅠ and SUMO-Bt-HepⅠ. Heparin sodium was used as substrate to study the enzymatic properties of the recombinant proteins. [Results] SDS-PAGE analysis showed that the molecular weights of Bt-HepⅠ and SUMO-Bt-HepⅠ were about 42.5 kDa and 55 kDa, respectively. Compared with Bt-HepⅠ, the specific enzyme activity of Heparinase I increased by 48.9% after fusion SUMO-Tag. The enzymological properties showed that the optimum pH and temperature of Bt-HepⅠ and SUMO-Bt-HepⅠ were pH 9 and 45 °C, and both recombinant enzymes were stable at pH 5–9, while the acid resistance of SUMO-Bt-HepⅠ was obviously higher than Bt-HepⅠ when the pH value was lower than 5. Besides, SUMO-Bt-HepⅠ also showed higher activities than Bt-HepⅠ under50 °C. In addition, Ca~(2+) and Mg~(2+) have obvious promoting effect on the recombinant heparinase I, while Cu~(2+),Mn~(2+) and Zn~(2+) show certain inhibiting effect, suggesting that in addition to the well-known Ca~(2+) binding site, Mg~(2+)binding sites may aslo exist in the structure of B. thetaiotaomicron Heparinase I. [Conclusion] Recombinant Heparinase I in B. thetaiotaomicron using SUMO fusion system significantly improved its specific enzyme activity for potential production and application.
[Objective] To clone and recombinant express the gene Heparinase I from Bacteroides thetaiotaomicron,and then characterize the recombinant SUMO-Bt-HepⅠ and Bt-HepⅠ. [Methods] Codon optimization was done on the gene sequence of B. thetaiotaomicron heparinase I. The target gene was obtained by PCR amplification, inserted into the expression vectors pET-28 a and pE-SUMO, and then transformed into E. coli Rosetta(DE3) to obtain recombinant products Bt-HepⅠ and SUMO-Bt-HepⅠ. Heparin sodium was used as substrate to study the enzymatic properties of the recombinant proteins. [Results] SDS-PAGE analysis showed that the molecular weights of Bt-HepⅠ and SUMO-Bt-HepⅠ were about 42.5 kDa and 55 kDa, respectively. Compared with Bt-HepⅠ, the specific enzyme activity of Heparinase I increased by 48.9% after fusion SUMO-Tag. The enzymological properties showed that the optimum pH and temperature of Bt-HepⅠ and SUMO-Bt-HepⅠ were pH 9 and 45 °C, and both recombinant enzymes were stable at pH 5–9, while the acid resistance of SUMO-Bt-HepⅠ was obviously higher than Bt-HepⅠ when the pH value was lower than 5. Besides, SUMO-Bt-HepⅠ also showed higher activities than Bt-HepⅠ under50 °C. In addition, Ca~(2+) and Mg~(2+) have obvious promoting effect on the recombinant heparinase I, while Cu~(2+),Mn~(2+) and Zn~(2+) show certain inhibiting effect, suggesting that in addition to the well-known Ca~(2+) binding site, Mg~(2+)binding sites may aslo exist in the structure of B. thetaiotaomicron Heparinase I. [Conclusion] Recombinant Heparinase I in B. thetaiotaomicron using SUMO fusion system significantly improved its specific enzyme activity for potential production and application.
引文
[1]Witczak ZJ,Nieforth KA.Carbohydrates in drug design.European Journal of Medicinal Chemistry,1997,32(11):842.
[2]Galliher PM,Cooney CL,Langer R,Linhardt RJ.Heparinase production by Flavobacterium heparinum.Applied and Environmental Microbiology,1981,41(2):360-365.
[3]Kim BT,Kim WS,Kim YS,Linhardt RJ,Kim DH.Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15.The Journal of Biochemistry,2000,128(2):323-328.
[4]Yoshida E,Sakai K,Tokuyama S,Miyazono H,Maruyama H,Morikawa K.Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans.Bioscience,Biotechnology,and Biochemistry,2002,66(5):1181-1184.
[5]Yang VC,Linhardt RJ,Bernstein H,Cooney CL,Langer R.Purification and characterization of heparinase from Flavobacterium heparinum.Journal of Biological Chemistry,1985,260(3):1849-1857.
[6]Sasisekharan R,Moses MA,Nugent MA,Cooney CL,Langer R.Heparinase inhibits neovascularization.Proceedings of the National Academy of Sciences of the United States of America,1994,91(4):1524-1528.
[7]Ernst S,Venkataraman G,Winkler S,Godavarti R,Langer R,Cooney CL,Sasisekharan R.Expression in Escherichia coli,purification and characterization of heparinase I from Flavobacterium heparinum.Biochemical Journal,1996,315(2):589-597.
[8]Hirsh J,Warkentin TE,Shaughnessy SG,Anand SS,Halperin JL,Raschke R,Granger C,Ohman EM,Dalen JE.Heparin and low-molecular-weight heparin mechanisms of action,pharmacokinetics,dosing,monitoring,efficacy,and safety.Chest,2001,119(1):64S-94S.
[9]Qureshi A,Perera A.Low molecular weight heparin versus unfractionated heparin in the management of cerebral venous thrombosis:A systematic review and meta-analysis.Annals of Medicine and Surgery,2017,17:22-26.
[10]Welsby IJ,Newman MF,Phillips-Bute B,Messier RH,Kakkis ED,Stafford-Smith M.Hemodynamic changes after protamine administration:association with mortality after coronary artery bypass surgery.Anesthesiology,2005,102(2):308-314.
[11]Guerrini M,Beccati D,Shriver Z,Naggi A,Viswanathan K,Bisio A,Capila I,Lansing JC,Guglieri S,Fraser B,Al-Hakim A,Gunay NS,Zhang ZQ,Robinson L,Buhse L,Nasr M,Woodcock J,Langer R,Venkataraman G,Linhardt RJ,Casu B,Torri G,Sasisekharan R.Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events.Nature Biotechnology,2008,26(6):669-675.
[12]Aich U,Shriver Z,Tharakaraman K,Raman R,Sasisekharan R.Competitive inhibition of heparinase by persulfonated glycosaminoglycans:a tool to detect heparin contamination.Analytical Chemistry,2011,83(20):7815-7822.
[13]Ameer GA,Harmon W,Sasisekharan R,Langer R.Investigation of a whole blood fluidized bed Taylor-Couette flow device for enzymatic heparin neutralization.Biotechnology and Bioengineering,1999,62(5):602-608.
[14]Sasisekharan R,Bulmer M,Moremen KW,Cooney CL,Langer R.Cloning and expression of heparinase I gene from Flavobacterium heparinum.Proceedings of the National Academy of Sciences of the United States of America,1993,90(8):3660-3664.
[15]Shpigel E,Goldlust A,Efroni G,Avraham A,Eshel A,Dekel M,Shoseyov O.Immobilization of recombinant heparinase I fused to cellulose-binding domain.Biotechnology and Bioengineering,1999,65(1):17-23.
[16]Ling CF,Zhang JY,Lin DQ,Tao AL.Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli.World Journal of Microbiology and Biotechnology,2015,31(5):681-690.
[17]Kapust RB,Waugh DS.Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.Protein Science,1999,8(8):1668-1674.
[18]Chen Y,Xing XH,Ye FC,Kuang Y,Luo MF.Production of MBP-HepA fusion protein in recombinant Escherichia coli by optimization of culture medium.Biochemical Engineering Journal,2007,34(2):114-121.
[19]Namvar S,Barkhordari F,Raigani M,Jahandar H,Nematollahi L,Davami F.Cloning and soluble expression of matureα-luffin from Luffa cylindrica in E.coli using SUMOfusion protein.Turkish Journal of Biology,2018,42:23-32.
[20]Li JH,Han QX,Zhang T,Du J,Sun QQ,Pang YL.Expression of soluble native protein in Escherichia coli using a cold-shock SUMO tag-fused expression vector.Biotechnology Reports,2018,19:e00261.
[21]Zhang J,Sun AY,Dong YG,Wei DZ.Recombinant production and characterization of SAC,the core domain of Par-4,by SUMO fusion system.Applied Biochemistry and Biotechnology,2018,184(4):1155-1167.
[22]Hartwig S,Frister T,Alemdar S,Li Z,Scheper T,Beutel S.SUMO-fusion,purification,and characterization of a(+)-zizaene synthase from Chrysopogon zizanioides.Biochemical and Biophysical Research Communications,2015,458(4):883-889.
[23]Bernstein H,Yang VC,Cooney CL,Langer R.Immobilized heparin lyase system for blood deheparinization.Methods in Enzymology,1988,137:515-529.
[24]Huang J,Cao L,Guo WH,Yuan RX,Jia ZJ,Huang KH.Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners.Protein Expression and Purification,2012,83(2):169-176.
[25]Xu SQ,Zhang XY,Qiu ML,Chen JH.Design and expression of recombinant heparanase I fused to chitin binding domain and SUMO-Tag.Chemical Industry and Engineering Progress,2016,35(S2):315-318.(in Chinese)许淑琴,张轩月,邱美玲,陈敬华.ChBD和SUMO双功能融合肝素酶I的设计与表达.化学进展,2016,35(S2):315-318.
[26]Xu J,Bjursell MK,Himrod J,Deng S,Carmichael LK,Chiang HC,Hooper LV,Gordon JI.A genomic view of the human-Bacteroides thetaiotaomicron symbiosis.Science,2003,229(5615):2074-2076.
[27]Luo YD,Huang XQ,McKeehan WL.High yield,purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli.Archives of Biochemistry and Biophysics,2007,460(1):17-24.
[28]Han YH,Garron ML,Kim HY,Kim WS,Zhang ZQ,Ryu KS,Shaya D,Xiao ZP,Cheong C,Kim YS,Linhardt RJ,Jeon YH,Cygler M.Structural snapshots of heparin depolymerization by heparin lyase I.Journal of Biological Chemistry,2009,284(49):34019-34027.
[29]Ma XL,Wang ZS,Li SX,Shen Q,Yuan QS.Effect of CaCl2as activity stabilizer on purification of heparinase I from Flavobacterium heparinum.Journal of Chromatography B,2006,843(2):209-215.
[2]Galliher PM,Cooney CL,Langer R,Linhardt RJ.Heparinase production by Flavobacterium heparinum.Applied and Environmental Microbiology,1981,41(2):360-365.
[3]Kim BT,Kim WS,Kim YS,Linhardt RJ,Kim DH.Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15.The Journal of Biochemistry,2000,128(2):323-328.
[4]Yoshida E,Sakai K,Tokuyama S,Miyazono H,Maruyama H,Morikawa K.Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans.Bioscience,Biotechnology,and Biochemistry,2002,66(5):1181-1184.
[5]Yang VC,Linhardt RJ,Bernstein H,Cooney CL,Langer R.Purification and characterization of heparinase from Flavobacterium heparinum.Journal of Biological Chemistry,1985,260(3):1849-1857.
[6]Sasisekharan R,Moses MA,Nugent MA,Cooney CL,Langer R.Heparinase inhibits neovascularization.Proceedings of the National Academy of Sciences of the United States of America,1994,91(4):1524-1528.
[7]Ernst S,Venkataraman G,Winkler S,Godavarti R,Langer R,Cooney CL,Sasisekharan R.Expression in Escherichia coli,purification and characterization of heparinase I from Flavobacterium heparinum.Biochemical Journal,1996,315(2):589-597.
[8]Hirsh J,Warkentin TE,Shaughnessy SG,Anand SS,Halperin JL,Raschke R,Granger C,Ohman EM,Dalen JE.Heparin and low-molecular-weight heparin mechanisms of action,pharmacokinetics,dosing,monitoring,efficacy,and safety.Chest,2001,119(1):64S-94S.
[9]Qureshi A,Perera A.Low molecular weight heparin versus unfractionated heparin in the management of cerebral venous thrombosis:A systematic review and meta-analysis.Annals of Medicine and Surgery,2017,17:22-26.
[10]Welsby IJ,Newman MF,Phillips-Bute B,Messier RH,Kakkis ED,Stafford-Smith M.Hemodynamic changes after protamine administration:association with mortality after coronary artery bypass surgery.Anesthesiology,2005,102(2):308-314.
[11]Guerrini M,Beccati D,Shriver Z,Naggi A,Viswanathan K,Bisio A,Capila I,Lansing JC,Guglieri S,Fraser B,Al-Hakim A,Gunay NS,Zhang ZQ,Robinson L,Buhse L,Nasr M,Woodcock J,Langer R,Venkataraman G,Linhardt RJ,Casu B,Torri G,Sasisekharan R.Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events.Nature Biotechnology,2008,26(6):669-675.
[12]Aich U,Shriver Z,Tharakaraman K,Raman R,Sasisekharan R.Competitive inhibition of heparinase by persulfonated glycosaminoglycans:a tool to detect heparin contamination.Analytical Chemistry,2011,83(20):7815-7822.
[13]Ameer GA,Harmon W,Sasisekharan R,Langer R.Investigation of a whole blood fluidized bed Taylor-Couette flow device for enzymatic heparin neutralization.Biotechnology and Bioengineering,1999,62(5):602-608.
[14]Sasisekharan R,Bulmer M,Moremen KW,Cooney CL,Langer R.Cloning and expression of heparinase I gene from Flavobacterium heparinum.Proceedings of the National Academy of Sciences of the United States of America,1993,90(8):3660-3664.
[15]Shpigel E,Goldlust A,Efroni G,Avraham A,Eshel A,Dekel M,Shoseyov O.Immobilization of recombinant heparinase I fused to cellulose-binding domain.Biotechnology and Bioengineering,1999,65(1):17-23.
[16]Ling CF,Zhang JY,Lin DQ,Tao AL.Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli.World Journal of Microbiology and Biotechnology,2015,31(5):681-690.
[17]Kapust RB,Waugh DS.Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.Protein Science,1999,8(8):1668-1674.
[18]Chen Y,Xing XH,Ye FC,Kuang Y,Luo MF.Production of MBP-HepA fusion protein in recombinant Escherichia coli by optimization of culture medium.Biochemical Engineering Journal,2007,34(2):114-121.
[19]Namvar S,Barkhordari F,Raigani M,Jahandar H,Nematollahi L,Davami F.Cloning and soluble expression of matureα-luffin from Luffa cylindrica in E.coli using SUMOfusion protein.Turkish Journal of Biology,2018,42:23-32.
[20]Li JH,Han QX,Zhang T,Du J,Sun QQ,Pang YL.Expression of soluble native protein in Escherichia coli using a cold-shock SUMO tag-fused expression vector.Biotechnology Reports,2018,19:e00261.
[21]Zhang J,Sun AY,Dong YG,Wei DZ.Recombinant production and characterization of SAC,the core domain of Par-4,by SUMO fusion system.Applied Biochemistry and Biotechnology,2018,184(4):1155-1167.
[22]Hartwig S,Frister T,Alemdar S,Li Z,Scheper T,Beutel S.SUMO-fusion,purification,and characterization of a(+)-zizaene synthase from Chrysopogon zizanioides.Biochemical and Biophysical Research Communications,2015,458(4):883-889.
[23]Bernstein H,Yang VC,Cooney CL,Langer R.Immobilized heparin lyase system for blood deheparinization.Methods in Enzymology,1988,137:515-529.
[24]Huang J,Cao L,Guo WH,Yuan RX,Jia ZJ,Huang KH.Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners.Protein Expression and Purification,2012,83(2):169-176.
[25]Xu SQ,Zhang XY,Qiu ML,Chen JH.Design and expression of recombinant heparanase I fused to chitin binding domain and SUMO-Tag.Chemical Industry and Engineering Progress,2016,35(S2):315-318.(in Chinese)许淑琴,张轩月,邱美玲,陈敬华.ChBD和SUMO双功能融合肝素酶I的设计与表达.化学进展,2016,35(S2):315-318.
[26]Xu J,Bjursell MK,Himrod J,Deng S,Carmichael LK,Chiang HC,Hooper LV,Gordon JI.A genomic view of the human-Bacteroides thetaiotaomicron symbiosis.Science,2003,229(5615):2074-2076.
[27]Luo YD,Huang XQ,McKeehan WL.High yield,purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli.Archives of Biochemistry and Biophysics,2007,460(1):17-24.
[28]Han YH,Garron ML,Kim HY,Kim WS,Zhang ZQ,Ryu KS,Shaya D,Xiao ZP,Cheong C,Kim YS,Linhardt RJ,Jeon YH,Cygler M.Structural snapshots of heparin depolymerization by heparin lyase I.Journal of Biological Chemistry,2009,284(49):34019-34027.
[29]Ma XL,Wang ZS,Li SX,Shen Q,Yuan QS.Effect of CaCl2as activity stabilizer on purification of heparinase I from Flavobacterium heparinum.Journal of Chromatography B,2006,843(2):209-215.