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  • ISSN 1006-3080
  • CN 31-1691/TQ

溶组织梭菌胶原酶H在大肠杆菌中的分泌表达

赵钱山 刘晓 李素霞

赵钱山, 刘晓, 李素霞. 溶组织梭菌胶原酶H在大肠杆菌中的分泌表达[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.2021092400
引用本文: 赵钱山, 刘晓, 李素霞. 溶组织梭菌胶原酶H在大肠杆菌中的分泌表达[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.2021092400
Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.2021092400
Citation: Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.2021092400

溶组织梭菌胶原酶H在大肠杆菌中的分泌表达

doi: 10.14135/j.cnki.1006-3080.2021092400
详细信息
    作者简介:

    赵钱山(1995—),男,安徽合肥人,硕士生,研究方向重组蛋白酶。E-mail:3170430889@qq.com

    通讯作者:

    李素霞,E-mail: lisuxia@ecust.edu.cn

  • 中图分类号: Q78

Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli

  • 摘要: 溶组织梭菌Clostridium histolyticum胶原酶H(ColH)作用于胶原蛋白的Y-Gly处,将其水解成小分子肽。通过在colH基因中融合大肠杆菌外膜蛋白A的信号肽序列,成功构建了分泌高分子量的ColH(116 kDa)的菌株,结果发现信号肽在N端的位置影响其分泌效果,N端若有多余的氨基酸片段会显著降低信号肽引导胶原酶的分泌功能。通过正交实验与单因素实验对诱导条件和培养基添加剂优化提高其分泌表达量, 结果为:在诱导温度为25 ℃、诱导菌浓OD600=0.9、IPTG浓度为0.1 mmol/L、装液量为20%,镁离子浓度为10 mmol/L,诱导2.5 h后添加2%甘氨酸条件下,诱导培养20 h,胞外胶原酶活性最高为0.68 U/mL,是优化前的38.1倍,显著提高了分泌表达量。研究结果还发现,镁离子的添加可增加ColH的胞外分泌。

     

  • 图  1  构建的两种ColH分泌质粒结构示意图

    Figure  1.  Schematic representation of two ColH secretory expression plasmids.

    图  2  三种重组菌株胶原酶的胞外分泌情况分析

    Figure  2.  Extracellular secretion of collagenase of the three strains. A: SDS-PAGE analysis of the extracellular component at 10% gel concentration; B: enzymatic activity of the extracellular component hydrolyzing PZ peptide. 1: protobacteria, 2: strain A, 3: strain B.

    图  3  正交实验的因素指标趋势图

    Figure  3.  Factor index trend chart of orthogonal experiment

    图  4  SDS-PAGE电泳分析胶原酶ColH的分泌表达鉴定(A,胞外分泌蛋白分析;B,胞内蛋白分析)

    Figure  4.  SDS-PAGE analysis of culture medium components and intracellular soluble components

    图  5  甘氨酸添加量对ColH表达和分泌的影响

    Figure  5.  Effects of amount of glycine added on ColH expression and secretion

    图  6  甘氨酸添加时间对ColH表达和分泌的影响

    Figure  6.  Effect of time of glycine added on ColH expression and secretion

    图  7  钙离子对ColH表达和分泌的影响

    Figure  7.  Effects of calcium ion on ColH expression and secretion.

    图  8  镁离子对ColH表达和分泌的影响

    Figure  8.  Effects of magnesium ion on ColH expression and secretion.

    图  9  诱导时间对ColH表达和分泌的影响

    Figure  9.  Effect of inducing time on ColH expression and secretion.

    表  1  本研究使用的引物

    Table  1.   Primers used in this work. The underlined part is the restriction site, the oblique part is the signal peptide sequence, and the bold part is the homologous sequence of primer and carrier terminal.

    Primer namesPrimer sequence(5′–3′)
    F1ATCGAGGATCCATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGG
    TTTCGCTACCGTAGCGCAGGCCGTCCAGAACGAAAGCAAACGCTAC
    R1ATCGACTCGAGACGACCAACGCTGCCTTC
    F2AAGGAGATATACCATGAAAAAGACAGCTATCGCGATTGC
    R2GGTGGTGGTGCTCGAGACGAC
    下载: 导出CSV

    表  2  因素水平表

    Table  2.   Factor level table

    FactorsLevels
    Inducing
    temperature/℃
    C(IPTG)/
    (mmol·L−1)
    OD600Loading
    volume/%
    1200.10.4510
    2250.50.9020
    3301.01.8030
    下载: 导出CSV
  • [1] KIRKNESS M W H, LEHMANN K, FORDE N R. Mechanics and structural stability of the collagen triple helix[J]. Current Opinion in Chemical Biology, 2019, 53: 98-105. doi: 10.1016/j.cbpa.2019.08.001
    [2] BELLA J. Collagen structure: New tricks from a very old dog[J]. Biochemical Journal, 2016, 473: 1001-1025. doi: 10.1042/BJ20151169
    [3] HOLMBECK K, BIANCO P, BIRKEDAL-HANSEN H. MT1-mmp: A collagenase essential for tumor cell invasive growth[J]. Cancer Cell, 2003, 4(2): 83-84. doi: 10.1016/S1535-6108(03)00196-X
    [4] BHAGWAT P K, DANDGE P B. Collagen and collagenolytic proteases: A review[J]. Biocatalysis and Agricultural Biotechnology, 2018, 15: 43-55. doi: 10.1016/j.bcab.2018.05.005
    [5] DUARTE A S, CORREIA A, ESTEVES A C. Bacterial collagenases-a review[J]. Critical Reviews in Microbiology, 2016, 42(1): 106-126. doi: 10.3109/1040841X.2014.904270
    [6] YU M S, LEE C Y. Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli[J]. Microbiology, 1999, 145(1): 143-150. doi: 10.1099/13500872-145-1-143
    [7] JORDAN G H. The use of intralesional clostridial collagenase injection therapy for Peyronie's disease: A prospective, single-center, non-placebo-controlled study[J]. Journal of Sexual Medicine, 2010, 5(1): 180-187.
    [8] ERDEVE O, ATASAY B, ARSAN S. Collagenase application for amputation in a preterm[J]. Pediatric Dermatology, 2010, 24(2): 195-196.
    [9] BRANDHORST H, BRANDHORST D, HESSE F, et al. Successful human islet isolation utilizing recombinant collagenase[J]. Diabetes, 2003, 52(5): 1143-1146. doi: 10.2337/diabetes.52.5.1143
    [10] BOND M, WART H V. Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography[J]. Biochemistry, 1984, 23(13): 3077-3085. doi: 10.1021/bi00308a035
    [11] YOSHIHARA K, MATSUSHITA O, MINAMI J, et al. Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase[J]. Journal of Bacteriology, 1994, 176(21): 6489-6496. doi: 10.1128/jb.176.21.6489-6496.1994
    [12] MATSUSHITA O, JUNG C M, KATAYAMA S, et al. Gene duplication and multiplicity of collagenases in Clostridium histolyticum[J]. Journal of Bacteriology, 1999, 181(3): 923-933. doi: 10.1128/JB.181.3.923-933.1999
    [13] TAMAI E, MIYATA S, TANAKA H, et al. High-level expression of his-tagged clostridial collagenase in Clostridium perfringens[J]. Appl Microbiol Biotechnol, 2008, 80(4): 627-635. doi: 10.1007/s00253-008-1592-1
    [14] JUNG C I, MATSUSHITA O, KATAYAMA S, et al. Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification[J]. Microbiology and Immunology, 2013, 40(12): 923-929.
    [15] HESSE F, BURTSCHER H, POPP F, et al. Recombinant enzymes for islet isolation: Purification of a collagenase from Clostridium histolyticum and cloning/expression of the gene[J]. Transplantation Proceedings, 1995, 27(6): 3287-3289.
    [16] DUCKA P, ECKHARD U, SCHöNAUER E, et al. A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli[J]. Applied Microbiology and Biotechnology, 2009, 83(6): 1055-1065. doi: 10.1007/s00253-009-1953-4
    [17] MERGULHãO F, SUMMERS D K, MONTEIRO G A. Recombinant protein secretion in Escherichia coli[J]. Biotechnology Advances, 2005, 23(3): 177-202. doi: 10.1016/j.biotechadv.2004.11.003
    [18] 华烨, 刘鹏, 王永红. 不同来源寡聚-1, 6-葡糖苷酶异源表达及酶学特性[J]. 华东理工大学学报(自然科学版), 2021, 47(04): 445-454.
    [19] WÜNSCH E, HEIDRICH H G. Zur quantitativen bestimmung der kollagenase[J]. Hoppe-Seyler´ s Zeitschrift für Physiologische Chemie, 1963, 333(1): 149-151.
    [20] 刘剑平. 应用数理统计[M]. 第3版. 华东理工大学出版社: 2019.
    [21] 盖霞, 陆兵, 夏杰, 等. 藻粉中叶黄素提取工艺的优化[J]. 华东理工大学学报(自然科学版), 2015, 41(01): 43-47. doi: 10.3969/j.issn.1006-3080.2015.01.007
    [22] LIU Z, TIAN L, CHEN Y, et al. Efficient extracellular production of κ-carrageenase in Escherichia coli: Effects of wild-type signal sequence and process conditions on extracellular secretion[J]. Journal of Biotechnology, 2014, 185: 8-14. doi: 10.1016/j.jbiotec.2014.06.007
    [23] SUN X, WEI S, GAO Y, et al. Heterologous expression and purification of a marine alginate lyase in Escherichia coli[J]. Protein Expression and Purification, 2018, 153: 97-104.
    [24] GUMPERT J, HOISCHEN C. Use of cell wall-less bacteria (L-forms) for efficient expression and secretion of heterologous gene products[J]. Current Opinion in Biotechnology, 1998, 9(5): 506-509. doi: 10.1016/S0958-1669(98)80037-2
    [25] PENG Y, ARISTIDOU A A, SAN K Y. Synergistic effect of glycine and bacteriocin release protein in the release of periplasmic protein in recombinant E. coli[J]. Biotechnology Letters, 1991, 13(5): 311-316. doi: 10.1007/BF01027674
    [26] HAMMES W, SCHLEIFER K H, KANDLER O. Mode of action of glycine on the biosynthesis of peptidoglycan[J]. Journal of Bacteriology, 1973, 116(2): 1029-1053. doi: 10.1128/jb.116.2.1029-1053.1973
    [27] LI Z F, LI B, LIU Z G, et al. Calcium leads to further increase in glycine-enhanced extracellular secretion of recombinant α-Cyclodextrin glycosyltransferase in Escherichia coli[J]. Journal of Agricultural and Food Chemistry, 2009, 57(14): 6231-6237. doi: 10.1021/jf901239k
    [28] OHBAYASHI N, MATSUMOTO T, SHIMA H, et al. Solution structure of clostridial collagenase H and its calcium-dependent global conformation change[J]. Biophysical Journal, 2013, 104(7): 1538-1545. doi: 10.1016/j.bpj.2013.02.022
    [29] YAMABHAI M, EMRAT S, SUKASEM S, et al. Secretion of recombinant bacillus hydrolytic enzymes using Escherichia coli expression systems[J]. Journal of Biotechnology, 2008, 133(1): 50-57. doi: 10.1016/j.jbiotec.2007.09.005
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出版历程
  • 收稿日期:  2021-09-24
  • 网络出版日期:  2022-04-12

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