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Endolysin Expression, Purification and Activity Determination by Zymography
酶谱法进行细胞内溶素表达、纯化和活性测定   

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Abstract

Endolysins are peptidoglycan-degrading (muralytic) enzymes produced by many bacteriophages for cell lysis of the host bacterium. The enzymatic activity of muralytic enzymes can be assayed qualitatively using a zymogram containing incorporated peptidoglycan. This protocol describes the expression of a recombinant 6x His-tagged endolysin using an Escherichia coli (E. coli) expression system and native affinity purification of the protein using Ni-NTA agarose. For the zymogram, the protocol details isolation of crude peptidoglycan from the Gram-negative bacterium Rhodobacter capsulatus and the zymography of purified protein and crude cell lysate. Construction of an E. coli BL21 (DE3) pET28-a(+)-derived endolysin-expression system is briefly described.
The protocol described here was developed and optimized for the endolysin 555 utilized by the Rhodobacter capsulatus bacteriophage-like gene transfer agent (RcGTA) (Westbye et al., 2013) and to study the muralytic activities of protein P14 of RcGTA (Fogg et al., 2012), but should be transferrable as a general protocol to express and study a variety of endolysins.

Keywords: Endolysin(素), Lysozyme(溶菌酶), Zymogram(酶谱), Peptidoglycan(肽聚糖), Muralytic(muralytic)

Materials and Reagents

  1. Protein purification
    1. pET28-a(+) (EMD Millipore, catalog number: 69864 ) or similar IPTG-inducible T7-based protein expression plasmid incorporating a 6x His tag
    2. Standard reagents and tools for molecular cloning (see Hasmann et al., 2011)
    3. E. coli BL21 (DE3) (New England BioLabs, catalog number: C2527I , or other source), or similar E. coli T7-based protein overexpression strain
    4. Kanamycin sulfate (50 mg/ml in dH2O, filter sterilized) or appropriate antibiotic if using different plasmid
    5. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (100 mM in dH2O, filter sterilized)
    6. Ni-NTA Agarose (QIAGEN, catalog number: 30210 )
    7. Imidazole (2 M in dH2O)
    8. Lysogeny broth (LB) (see Recipes)
    9. Lysis buffer (see Recipes)

  2. SDS-PAGE and zymogram
    1. SDS-PAGE gel (12% separation and 4% stacking layer, He, 2011)
    2. Laemmli buffer/sample loading buffer (He, 2011)
    3. 2-propanol
    4. Renaturation buffer (see Recipes)
    5. Coomassie brilliant blue protein stain solution (see Recipes)
    6. YPS broth (see Recipes)
    7. Destain solution (see Recipes)
    8. Phosphate buffer (see Recipes)

Equipment

  1. 250 ml culture flasks
  2. Incubator with shaker for culture flask (temperature adjustable) (or similar)
  3. French press for cell lysis (see Note on alternative lysis methods)
  4. Rotor JA-20 (Beckman Coulter) or equivalent
  5. Centrifuge suitable for rotor (Beckman Coulter, model: J2-HS or similar)
  6. Gravity column for Ni-NTA agarose
  7. SDS-PAGE apparatus with power supply (Mini-Protean and PowerPac, Bio-Rad Laboratories or equivalent)
  8. Microtube centrifuge (table-top)
  9. Microwave (optional)
  10. Flat (roux, or tissue culture) bottles (1 L for photoheterotrophic growth)
  11. Light-emitting incubator for photoheterotrophic growth (see Note 6)
  12. Erlenmeyer flask (500 ml) (pyrex, or other heat resistant glass)
  13. Glass beaker (1 L or large enough to encompass a 500 ml flask) (pyrex, or other heat resistant glass)
  14. Bunsen burner (or similar heat source)
  15. Vacuum concentrator (Savant SpeedVac SC110A concentrator with UVS400 vacuum system, or similar) (optional)

Procedure

  1. Construction of IPTG-inducible 6x His-tagged recombinant endolysin expression system
    1. The open reading frame of the putative endolysin is cloned into the vector pET28-a (+) using the unique NcoI (C-terminal 6x His tag) or NdeI (N-terminal 6x His tag) and XhoI restriction sites by standard molecular cloning methods. The resultant plasmid should encode a recombinant protein incorporating a terminal 6x His tag driven by a T7-based promoter (Note 1).
    2. Transform the resultant expression plasmid into E. coli BL21 (DE3) cells, to create an IPTG-inducible 6x His tagged endolysin expression system (Note 2).

  2. Native batch purification of 6x His tagged protein from E. coli
    1. Grow culture of BL21 (DE3) containing the expression plasmid in 10 ml LB containing appropriate antibiotic at 37 °C.
    2. Inoculate starter culture into 100 ml LB containing appropriate antibiotic in 250 ml culture flask to an optical density at 600 nm (OD600) of ~0.01 to 0.05. Incubate at 37 °C with shaking (200 rpm) to an OD600 of 0.5.
    3. Induce expression by addition of IPTG to 1 mM, shift culture to 30 °C (optional) and incubate for 4 h (200 rpm) (Notes 3 and 4).
    4. Harvest cells by centrifugation at 3,500 rcf at 4 °C and resuspend pellet in 2 to 5 ml lysis buffer.
    5. Lyse cells by passing through a French press at 900 psi, alternatively use sonicator. Clear lysate by centrifugation 16,000 rcf for 10 min and collect supernatant.
      Note: A French press should only be operated by trained personnel. Ensure that maximum pressure for cell is not exceeded.
    6. Add 1 to 3 ml Ni-NTA agarose to an empty gravity protein column. Wash with three column volumes of lysis buffer to equilibrate beads.
    7. Load cleared supernatant on column (see Figure 1), and allow to drain, collecting flow through. Wash with 5 column-volumes of lysis buffer.


      Figure 1. Column for Ni-NTA affinity purification. Gravity column for affinity purification using Ni-NTA agarose. Flow is controlled by clamping the short rubber tube on bottom of column. (The Ni-NTA agarose is stored in a solution of 20% ethanol.)

    8. Elute protein with ten 0.5 to 1 ml aliquots lysis buffer supplemented with 450 mM imidazole (final concentration), collecting each fraction (Note 5).
    9. Run a standard SDS-PAGE gel containing crude lysate, cleared lysate, flowthrough, wash and eluates (typically 5 μl). Stain gel using Coomassie brilliant blue and destain using water or Destain solution.
      Pool eluate fractions containing pure protein.
      Note: This step is required to determine the purity of isolated protein. The eluate should contain a single band of predicted size. Presence of several bands requires optimization of binding or wash conditions, or could indicate proteolysis of the protein. Gels can be rapidly stained and destained by heating in microwave, taking care to avoid boiling.

  3. Isolation of crude peptidoglycan from R. capsulatus
    1. Inoculate R. capsulatus into 1 L capped (roux or tissue culture) flat bottles filled with YPS medium.
    2. Incubate photoheterotrophically at 30 °C with illumination until stationary phase (2 to 3 days) (Note 6). Harvest cells by centrifugation at 6,800 rcf and resuspend cell pellet in 100 ml 4% SDS in water.
    3. Boil suspension vigorously in heat-resistant glass flask by holding flask over Bunsen burner for 15 min, swirling occasionally. Keep beaker at hand.
      Note: If suspension starts to foam and threaten to flow out of flask, move flask into beaker to collect and contain any spill. Wear appropriate safety equipment (goggles, gloves and lab coat advised as minimum).
    4. Cool cell suspension, centrifuge at 10,000 rcf for 10 min. Gently decant supernatant.
    5. Wash pellet 5 times by resuspending in 10 ml dH2O followed by centrifugation (10,000 rcf 10 min).
    6. Resuspend pellet in a small volume dH2O (~3 ml).
      Note: Crude peptidoglycan can be stored at 4 °C until use.
    7. To estimate mass of purified peptidoglycan material, dry material in pre-weighted microcentrifuge tubes using a vacuum concentrator. Resuspend material in small volume dH2O.

  4. Zymography of endolysin - protein separation
    1. Prepare SDS-PAGE zymogram by incorporating 5% (w/v) peptidoglycan into a standard 12% SDS-PAGE separating layer mixture before pouring into gel casting apparatus assembled according to manufacturer’s instructions. Overlay with 2-propanol and allow to set (20-40 min). Rinse 2-propanol off with dH2O, then overlay with standard 4% SDS-PAGE stacking layer and insert well-comb. Allow to set (20 - 40 min).
    2. Prepare (dilutions) of sample(s) by mixing with Laemmli loading buffer (5 μl Laemmli buffer per 10 μl sample).
    3. Briefly denature proteins by boiling for 5 min.
      Note: It is advisable to perform an initial titration of denaturation temperature (and time) required for the protein to enter gel. Extensive heating (boiling) may prevent efficient protein re-folding required for the functional assay.
    4. Load samples on gel and separate at 120 V until markers are sufficiently separated (~2 h). Dissemble gel apparatus, discard stacking layer.

  5. Zymography of endolysin - enzymatic assay
    1. Transfer resolving gel to plastic container, rinse extensively with dH2O to remove SDS-PAGE running buffer (Note 7).
    2. Renature proteins by washing gel twice in 5% Triton X-100 (v/v) in dH2O for 20 min with gentle shaking.
    3. For the enzymatic assay, decant Triton-X solution from gel and incubate the gel overnight (see Note 9) in 100 mM phosphate buffer at 30 °C or 37 °C with gentle shaking.
    4. To visualize, stain gels with Coomassie brilliant blue until bright blue. Destain using destain solution (Note 8).
    5. Enzyme activity is visible as a clearing against a blue background (see Figure 2).
      Note: Clearing should only be observed at the correct molecular weight of the protein in samples of purified protein or crude lysate of cells expressing protein. Activity should be absent in crude lysate of empty vector-control strains.


      Figure 2. Zymograms of muralytic proteins. A. The endolysin 555 from R. capsulatus required for RcGTA release. Zymogram of E. coli total cell lysate (lane 1) and purified protein (lane 2), and SDS-PAGE gel of purified protein (lane 3). B. The muralytic enzyme P14 encoded in the RcGTA gene cluster. E. coli total cell lysate of empty vector control (-) and P14-expressing vector (+). Panel A is Copyright © American Society for Microbiology, Journal of Bacteriology, 195(22), 2013, p. 5025 - 5040 and DOI: 10.1128/JB.00669-13 and reused with permission from Reference 4. Panel B is adapted from Reference 1 in accordance with the Creative Commons Attribution License (CC BY).

Notes

  1. Several other unique downstream restriction sites in addition to XhoI are present in pET28-a(+). For affinity purification using Ni-NTA, it is essential that the recombinant protein contains a 6xHis tag. The ATG-start codon of the endolysin gene should form part of the NdeI or NcoI restriction site. If using NcoI, the second codon may require optimization. The restriction sites are typically introduced in the PCR primer sequences. For DNA cloning work, we recommend the use of an E. coli strain optimized for cloning, such as DH5alpha.
  2. For high level T7-based expression of proteins such as the endolysin 555 from R. capsulatus in E. coli, the E. coli strain BL21 (DE3) or similar strains optimized for protein expression encoding IPTG-inducible T7 RNA polymerase works well. Be aware some E. coli protein expression strains contain the plasmids pLysS or pLysE, which express the T7 lysozyme (an endolysin).
  3. Expression conditions such as temperature should be determined empirically for each protein to be tested as many proteins are difficult to overexpress stably. For example, temperatures may be decreased to as low as 16 °C and IPTG induction concentrations can be decreased substantially.
  4. For initial expression, it is advisable to collect samples (1 ml) at several time points (0, 0.5, 1, 2 and 4 h) to optimize the time course of expression. Centrifuge and resuspend samples in Laemmli buffer and run on SDS-PAGE, followed by Coomassie brilliant blue staining to visualize.
  5. The binding affinity of proteins to Ni-NTA differs. The imidazole concentration required to elute a specific protein can be established by eluting protein using increasing concentration of imidazole. Run samples on SDS-PAGE and stain with Coomassie brilliant blue, to establish the concentration required to elute the protein from the Ni-NTA.
  6. A low cost incubator for photoheterotrophic growth of R. capsulatus can been constructed from a fishtank (“aquarium”) illuminated from the side by incandescent light bulbs, the temperature kept constant using a circulating water bath heater and immersing the culture vessel in the tank. Alternatively, R. capsulatus may be cultured chemoheterotrophically to lower cell density.
  7. A container lacking protruding internal edges is preferred to avoid damage to gel.
  8. If destained with water, the gel will destain slower, the color will change due to pH changes, and the intensity of background reduced.
  9. If no degradation is observed, possible reasons are:
    1. Absence of purified protein or cleavage of protein. Presence of purified protein of correct size should be verified by SDS-PAGE gel. If degradation/cleavage is expected, include protease inhibitors in lysis buffer.
    2. Too short incubation time. Increase incubation time.
    3. Low efficiency of protein renaturation. Subject samples to less intense heating before loading of gel, include low concentration (~0.5%) of Triton X-100 in phosphate buffer, or attempt using different detergent than Triton X-100 for renaturation. Alternatively, attempt a native gel using purified protein.
    4. Reducing agent may interfere with activity. Exclude beta-mercaptoethanol from Laemmli buffer.

Recipes

  1. Lysogeny broth (LB)
    10 g Bacto tryptone
    5 g yeast extract
    10 g NaCl
    Dissolved in 900 ml dH2O
    Adjust pH to 7.0 using NaOH or HCl
    Adjust to 1,000 ml final volume using dH2O
    Autoclave
  2. YPS broth
    3 g yeast extract
    3 g Bacto peptone
    2 ml of 1 M MgSO4
    2 ml of 1 M CaCl2
    Dissolved in 900 ml dH2O
    Adjust pH to 6.8 using NaOH or HCl
    Adjust to 1,000 ml final volume using dH2O
    Autoclave
  3. Lysis buffer
    20 mM NaCl
    20 mM Tris-HCl (pH 8.0)
    20 mM Imidazole
  4. Renaturation buffer
    5% (v/v) Triton X-100 in dH2O
  5. Coomassie brilliant blue protein stain solution
    0.1% (w/v) Coomassie brilliant blue R-250
    10% (v/v) acetic acid (glacial)
    40% (v/v) methanol
    dH2O
  6. Destain solution
    10% (v/v) acetic acid (glacial)
    40% (v/v) methanol
    dH2O
  7. Phosphate buffer (pH 7.5)
    83.4 ml of 1 M K2HPO4
    16.6 ml of 1 M KH2PO4
    dH2O to 1,000 ml final volume
    Note: Detailed SDS-PAGE gel buffers and sample loading/Laemmli buffers are described in He (2011).

Acknowledgments

The zymogram protocol was originally developed from Rosenthal and Dziarski (1994) and Hasmann et al. (2011).

References

  1. Fogg, P. C., Westbye, A. B. and Beatty, J. T. (2012). One for all or all for one: heterogeneous expression and host cell lysis are key to gene transfer agent activity in Rhodobacter capsulatus. PloS One 7(8): e43772.
  2. Hasmann, A., Wehrschuetz-Sigl, E., Kanzler, G., Gewessler, U., Hulla, E., Schneider, K. P., Binder, B., Schintler, M. and Guebitz, G. M. (2011). Novel peptidoglycan-based diagnostic devices for detection of wound infection. Diagn Microbiol Infect Dis 71(1): 12-23.
  3. He, F. L. (2011). Standard DNA Cloning. Bio-protocol 1(7): e52.
  4. He, F. L. (2011). Laemmli-SDS-PAGE. Bio-protocol 1(11): e80.
  5. Rosenthal, R. S. and Dziarski, R. (1994). Isolation of peptidoglycan and soluble peptidoglycan fragments. Methods Enzymol 235: 253–285.
  6. Westbye, A. B., Leung, M. M., Florizone, S. M., Taylor, T. A., Johnson, J. A., Fogg, P. C. and Beatty, J. T. (2013). Phosphate concentration and the putative sensor kinase protein CckA modulate cell lysis and release of the Rhodobacter capsulatus gene transfer agent. J Bacteriol 195(22): 5025-5040.

简介

内溶素是由许多噬菌体产生的用于细菌裂解宿主细菌的肽聚糖降解(muralytic)酶。可以使用含有掺入的肽聚糖的酶谱定性测定壁溶酶的酶活性。该方案描述了使用大肠杆菌(大肠杆菌)表达系统和使用Ni-NTA琼脂糖的蛋白质的天然亲和纯化的重组6x His-标记的细胞内溶素的表达。对于酶谱,方案详述了粗制肽聚糖从革兰氏阴性细菌红细菌菌体的分离和纯化蛋白质和粗细胞裂解物的酶谱。建筑。简要描述了大肠杆菌BL21(DE3)pET28-a(+)衍生的内溶素表达系统。本文所述的方案针对由荚膜红细菌使用的细胞内溶素555开发和优化,噬菌体样基因转移试剂(RcGTA)(Westbye等人,2013),并研究RcGTA的蛋白P14的干扰活性(Fogg等人, ,2012),但应该是可转移的,作为一个一般的协议,以表达和研究各种细胞内溶素。

关键字:素, 溶菌酶, 酶谱, 肽聚糖, muralytic

材料和试剂

  1. 蛋白纯化
    1. pET28-a(+)(EMD Millipore,目录号:69864)或含有6×His标签的类似的基于IPTG诱导型T7的蛋白质表达质粒。
    2. 用于分子克隆的标准试剂和工具(参见Hasmann等人,2011)
    3. 大肠杆菌BL21(DE3)(New England BioLabs,目录号:C2527I,或其他来源)或类似的E。 大肠杆菌 T7基蛋白质过表达株
    4. 如果使用不同的质粒,硫酸卡那霉素(50mg/ml,在dH 2 O中,过滤灭菌)或适当的抗生素。
    5. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(100mM,在dH 2 O中,过滤除菌)
    6. Ni-NTA琼脂糖(QIAGEN,目录号:30210)
    7. 咪唑(2M,在dH 2 O中)
    8. 溶菌酶肉汤(LB)(参见食谱)
    9. 裂解缓冲液(见配方)

  2. SDS-PAGE和酶谱
    1. SDS-PAGE凝胶(12%分离和4%堆积层,He,2011)
    2. Laemmli缓冲液/样品上样缓冲液(He,2011)
    3. 2-丙醇
    4. 复性缓冲液(参见配方)
    5. 考马斯亮蓝蛋白染色溶液(见配方)
    6. YPS肉汤(见配方)
    7. Destain解决方案(参见配方)
    8. 磷酸盐缓冲液(参见配方)

设备

  1. 250 ml培养瓶
  2. 带培养瓶振荡器(温度可调)(或类似)的培养箱
  3. 法语压力器用于细胞裂解(参见关于替代性裂解方法的注释)
  4. Rotor JA-20(Beckman Coulter)或等同物
  5. 适用于转子(Beckman Coulter,型号:J2-HS或类似)的离心机
  6. Ni-NTA琼脂糖的重力柱
  7. 具有电源(Mini-Protean和PowerPac,Bio-Rad Laboratories或等同物)的SDS-PAGE装置
  8. Microtube离心机(台式)
  9. 微波(可选)
  10. 平(瓶或组织培养)瓶(1 L用于光异养生长)
  11. 用于光异养生长的发光孵化器(见注6)
  12. 锥形瓶(500ml)(pyrex或其它耐热玻璃)
  13. 玻璃烧杯(1升或大到足以容纳500ml烧瓶)(pyrex或其他耐热玻璃)
  14. 本生灯(或类似的热源)
  15. 真空浓缩器(带UVS400真空系统的Savant SpeedVac SC110A浓缩器或类似设备)(可选)

程序

  1. 构建IPTG诱导的6×His标记的重组内溶素表达系统
    1. 使用独特的NcoI(C末端6×His标签)或NdeI(N末端6×His标签)和XhoI限制性位点通过标准分子克隆方法将推定的细胞内溶素的开放阅读框克隆入载体pET28-a(+) 。 所得质粒应编码掺入由基于T7的启动子驱动的末端6×His标签的重组蛋白质(注1)。
    2. 将所得的表达质粒转化为E。 大肠杆菌BL21(DE3)细胞,以产生IPTG诱导的6x His标记的细胞内溶素表达系统(注释2)。

  2. 来自e的6x His标签蛋白的天然批次纯化。 大肠杆菌
    1. 在37℃下,在含有适当抗生素的10ml LB中培养含有表达质粒的BL21(DE3)
    2. 在250ml培养瓶中将起始培养物接种到含有合适抗生素的100ml LB中,达到〜0.01-0.05的600nm光密度(OD 600)。 在37℃下振荡(200rpm)孵育至OD 600为0.5。
    3. 通过加入IPTG至1mM诱导表达,将培养物转移至30℃(任选)并孵育4小时(200rpm)(注3和4)。
    4. 通过在4℃下以3500 rcf离心收获细胞,并在2至5 ml裂解缓冲液中重悬沉淀。
    5. 通过在900psi下通过French压片机裂解细胞,或者使用超声波仪。 通过离心16,000 rcf清洗裂解物10分钟,收集上清液 注意:法语新闻只应由经过培训的人员操作。确保不超过电池的最大压力。
    6. 加入1到3毫升Ni-NTA琼脂糖到空重力蛋白柱。用三倍柱体积的裂解缓冲液洗涤以平衡珠子
    7. 在柱上加载澄清的上清液(见图1),并允许排放,收集流过。用5倍柱体积的裂解缓冲液洗涤

      图1. Ni-NTA亲和纯化柱。使用Ni-NTA琼脂糖进行亲和纯化的重力柱。通过将短橡胶管夹在色谱柱底部来控制流量。 (Ni-NTA琼脂糖贮存在20%乙醇的溶液中)
    8. 用补充有450mM咪唑(最终浓度)的10个0.5至1ml等分试样溶解缓冲液洗脱蛋白质,收集各级分(注释5)。
    9. 运行包含粗裂解液,澄清裂解液,流过液,洗涤液和洗脱液(通常为5μl)的标准SDS-PAGE凝胶。使用考马斯亮蓝染色凝胶并使用水或脱色溶液脱色 含有纯蛋白的池洗脱级分 注意:该步骤是确定分离蛋白纯度所必需的。洗脱液应包含预测大小的单个条带。存在几个条带需要优化结合或洗涤条件,或可以指示蛋白质的蛋白水解。凝胶可以通过微波加热快速染色和脱色,注意避免沸腾。

  3. 从R中分离粗制肽聚糖。 capsulatus
    1. 接种 R。荚膜囊泡装入装有YPS培养基的1L封盖(胚乳或组织培养)扁瓶中。
    2. 在30℃下照明直至静止期(2至3天)孵育光异源性(注6)。通过在6,800 rcf离心收获细胞,并重悬细胞沉淀在100 ml 4%SDS水中
    3. 在耐热玻璃烧瓶中通过将烧瓶在本生灯上保持15分钟,偶尔涡旋而剧烈地煮沸悬浮液。保持烧杯在手。
      注意:如果悬浮液开始起泡并有可能从烧瓶中流出,将烧瓶移入烧杯中收集并包含任何溢出物。 穿戴适当的安全设备(护目镜,手套和实验服)。
    4. 冷却细胞悬浮液,10,000 rcf离心10分钟。 轻轻倒出上清液。
    5. 通过重悬于10ml dH 2 O中然后离心(10,000rcf 10分钟)来洗涤沉淀5次。
    6. 在小体积dH 2 O(〜3ml)中重悬沉淀 注意:粗制肽聚糖可以在4°C储存备用。
    7. 为了估计纯化的肽聚糖材料的质量,使用真空浓缩器在预加重的微量离心管中干燥材料。 以小体积dH 2 O重悬材料。

  4. 内溶素 - 蛋白质分离的酶谱
    1. 通过将5%(w/v)肽聚糖掺入标准的12%SDS-PAGE分离层混合物中制备SDS-PAGE酶谱,然后倒入根据制造商的说明书组装的凝胶铸造设备中。用2-丙醇覆盖并允许凝固(20-40分钟)。用dH 2 O冲洗2-丙醇,然后用标准的4%SDS-PAGE堆积层和插入孔梳覆盖。允许设置(20 - 40分钟)。
    2. 通过与Laemmli上样缓冲液(每10μl样品5μlLaemmli缓冲液)混合来制备(稀释)样品。
    3. 通过煮沸5分钟使蛋白质短暂变性。
      注意:建议对蛋白质进入凝胶所需的变性温度(和时间)进行初始滴定。大量加热(沸腾)可能会妨碍功能测定所需的蛋白质重折叠。
    4. 加载样品在凝胶上,在120 V分离,直到标记充分分离(〜2小时)。拆卸凝胶装置,丢弃堆积层。

  5. 内溶素酶法测定
    1. 将分离的凝胶转移到塑料容器中,用dH 2 O彻底冲洗以除去SDS-PAGE运行缓冲液(注7)。
    2. 通过在dH 2 O中在5%Triton X-100(v/v)中洗涤凝胶两次,轻轻摇动20分钟来使蛋白质再生。
    3. 对于酶测定,从凝胶中倾析Triton-X溶液,并在30℃或37℃下,在轻微摇动的情况下,在100mM磷酸盐缓冲液中孵育凝胶过夜(见注9)。
    4. 为了可视化,用考马斯亮蓝染色凝胶直至亮蓝色。使用脱色溶液脱色(注8)。
    5. 酶活性作为蓝色背景的清除是可见的(参见图2) 注意:只有在纯化蛋白质样品或表达蛋白质的细胞粗提物的蛋白质分子量正确的情况下才能观察到清除。空载体对照的粗裂解物中应不存在活性 应变。


      图2.溶壁蛋白的酶谱。 A.来自R的细胞内溶素555。 capsulatus 所需的RcGTA版本。酶的酶谱。 (泳道1)和纯化的蛋白质(泳道2),和纯化的蛋白质的SDS-PAGE凝胶(泳道3)。 B.在RcGTA基因簇中编码的Muralytic酶P14。 E。空载体对照( - )和表达P14的载体(+)的大肠杆菌总细胞裂解物。面板A版权所有American Society for Microbiology,Journal of Bacteriology,195(22),2013,p。 5025 - 5040和DOI:10.1128/JB.00669-13并且在参考文献4的许可下重复使用。根据知识共享署名许可(CC BY)。

笔记

  1. pET28-a(+)中存在除XhoI外的几个其它独特的下游限制性位点。对于使用Ni-NTA的亲和纯化,重要的蛋白质含有6xHis标签是必要的。内溶素基因的ATG起始密码子应形成NdeI或NcoI限制性位点的一部分。如果使用NcoI,第二密码子可能需要优化。通常将限制性位点引入PCR引物序列中。对于DNA克隆工作,我们建议使用 E。大肠杆菌菌株,例如DH5α
  2. 对于蛋白质如来自R的细胞内溶素555的高水平基于T7的表达。 capsulatus 在 大肠杆菌, 大肠杆菌菌株BL21(DE3)或针对编码IPTG诱导型T7RNA聚合酶的蛋白质表达而优化的类似菌株工作良好。注意一些 E。大肠杆菌蛋白质表达菌株含有表达T7溶菌酶(内溶素)的质粒pLysS或pLysE。
  3. 对于待测试的每种蛋白质,应该根据经验确定表达条件例如温度,因为许多蛋白质难以稳定地过表达。例如,温度可以降低至低至16℃,并且IPTG诱导浓度可以显着降低。
  4. 对于初始表达,建议在几个时间点(0,0.5,1,2和4小时)收集样品(1ml)以优化表达的时间过程。离心并将样品重悬于Laemmli缓冲液中并在SDS-PAGE上运行,随后进行考马斯亮蓝染色以显现。
  5. 蛋白质与Ni-NTA的结合亲和力不同。洗脱特定蛋白质所需的咪唑浓度可以通过使用增加浓度的咪唑洗脱蛋白质来建立。在SDS-PAGE上运行样品并用考马斯亮蓝染色,以建立从Ni-NTA中洗脱蛋白所需的浓度。
  6. 用于R的光异养生长的低成本培养箱。可以由从白炽灯泡的侧面照明的鱼缸("水族馆")构造,温度保持恒定,使用 循环水浴加热器并将培养容器浸入槽中。 或者,R。 荚膜囊泡可以化学诱导培养以降低细胞密度
  7. 优选容器没有突出的内部边缘,以避免损坏凝胶
  8. 如果用水脱色,凝胶将脱色较慢,颜色将由于pH变化而改变,并且背景的强度降低。
  9. 如果没有观察到降解,可能的原因是:
    1. 缺乏纯化的蛋白质或蛋白质的切割。 存在正确大小的纯化蛋白应通过SDS-PAGE凝胶验证。 如果预期降解/裂解,在裂解缓冲液中包括蛋白酶抑制剂
    2. 孵育时间过短。 增加孵育时间。
    3. 低效率的蛋白复性。 受试样品在加载凝胶之前进行不太强烈的加热,包括在磷酸盐缓冲液中的低浓度(〜0.5%)Triton X-100,或尝试使用与Triton X-100不同的去垢剂用于复性。 或者,尝试使用纯化的蛋白质的天然凝胶
    4. 还原剂可能会干扰活性。 从Laemmli缓冲液中排除β-巯基乙醇。

食谱

  1. 溶菌酶肉汤(LB)
    10克细菌胰蛋白酶
    5g酵母提取物
    10克NaCl
    溶于900ml dH 2 O中 使用NaOH或HCl调节pH至7.0 使用dH 2 O/dm 2调节至1000ml最终体积 高压灭菌器
  2. YPS肉汤
    3g酵母提取物
    3克细菌蛋白胨
    加入2ml 1M MgSO 4 加入2ml 1M CaCl 2 溶于900ml dH 2 O中 使用NaOH或HCl调节pH至6.8 使用dH 2 O/dm 2调节至1000ml最终体积 高压灭菌器
  3. 裂解缓冲液
    20mM NaCl 20mM Tris-HCl(pH8.0) 20mM咪唑
  4. 复性缓冲区
    5%(v/v)Triton X-100,在dH 2 O中
  5. 考马斯亮蓝蛋白染色溶液
    0.1%(w/v)考马斯亮蓝R-250
    10%(v/v)乙酸(冰) 40%(v/v)甲醇
    dH 2 2 O
  6. 解除解决方案
    10%(v/v)乙酸(冰) 40%(v/v)甲醇
    dH 2 2 O
  7. 20mM咪唑
  8. 复性缓冲区
    5%(v/v)Triton X-100,在dH 2 O中
  9. 考马斯亮蓝蛋白染色溶液
    0.1%(w/v)考马斯亮蓝R-250
    10%(v/v)乙酸(冰) 40%(v/v)甲醇
    dH 2 2 O
  10. 解除解决方案
    10%(v/v)乙酸(冰) 40%(v/v)甲醇
    dH 2 2 O
  11. ... Fogg,P.C.,Westbye,A.B.and Beatty,J.T。(2012)。 一个或所有的:异质表达和宿主细胞裂解是基因转移剂活性的关键in Rhodobacter capsulatus 。 PloS One 7(8):e43772。
  12. Hasmann,A.,Wehrschuetz-Sigl,E.,Kanzler,G.,Gewessler,U.,Hulla,E。,Schneider,K.P.,Binder,B.,Schintler,M.and Guebitz, 用于检测伤口感染的新型基于肽聚糖的诊断装置。 Diagn Microbiol Infect Dis 71(1):12-23。
  13. 他,F.L。(2011)。 标准DNA克隆 生物协议 1(7):e52。
  14. 他,F.L。(2011)。 Laemmli-SDS-PAGE。生物协议 1(11): e80。
  15. Rosenthal,R.S.and Dziarski,R。(1994)。 肽聚糖和可溶性肽聚糖片段的分离方法Enzymol 235:253-285。
  16. Westbye,A.B.,Leung,M.M.,Florizone,S.M.,Taylor,T.A.,Johnson,J.A.,Fogg,P.C.and Beatty,J.T。(2013)。 磷酸盐浓度和 假定的传感器激酶蛋白CckA调节细胞裂解并释放耻垢红细菌基因转移剂。细菌195(22):5025-5040。 >
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Westbye, A. B., Fogg, P. C. and Beatty, J. T. (2014). Endolysin Expression, Purification and Activity Determination by Zymography. Bio-protocol 4(16): e1208. DOI: 10.21769/BioProtoc.1208.
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