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Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor
由弧菌群体感应受体锚定的腺苷酸环化酶膜的催化结构域的蛋白表达方案   

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Abstract

The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.

Keywords: Adenylate cyclase (AC)(腺苷酸环化酶(AC)), Quorum sensing (QS)(群体感应(QS)), CqsS(CqsS), Membrane protein(膜蛋白), Protein expression(蛋白表达), French press(弗氏压碎器)

Background

Membrane-delimited mammalian adenylate cyclases (ACs) are class IIIa ACs. Regulation is indirectly via stimulatory (or inhibitory) Gα-proteins which are released intracellularly upon extracellular stimulation of G-protein-coupled-receptors (GPCR) by first messengers. ACs generate the universal second messenger cAMP using ATP as a substrate. The size of the two hexahelical membrane domains in vertebrate ACs by far exceeds the requirements for a simple membrane anchorage. Yet, regulatory features of this intrinsic membrane anchor/receptor domain are unknown. To investigate a potential function the canonical class IIIa AC Rv1625c from Mycobacteria was chosen which can be easily expressed in bacteria (Guo et al., 2001 and 2005) in contrast to mammalian AC isoforms. We replaced the hexahelical membrane anchor of the Rv1625c AC by the receptor domain of the hexahelical quorum sensing (QS) receptor from V. harveyi, CqsS, to examine whether we can confer a direct regulation of the AC by the QS-ligand ‘cholera autoinducer-1’, CAI-1. The design of the QS-receptor and the class IIIa membrane anchors are highly similar, i.e., minimal transmembrane α-helices and exceptionally short connecting loops.

We have demonstrated a direct regulation of a class IIIa AC by an extracellular signal. This considerably supports the hypothesis of a receptor function for the membrane anchor. Indeed, it raises the possibility that in addition to the well-established indirect GPCR-Gα-protein regulation of mammalian ACs a second, rather different set of signals directly impinge on this most important enzyme.

Materials and Reagents

  1. Expression
    1. FisherbrandTM syringe filter, 25 mm, 0.22 µm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09719A )
      Note: This product has been discontinued.
    2. Glycerol stock of Escherichia coli BL21 (DE3) (F-ompT hsdSB [rB-mB-] gal dcm [DE3]) transformed with plasmid pQE80L or pETDuet-3:
      1. pQE80L [QIAGEN, ΔXhoI, ΔNcoI]
        Encodes a lacIq repression module; N-terminal RGS- His6-tag
      2. pETDuet-3 (Figure 1)
        MCS1 of pETDuet-1 [Novagen] is converted to MCS of pQE30 [QIAGEN]
        MCS1: N-terminal RGS-His6-tag, MCS2: C-terminal S-tag
      Note: Expression of both plasmids is controlled by an IPTG-inducible T5 (pQE80L) or T7 (pETDuet-3) promoter. pQE80L has only one MCS. If you want to co-express a second protein from the same plasmid, pETDuet expression vectors are suitable.
    3. Ampicillin sodium salt (Carl Roth, catalog number: K029 )
    4. IPTG (isopropyl β-D-thiogalactopyranoside) (AppliChem, catalog number: A1008 )
    5. LB broth (Lennox) (Carl Roth, catalog number: X964 )
    6. Ampicillin solution (see Recipes)
    7. IPTG solution (see Recipes)
    8. LB-medium (see Recipes)


      Figure 1. Comparison of the two MCS of pETDuet-1 (A) and pETDuet-3 (B)
      Note: This sequence map is generated using the program DNA5 (https://pga.mgh.harvard.edu/web_apps/web_map/start).

  2. Cell harvesting
    1. Culture from expression
    2. Tris (AppliChem, catalog number: A1086 )
    3. HCl (37%)
    4. EDTA (Sigma-Aldrich, catalog number: E5134 )
    5. Wash-buffer (see Recipes)

  3. Membrane preparation
    1. 1.5 ml Eppendorf tube
    2. Frozen cell pellet from expression
    3. Thioglycerol (Sigma-Aldrich, catalog number: M1753 )
    4. Sodium chloride, NaCl (EMD Millipore, catalog number: 106404 )
    5. cOmpleteTM EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 05056489001 )
    6. Glycerol (EMD Millipore, catalog number: 104094 )
    7. Liquid N2
    8. Lysis-buffer (see Recipes)
    9. Membrane-buffer (see Recipes)

  4. Data analysis
    1. Tris (AppliChem, catalog number: A1086)
    2. HCl (37%)
    3. Sodium dodecyl sulfate, SDS (Sigma-Aldrich, catalog number: 71729 )
    4. β-mercaptoethanol (EMD Millipore, catalog number: 805740 )
    5. Glycerol (EMD Millipore, catalog number: 104094)
    6. Bromophenol blue (Sigma-Aldrich, catalog number: B8026 )
    7. RGSHis-antibody (QIAGEN, catalog number: 34650 )
    8. S-tag-antibody (EMD Millipore, catalog number: 71549 )
    9. Protein marker IV prestained (VWR, catalog number: 27-2110 )
    10. Protein marker I unstained (VWR, catalog number: PEQL27-1010 )
    11. ECL Plex goat-anti-mouse IgG-Cy3-antibody (GE Healthcare, catalog number: PA43010 )
    12. 4x SDS-loading dye (see Recipes)

Equipment

  1. Expression
    1. 1 L Erlenmeyer flask
    2. Eppendorf Biophotometer
    3. Cuvettes  
    4. Shaker incubator (37 °C and 22 °C)

  2. Cell harvesting
    1. Centrifuges (supplier: Thermo Fisher Scientific)
      1. Sorvall RC5B Plus
        Rotor: Kontron-Hermle A6.14 (Sorvall, catalog number: 202200 )
      2. Heraeus Megafuge 1.0R
        Rotor: Heraeus Sepatech BS4402/A (Heraeus, catalog number: 3360 )
    2. 250 ml Sorvall metal rotor tubes (Sorvall, catalog number: 522 )
    3. 50 ml centrifuge tube (Greiner Bio One, catalog number: 227261 )
    4. Vortex Genie-2 (VWR, catalog number: 4445900 )
    5. Box with water-ice-mix
    6. Freezer (-80 °C)

  3. Membrane preparation
    1. Vortex Genie-2 (Scientific Industries, model: Vortex-Genie 2 )
    2. French® Pressure Cell Press (SLM Instruments, SLM Aminco®, model: FA-078-E1 )
      Alternative supplier: Glen Mills Inc. (USA) or G.Heinemann Ultraschall- und Labortechnik (Germany)
    3. Aminco® French Pressure Cell (SLM Instruments, SLM Aminco®, catalog number: FA-073 , serial number: 9110668)
      Alternative supplier: Glen Mills Inc. (USA) or G.Heinemann Ultraschall- und Labortechnik (Germany)
    4. Centrifuges
      1. Heraeus Megafuge 1.0R
        1. Rotor: Heraeus Sepatech BS4402/A (Heraeus, catalog number: 3360 )
      2. Beckmann L-60
        1. Rotor: Beckman Coulter, model: Type 50.2 Ti
    5. Box with water-ice-mix
    6. Polycarbonate ultracentrifuge-tubes
    7. 7 ml Dounce Tissue Grinder (WHEATON, catalog number: 357542 )
    8. Freezer (-80 °C)

  4. Data analysis
    1. Ettan DIGE Imager (GE Healthcare, catalog number: 63005642 or 29-0834-61 )

Software

  1. Program DNA5 (https://pga.mgh.harvard.edu/web_apps/web_map/start)

Procedure

  1. Expression
    1. Inoculate a flask containing 200 ml LB-medium and 200 µl ampicillin (final concentration: 100 µg/ml) with approximately 5 ml overnight culture with the desired construct (see Materials and Reagents A1. point) to an OD600 of 0.1.
    2. Incubate the culture under shaking (200 rpm) at 37 °C up to an OD600 of 0.2-0.3 (approx. 45-90 min).
    3. Lower temperature to 22 °C (shaker incubator).
    4. At an OD600 of 0.4-0.6 induce expression by 500 µM IPTG (100 µl 1 M IPTG/200 ml culture).

  2. Cell harvesting
    1. Harvest the cells at an OD600 of 2.0-2.8 (120-150 min after induction).
      Collect cells at 3,200 x g for 10 min at 4 °C (Sorvall centrifuge).
    2. Add 25 ml of wash-buffer (4 °C) to the pellet, suspend by vortexing and pellet at 4,300 x g for 30 min at 4 °C (Heraeus centrifuge).
    3. Discard supernatant and store cells at -80 °C or continue with the membrane preparation.

  3. Membrane preparation
    1. Thaw frozen cells on ice and suspend in 25 ml of lysis-buffer (4 °C) by vortexing.
      Notes:
      1. The cell pellet should be completely dissolved to avoid clogging the outlet of the Aminco® French Pressure Cell.
      2. Add always the cOmpleteTM EDTA-free protease inhibitor tablet just before using the lysis-buffer.
    2. Lyse cells mechanically by French press (1,100 psi) twice (Figure 2).
      Notes:
      1. Aminco® French Pressure Cell should be kept pre-cooled at 4 °C.
      2. Open outlet of the Aminco® French Pressure Cell such that a flow drop by drop is visible.
      3. Make sure that samples are continuously cooled in ice-water.
    3. Centrifuge homogenate for 30 min, 4 °C, 4,300 x g (Heraeus centrifuge) and discard pellet (cell debris).
    4. Transfer supernatant to an ultracentrifuge-tube and pellet membranes at 100,000 x g, 4 °C for 60 min (Beckman L-60 centrifuge).
    5. Decant supernatant, take up membranes (pellet) in 1-2 ml membrane-buffer and gently suspend in a homogenizer (Dounce Tissue Grinder). Transfer the membrane preparation into a 1.5 ml Eppendorf tube.
      Note: The amount of membrane-buffer to be used depends on the size of the pellet. Suspend a pellet of approximately 1 cm in diameter at the bottom of the centrifuge tube in 2 ml membrane-buffer.
    6. Freeze membrane preparation in liquid N2 and store at -80 °C.


      Figure 2. Short French press protocol. A. Equipment of the French Pressure Cell; B. Aminco French Pressure Cell (taken out of the fridge 4 °C); C. Attention! Before use: Grease the O-rings and back-up rings of the stamp with glycerol! Insert the stamp into the cell body so that the inner cell body surface is covered with glycerol as well! D. Place the cell body with the stamp upside down into the stand with the opening facing upwards. E. Place the flow valve into the hole. F. Fill in the cell solution (looks milky). G. Put the lid on top. H. Close the flow valve. I. Place the French Pressure Cell into the gadget and… (J) close the bracket. K. Start the French press by turning the hand gear on ‘high’ and (L) switch on the pump. M. When the French press set up 1,100 psi… (N) open carefully the flow valve such that… (O) a flow drop by drop is visible. P. Attention! Stop in time so you can easily open the bracket and the stamp does not hit the ground! Q. When you are done, flip the switch to ‘down’ and turn on the pump again. The hydraulic lift moves down and you can take out the French Pressure Cell. R. Your cells are lysed properly when you can see the scale through the tube. S. Put your sample back on ice for the next step.

Data analysis

The expression of the proteins is verified by SDS-PAGE (Laemmli, 1970) and Western blot. The isolated membranes are incubated in 4x SDS-loading dye at room temperature for at least 30 min prior to application to SDS-PAGE (do not boil sample). Dilute the sample (membrane preparation) in MilliQ H2O to get 2.5-5 µg of protein in a final volume of 15 µl. Add 5 µl 4x SDS-loading dye. Load 20 µl of the mixture into one slot of the SDS-PAGE. The membranes are incubated for 1 h with each antibody (first antibody at 4 °C, second antibody at room temperature). The first antibody is either the RGSHis- (Figures 3A and 3C) or the S-tag-antibody (Figure 3B). In both cases, the ECL Plex goat-anti-mouse IgG-Cy3-antibody is used as a secondary antibody (dilution 1:2,500). Western blot evaluation is carried out using an Ettan DIGE Imager.
Note: In contrast to soluble proteins, membrane proteins are NOT boiled (95 °C, 5-10 min).


Figure 3. Western blots (A-C) and SDS-PAGE (D)

Recipes

  1. Ampicillin solution (100 mg/ml)
    Dissolve 100 mg ampicillin in 1 ml MilliQ H2O (filter to sterilize)
  2. 1 M IPTG solution
    Dissolve 238.3 mg IPTG in 1 ml MilliQ H2O (filter to sterilize)
  3. LB-medium
    Dissolve 20 g LB in 1 L demineralized H2O (autoclave 20 min at 121 °C)
  4. Wash-buffer
    50 mM Tris/HCl (pH 8.0 at room temperature)
    1 mM EDTA
  5. Lysis-buffer
    50 mM Tris/HCl (pH 8.0 at room temperature)
    2 mM thioglycerol
    50 mM NaCl
    1 tablet cOmpleteTM EDTA-free protease inhibitor/50 ml lysis-buffer
  6. Membrane-buffer
    40 mM Tris/HCl (pH 8.0 at room temperature)
    1.6 mM thioglycerol
    20% glycerol (85%)
  7. 4x SDS-loading dye
    130 mM Tris/HCl (pH 6.8)
    10% SDS
    10% β-mercaptoethanol
    20% glycerol (85%)
    0.06% bromophenol blue

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 766; TP B08).

References

  1. Beltz, S., Bassler, J. and Schultz, J. E. (2016). Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases. Elife 5: e13098.
  2. Guo, Y. L., Kurz, U., Schultz, A., Linder, J. U., Dittrich, D., Keller, C., Ehlers, S., Sander, P. and Schultz, J. E. (2005). Interaction of Rv1625c, a mycobacterial class IIIa adenylyl cyclase, with a mammalian congener. Mol Microbiol 57(3): 667-677.
  3. Guo, Y. L., Seebacher, T., Kurz, U., Linder, J. U. and Schultz, J. E. (2001). Adenylyl cyclase Rv1625c of Mycobacterium tuberculosis: a progenitor of mammalian adenylyl cyclases. EMBO J 20(14): 3667-3675.
  4. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.

简介

最近报道了通过其群岛感应受体CqsS(Vibrio harveyi)交换其膜锚定点直接调节分枝杆菌腺苷酸环化酶(Rv1625c)(Beltz等,2016)。 该方案描述了这些嵌合蛋白的表达和膜制备。
【背景】膜分隔的哺乳动物腺苷酸环化酶(AC)是IIIa类AC。调节是间接通过第一信使细胞外刺激G蛋白偶联受体(GPCR)而在细胞内释放的刺激(或抑制性)Gα-蛋白。 AC产生使用ATP作为底物的通用第二信使cAMP。脊椎动物AC中两个六角膜结构域的大小远远超过了对简单膜锚固的要求。然而,这种内在的膜锚定/受体结构域的调节特征是未知的。为了研究潜在的功能,选择了可以容易地在细菌中表达的分类细菌的典型类IIIa AC Rv1625c(Guo等人,2001和2005)与哺乳动物AC同工型相反。我们用来自哈维氏弧菌CqsS的六价体积感知(QS)受体的受体结构域取代了Rv1625c AC的六角膜锚,以检查我们是否可以通过QS配体的霍乱自动诱导剂直接调节AC -1',CAI-1。 QS受体和IIIa类膜锚的设计是非常相似的,即最小的跨膜α-螺旋和极短的连接环。
我们已经证明了通过细胞外信号直接调节IIIa AC类。这大大支持了膜锚的受体功能的假说。事实上,它提出了除了已经确立的间接的GPCR-Gα-蛋白质调控哺乳动物AC之外,第二个相当不同的信号组直接影响这种最重要的酶的可能性。

关键字:腺苷酸环化酶(AC), 群体感应(QS), CqsS, 膜蛋白, 蛋白表达, 弗氏压碎器

材料和试剂

  1. 表达
    1. Fishersrand TM注射器过滤器,25mm,0.22μm(Thermo Fisher Scientific,Fisher Scientific,目录号:09719A)
      注意:本产品已停产。
    2. 大肠杆菌的甘油原液BL21(DE3)(F ompT hsdS [r B - m B - ] em> [DE3])转化质粒pQE80L或pETDuet-3:
      1. p>>>>>>> [>>>>>>>>>]>>>>>>>>>>>>>>>> 编码一个lacIq抑制模块; N-末端RGS-His 标签
      2. pETDuet-3(图1)
        pETDuet-1 [Novagen]的MCS1被转换为pQE30的MCS [QIAGEN]
        MCS1:N末端RGS-His 标签,MCS2:C-末端S标签
      注意:两种质粒的表达由IPTG诱导型T5(pQE80/L)或T7(pETDuet-3)启动子控制。 pQE80 L 只有一个MCS。如果您想从相同的质粒共表达第二个蛋白质,则pETDuet表达载体是合适的。
    3. 氨苄青霉素钠盐(Carl Roth,目录号:K029)
    4. IPTG(异丙基β-D-硫代吡喃半乳糖苷)(AppliChem,目录号:A1008)
    5. LB肉汤(Lennox)(Carl Roth,目录号:X964)
    6. 氨苄青霉素溶液(参见食谱)
    7. IPTG解决方案(见配方)
    8. LB-培养基(参见食谱)


      图1. pETDuet-1(A)和pETDuet-3(B)的两个MCS的比较
      注意:该序列图是使用程序DNA5生成的( https://pga.mgh.harvard.edu/web_apps/web_map/start )。

  2. 细胞收获
    1. 文化从表达
    2. Tris(AppliChem,目录号:A1086)
    3. HCl(37%)
    4. EDTA(Sigma-Aldrich,目录号:E5134)
    5. 洗涤缓冲液(参见食谱)

  3. 膜制备
    1. 1.5 ml Eppendorf管
    2. 冷冻细胞沉淀从表达
    3. 硫代甘油(Sigma-Aldrich,目录号:M1753)
    4. 氯化钠,NaCl(EMD Millipore,目录号:106404)
    5. cOmplete TM 无EDTA蛋白酶抑制剂(Roche Diagnostics,目录号:05056489001)
    6. 甘油(EMD Millipore,目录号:104094)
    7. 液体N 2
    8. 裂解缓冲液(见配方)
    9. 膜缓冲液(参见食谱)

  4. 数据分析
    1. Tris(AppliChem,目录号:A1086)
    2. HCl(37%)
    3. 十二烷基硫酸钠,SDS(Sigma-Aldrich,目录号:71729)
    4. β-巯基乙醇(EMD Millipore,目录号:805740)
    5. 甘油(EMD Millipore,目录号:104094)
    6. 溴苯酚蓝(Sigma-Aldrich,目录号:B8026)
    7. RGSHis-抗体(QIAGEN,目录号:34650)
    8. S标签抗体(EMD Millipore,目录号:71549)
    9. 蛋白质标记IV预分离(VWR,目录号:27-2110)
    10. 蛋白标记I未染色(VWR,目录号:PEQL27-1010)
    11. ECL Plex山羊抗小鼠IgG-Cy3抗体(GE Healthcare,目录号:PA43010)
    12. 4x SDS加载染料(见配方)

设备

  1. 表达
    1. 1升锥形瓶
    2. Eppendorf生物光度计
    3. 比武杯
    4. 摇床培养箱(37°C和22°C)

  2. 细胞收获
    1. 离心机(供应商:Thermo Fisher Scientific)
      1. Sorvall RC5B Plus
        转子:Kontron-Hermle A6.14(Sorvall,目录号:202200)
      2. Heraeus Megafuge 1.0R
        转子:Heraeus Sepatech BS4402/A(Heraeus,目录号:3360)
    2. 250毫升Sorvall金属转子管(Sorvall,目录号:522)
    3. 50ml离心管(Greiner Bio One,目录号:227261)
    4. Vortex Genie-2(VWR,目录号:4445900)
    5. 盒装水冰混合
    6. 冰箱(-80°C)

  3. 膜制备
    1. Vortex Genie-2(科学工业,型号:Vortex-Genie 2)
    2. 法国®压力容器压力机(SLM Instruments,SLM Aminco ®型号:FA-078-E1)
      替代供应商:Glen Mills Inc.(USA)或G.Heinemann Ultraschall- und Labortechnik(德国)
    3. Aminco ®法国压力传感器(SLM Instruments,SLM Aminco ®,目录号:FA-073,序列号:9110668)
      替代供应商:Glen Mills Inc.(USA)或G.Heinemann Ultraschall- und Labortechnik(德国)
    4. 离心机
      1. Heraeus Megafuge 1.0R
        1. 转子:Heraeus Sepatech BS4402/A(Heraeus,目录号:3360)
      2. 贝克曼L-60
        1. 转子:Beckman Coulter,型号:Type 50.2 Ti
    5. 盒装水冰混合
    6. 聚碳酸酯超速离心管
    7. 7毫升Dounce组织研磨机(WHEATON,目录号:357542)
    8. 冷冻柜(-80°C)

  4. 数据分析
    1. Ettan DIGE Imager(GE Healthcare,目录号:63005642或29-0834-61)

软件

  1. 计划DNA5( https://pga.mgh.harvard.edu/web_apps/web_map/start

程序

  1. 表达
    1. 将含有200ml LB-培养基和200μl氨苄青霉素(终浓度:100μg/ml)的烧瓶用约5ml过夜培养物接种到所需的构建体(参见材料和试剂A1。点)至OD 600, sub> 0.1。
    2. 在37℃振荡(200rpm)下孵育培养物至0.2-0.3(约45-90分钟)的OD 600。
    3. 降温至22°C(振荡培养箱)。
    4. 在0.6-0.64的OD 600中,通过500μMIPTG(100μl1M IPTG/200ml培养物)诱导表达。

  2. 细胞收获
    1. 在2.0-2.8的OD 600(诱导后120-150分钟)收获细胞。
      在4℃下(Sorvall离心机)以3,200×g收集细胞10分钟。
    2. 向沉淀物中加入25ml洗涤缓冲液(4℃),通过在4℃下(Heraeus离心机)以4300xg的速度涡旋悬浮30分钟悬浮。
    3. 弃去上清并将细胞储存在-80°C或继续进行膜制剂

  3. 膜制备
    1. 将冻结的细胞在冰上解冻并通过涡旋悬浮在25ml裂解缓冲液(4℃)中 注意:
      1. 细胞沉淀物应完全溶解,以免堵塞Aminco ®法国压力池的出口。
      2. 在使用裂解缓冲液之前,始终添加cOmplete TM 无EDTA蛋白酶抑制剂片。
    2. Lyse细胞通过法国压力机(1,100psi)机械两次(图2)。
      注意:
      1. 打开Aminco ® 法语压力单元格的出口,使流量逐滴可见。
      2. 确保样品在冰水中连续冷却。
    3. 离心机匀浆30分钟,4℃,4,300×g(Heraeus离心机)并弃去沉淀(细胞碎片)。
    4. 将上清液转移到超速离心管中,并以100,000xg,4℃沉淀60分钟(Beckman L-60离心机)沉淀膜。
    5. 去除上清液,在1-2ml膜缓冲液中吸收膜(沉淀),并轻轻悬浮在匀浆器(Dounce Tissue Grinder)中。将膜制剂转移到1.5ml Eppendorf管中 注意:使用的膜缓冲液的量取决于颗粒的大小。将离心管底部直径约1厘米的颗粒悬浮于2毫升膜缓冲液中
    6. 在液体N 2中冷冻膜制备,并储存在-80℃。


      图2.短法语新闻协议 A.法国压力传感器的设备; B. Aminco法国压力池(从冰箱中取出4°C); C.注意!使用前:用甘油润滑邮票的O形圈和后备环!将邮票插入细胞体,以使内细胞体表面也被甘油覆盖! D.将电池体倒置放置在支架上,开口朝上。 E.将流量阀放入孔中。 F.填充细胞溶液(看起来乳白色)。 G.将盖子放在上面。 H.关闭流量阀。 I.将法国压力容器放入小工具中,(J)关闭支架。 K.通过将手轮转到泵上的"高"和(L)开关,启动法国压机。 M.当法国压力机设置1,100 psi ...(N)时,请仔细打开流量阀,使得...(O)流量逐滴可见。 P.注意!停留在时间,所以你可以轻松打开支架,邮票不会撞到地面! Q.完成后,将开关翻到"下",再次打开泵。液压升降机向下移动,您可以取出法国压力传感器。当你可以看到通过管子的刻度时,你的细胞被正确地裂开。把你的样品放回冰上下一步。

数据分析

蛋白质的表达通过SDS-PAGE(Laemmli,1970)和Western印迹证实。将分离的膜在4x SDS-加载的染料中在室温下温育至少30分钟,然后施加到SDS-PAGE(不煮沸样品)。在MilliQ H 2 O中稀释样品(膜制备),得到最终体积为15μl的2.5-5μg蛋白质。加入5μl4x SDS-加载染料。将20μl混合物装入SDS-PAGE的一个槽中。将膜与每种抗体(4℃下的第一抗体,室温下的第二抗体)孵育1小时。第一抗体是RGSHis-(图3A和3C)或S-标签抗体(图3B)。在两种情况下,使用ECL Plex山羊抗小鼠IgG-Cy3抗体作为第二抗体(稀释1:2,500)。使用Ettan DIGE成像仪进行Western印迹评估。
注意:与可溶性蛋白质相反,膜蛋白质不煮沸(95℃,5-10分钟)。


图3.蛋白质印迹(A-C)和SDS-PAGE(D)

食谱

  1. 氨苄青霉素溶液(100mg/ml)
    将100毫克氨苄青霉素溶解于1毫升MilliQ H 2 O(过滤器灭菌)中
  2. 1 M IPTG解决方案
    将238.3mg IPTG溶解在1ml MilliQ H 2 O(过滤器灭菌)中
  3. LB-medium
    将20g LB溶解在1L软化的H 2 O(121℃,高压灭菌器20分钟)中
  4. 洗涤缓冲液
    50mM Tris/HCl(pH 8.0,室温)
    1 mM EDTA
  5. 裂解缓冲区
    50mM Tris/HCl(pH 8.0,室温)
    2mM硫甘油
    50 mM NaCl
    1片cOmplete TM 无EDTA蛋白酶抑制剂/50ml裂解缓冲液
  6. 膜缓冲器
    40mM Tris/HCl(pH 8.0,室温)
    1.6mM硫甘油
    20%甘油(85%)
  7. 4x SDS-负载染料
    130 mM Tris/HCl(pH 6.8)
    10%SDS
    10%β-巯基乙醇
    20%甘油(85%)
    0.06%溴酚蓝

致谢

这项工作得到了德意志民主共和国(SFB 766; TP B08)的支持。

参考文献

  1. Beltz,S.,Bassler,J.and Schultz,JE(2016)。  来自
    的弧形传感器的调节表示腺苷酸环化酶的膜锚的受体功能。
    5:e13098。 >
  2. Guo,YL,Kurz,U.,Schultz,A.,Linder,JU,Dittrich,D.,Keller,C.,Ehlers,S.,Sander,P。和Schultz,JE(2005) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16045612"target ="_ blank"> Rv1625c(分枝杆菌IIIa腺苷酸环化酶)与哺乳动物同源物的相互作用。/a>分子微生物 57(3):667-677。
  3. Guo,YL,Seebacher,T.,Kurz,U.,Linder,JU和Schultz,JE(2001)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih 20号(14号),"14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 ):3667-3675。
  4. Laemmli,UK(1970)。结构蛋白的切割噬菌体T4头部的组装。自然 227(5259):680-685。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Beltz and Schultz. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Beltz, S. and Schultz, J. E. (2017). Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor. Bio-protocol 7(2): e2112. DOI: 10.21769/BioProtoc.2112.
  2. Beltz, S., Bassler, J. and Schultz, J. E. (2016). Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases. Elife 5: e13098.
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