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Sep 2017

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Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses
用于定量比率荧光分析的大肠杆菌GO-Matryoshka生物传感器亲和纯化   

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Abstract

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating in vivo data. This protocol provides a robust methodology for in vitro binding assays of newly designed sensors. Here we present a detailed protocol for purification and in vitro characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP.

Keywords: Biosensors (生物传感器), GO-Matryoshka (GO-Matryoshka), In vitro binding assay (体外结合测定), Protein isolation (蛋白质分离), His-tag (His-标签), Calcium (钙), Ratiometric (比率测量), Fluorescence analysis (荧光分析)

Background

The green fluorescent protein (GFP) was identified in 1962 in the jellyfish Aequorea Victoria (Shimomura et al., 1962). Thirty years later, its first use as a reporter gene was described (Chalfie et al., 1994). Since their discovery, GFP variants and other fluorescent proteins have contributed greatly to the principal advancements in the biological sciences, and are now common tools in biomedical research (Frommer et al., 2009).

A large number of fluorescent proteins (FP) and FP variants have been used as reporters or fused to proteins in organisms of all kingdoms of life (Chudakov et al., 2010; Valeur and Berberan-Santos, 2012). Continuously, new fluorescent proteins with enhanced properties have been identified or are being engineered, further improving and extending the toolkit for visualization of in vivo processes. These novel fluorophores allow us to monitor a wide range of real-time processes, from structural organization of cells to dynamic processes in living organisms. Photoactivatable FPs have been successfully used to track molecules and cells in space and time (Misteli and Spector, 1997; March et al., 2003). Recently, FPs enabled the design of biosensors. Different categories of genetically encoded biosensors are commonly used, including single fluorophore intensity-based sensors, and two-fluorophore sensors based on Förster resonance energy transfer (FRET) (Frommer et al., 2009). Various approaches are being used to carry out multiplex sensor analyses in the same cells (Mehta et al., 2018).

The mechanisms by which sensors operate can rely on modifications of the FPs themselves, in response to their interaction with a ligand (as in pHluorin or Clomeleon for protons and chloride, respectively) (Miesenbock et al., 1998; Kuner and Augustine, 2000). Alternatively, FPs can be grafted onto a ligand-binding domain. Ligand binding triggers conformational rearrangements that affect the fluorescence properties or relative positioning of the fluorophores (Deuschle et al., 2006; Kaper et al., 2008). Nowadays, genetically encoded biosensors are widely used in vivo to monitor levels and dynamics of ions and metabolites, the activity of transporters or tension (Frommer et al., 2009). While biosensors that rely on changes in the fluorescence ratio caused by changes in the relative positioning of a pair of donor and acceptor FPs were successfully used, many of the first generation sensors are limited by their comparatively low dynamic range and signal-to-noise ratio [SNR; for definitions of dynamic range and SNR, see Perez Koldenkova and Nagai (2013)]. Some of the advanced single fluorophore intensiometric biosensors, which involve the use of conformational sensitive FPs (csFPs), can have impressively high dynamic ranges and high SNR. However, changes in the expression level of the biosensor will affect the readout, raising concerns about reliability and artifacts for in vivo use. Actual suitability for in vivo measurements requires comparison of different sensor variants in the target tissues (Perez Koldenkova and Nagai, 2013). Nevertheless, the in vitro properties of such sensors are important pieces of information, including quantitative information on affinity and also on kinetics in the case of rapid processes such as action potentials. To avoid artifacts, intensiometric sensors can be converted into ratiometric sensors by coupling csFPs with a reference FP.

Recently, a new technology, termed Matryoshka, provided a universal platform to create dual-FP biosensors with a large dynamic range, good stability across a wide range of pH and buffer conditions, and single wavelength excitation thanks to the use of a Large Stokes Shift (LSS) FP (Ast et al., 2017). Nesting such a reference FP (e.g., LSSmOrange) within a reporter FP (e.g., circularly permuted green FP), permits excitation of both FPs at a single excitation wavelength. The technology was successfully applied to generate cytosolic calcium and ammonium transport sensors, GO-MatryoshCaMP and AmTryoshka variants, respectively (Ast et al., 2017).

Here, we present a detailed protocol for the purification and characterization by in vitro binding assays of genetically encoded His-tagged Matryoshka biosensors from Escherichia coli K12. The protocol presented here was specifically developed for purification and characterization of GO-MatryoshCaMP6s, but can be used more generally for other fluorescence biosensors with minor modifications.

Materials and Reagents

  1. Micropipette tips (GilsonTM PIPETMAN ClassicTM, catalog number: Gilson F12360x and, Starlab, TipOne®, catalog numbers: 1112-1840 , 1110-1840 , 1110-3800 )
    Note: Pipetting accuracy is essential for the acquisition of reliable raw data. It may help to use of an electrical multichannel pipette (for 100 µl) (e.g., Sartorius AG)
  2. 200 µl TipOne® pipette tips (sterile), yellow, conical, rack (STARLAB, catalog number: S1111-6701-C )
  3. 0.22-μm filter
  4. Single-use plastic serological pipettes (Sarstedt, catalog number: 86.1254.001 )
  5. 1 and 2 liter glass flasks for cell culture (SciLabware, catalog numbers: 1135/26D and 1135/30D )
  6. 50 ml tubes (Sarstedt, catalog number: 62.547.004 )
  7. 15 ml tubes (Sarstedt, catalog number: 62.554.002 )
  8. 1.5 ml microtubes (Sarstedt, catalog number: 72.690.001 )
  9. Stellar Scientific Centrifuge Tubes, High Speed (20,000 x g), 50 ml, Red Screw Cap (Stellar Scientific, catalog number: T15-701 )
  10. Stellar Scientific Centrifuge Tubes, High Speed (20,000 x g), 15 ml, Red Screw Cap (Stellar Scientific, catalog number: T15-701 )
  11. Round 92 x 16 mm Petri dishes (Sarstedt, catalog number: 82.1473 )
  12. Disposable plastic columns (Thermo Scientific, catalog number: 29922 )
  13. Non-sterile 96-well flat bottom transparent plates (Corning, catalog number: 9017
  14. Non-sterile 96-well flat bottom black plates with transparent bottoms (Corning, catalog number: 3631 )
  15. ZebaTM Spin desalting column (Thermo Fisher Scientific, catalog number: 89882 )
  16. Purified plasmids containing His-tagged biosensors for lactose/IPTG inducible expression (e.g., the calcium sensor MatryoshCaMP6 (Ast et al., 2017) in the vector pRSET-B) for nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, as well as an empty vector, or the biosensor backbone protein lacking FPs (negative control) for expression in BL21 GOLD (DE3) cells. Plasmids and strains are described in more detail below
  17. Strains
    Escherichia coli BL21 GOLD (DE3) [fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS; λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5] (New England Biolabs, catalog number: C2527I )
  18. cOmpleteTM ULTRA Tablets, Mini, EDTA-free protease inhibitor cocktail (Merck, catalog number: 11836170001 )
  19. Ni-NTA Agarose Beads (Qiagen, catalog number: 30210 )
  20. Bradford assay kit (Bio-Rad, catalog number: 500-0006 )
  21. MOPS (3-(N-morpholino)propanesulfonic acid) buffer (Carl Roth, catalog number: 6979.3 )
  22. Imidazole (AppliChem, catalog number: A1073 ,0500; UN3263)
  23. D-(+) glucose monohydrate (Sigma-Aldrich, catalog number: 14431-43-7 )
  24. Lactose (Merk, catalog number: 7660.025 0)
  25. BSA (Bovine Serum Albumin; Carl Roth, catalog number: 0 163.3 )
  26. BactoTM Tryptone (BD BioSciences, catalog number: 211705 )
  27. BactoTM Yeast Extract (BD BioSciences, catalog number: 212750 )
  28. Sodium chloride (Carl Roth, catalog number: 3957.1 )
  29. BactoTM Agar (BD Biosciences, catalog number: 214030 )
  30. Antibiotic(s) required for plasmid selection. For example: ampicillin for vector pRSET-B containing MatryoshCaMP6 (Sigma-Aldrich, catalog number: 69-53-4 )
  31. Sodium hydroxide (Sigma-Aldrich, catalog number: 1310-73-2 )
  32. Calcium chloride (Sigma-Aldrich, catalog number: 10043-52-4 )
  33. 4-20% Mini-PROTEAN® TGXTM Precast Protein Gels, 10-well, 50 µl (Bio-Rad, catalog number: 4561094 )
  34. 4x SDS Sample buffer (MERK, catalog number: 70607-3
  35. PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, catalog number: 26616 )
  36. PierceTM 6xHis Protein Tag Stain Reagent Set (Thermo Fisher Scientific, catalog number: 24570 )
  37. Lysogeny broth (LB) liquid medium with antibiotics (see Recipes)
  38. LB solid medium (see Recipes)
  39. Auto-induction medium (for composition and explanations, see Recipes)
  40. Lysis buffer (see Recipes)
  41. Wash buffer (see Recipes)
  42. Elution buffer (see Recipes)
  43. Final buffer (see Recipes)
  44. Ligand binding assay buffer (see Recipes)
  45. Coomassie stain buffer (see Recipes)
  46. Stock solution of carbenicillin (see Recipes)
  47. Running Buffer (see Recipes)

Equipment

  1. Standard micropipette or multichannel (8 or 12 channel) pipette (for 100 µl) (e.g., Sartorius AG, catalog number: 725240 )
  2. pH meter (InoLab® pH Level1, catalog number: 72.690.001 )
  3. Sonicator (Branson Sonifier cell disruptor B15)
  4. Centrifuge (Hettich Rotanta 460/460R )
  5. Microplate reader with adjustable bandwidths (Spark®, Tecan)
  6. Fluorescence stereomicroscope for large fields observations (ZEISS, model: Axio Zoom.V16 )
  7. Eppendorf ThermoMixer® C (Eppendorf, catalog number: 5382000015 )
  8. Autoclave (Systec GmbH, model: Systec V-150 )
  9. Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (Bio-Rad, catalog number: 1658004 )
  10. NanoDrop 2000 (LabX, catalog number: LV40609601 )
  11. Eppendorf ThermoMixer® C (Eppendorf, catalog number: 5382000015 )
  12. Eppendorf Thermoblock SmartBlockTM 1.5 ml (Eppendorf, catalog number: 5360000038 )
  13. Gel Doc XR+ Gel Documentation System (Bio-Rad, catalog number: 1708195 )

Software

  1. ecan-related software (Spark®, Tecan Life Sciences)
  2. Excel (Microsoft, Microsoft Office Professional 2016)
  3. MyCurveFit 2019 (MyCurveFit, MyAssays Limited)

Procedure

  1. Preparation of purified ratiometric fluorescent biosensors
    1. Transformation of E. coli
      1. Thaw the DNA (minipreps of plasmids carrying the biosensors and negative controls. e.g., MatryoshCaMP6 (Ast et al., 2017) and the vector pRSET-B) and aliquots of BL21 GOLD (DE3) competent cells on ice for 10 min.
      2. Add 10-50 ng of plasmid to the cells and keep on ice for 30 min.
        Note: DNA quantification can be performed by using a NanoDrop device.
      3. Perform heat-shock by using a heat block at 42 °C for 45-60 s.
      4. Keep on ice for 2 min.
      5. Recover cells for 1 h with 800 µl of LB medium by shaking at 200 rpm at 37 °C.
        Note: SOC (Super optimal broth) medium, a nutrient-rich bacterial growth medium, can be used instead of LB medium for higher recovery after transformation.
      6. Centrifuge at 11,000 x g for 5-10 s.
      7. Remove most of the supernatant and keep about 100-200 µl.
      8. Resuspend sedimented cells in remaining LB medium and plate dilutions onto solid LB plates (dilutions depend on transformation efficacy) containing the respective antibiotic, here carbenicillin at 100 μg/ml (see Recipes).
        Note: To optimize colony density, plate different volumes (i.e., 30 and 170 µl) or dilute.
      9. Allow bacteria to grow on solid agar overnight at 37 °C.
      10. Store plates at 4 °C overnight to achieve fluorophore maturation.
    2. Clone selection and cell culture
      1. Visualize cpGFP fluorescence under a fluorescence stereomicroscope and pick bright colonies (Figure 1).
        Note: Leaky expression is observed on LB solid plates. Brightness of colonies can be used as a criterion to evaluate the sensor expression levels.


        Figure 1. E. coli cells transformed with empty vector and GO-MatryoshCaMP6s on solid LB plates. A-B. Colonies obtained after transformation with empty vector. C-D. Cells transformed with GO-MatryoshCaMP6s sensor. Fluorescence of the E. coli cells was recorded using a ZEISS Axio Zoom.V16 fluorescence stereo zoom microscope and GFP filter settings (λex 470/40 nm and λem 525/50 nm, with a beam splitter at λem 495 nm). Colonies were visualized using bright-field illumination (A, C) and GFP excitation for fluorescence detection (B, D). Scale bars = 1 mm.

      2. For each of the 3 biological replicate, inoculate 5 ml of LB liquid medium (with antibiotics for plasmid selection) with 1 independent bright colony and grow overnight in LB plus antibiotic at 37 °C and at 200 rpm shaking (Figure 2).
      3. For each biological replicate, inoculate 200 ml of Auto-induction medium containing antibiotics (see Recipes) with the 5 ml preculture in a large flask and grow at 37 °C for 2 h in the dark on a shaker at 220 rpm (Figure 2). Use sample from the remaining preculture for generating glycerol stocks.
        Notes:
        1. To get optimal protein yield and avoid toxicity, Auto-induction media (details available here) should be used instead of IPTG, and cells expressing biosensors should be grown at 20 °C rather than 37 °C. The Auto-induction medium is used for obtaining high-levels of recombinant protein expression of lactose-inducible expression-systems. It does not require the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) and it avoids the constraints of monitoring optical density (O.D.) for the determination of the IPTG supplementation time. The medium contains glucose and lactose that are metabolized at different rates. Glucose is favored initially and represses promoter activity. Once glucose is consumed, repression will be relieved and lactose leads to induction of transcription.
        2. Auto-induction media are used for lactose/IPTG inducible expression systems otherwise this part of the protocol has to be customized.
        3. Glycerol stocks are generated by mixing cell culture 1:1 with LB medium containing 50% w/v glycerol.
        4. When growing 200 ml cultures, flasks should have a volume of 1 or 2 L. Do not fill flasks beyond 20% of total volume to ensure optimal oxygenation.
        5. The initial incubation at 37 °C is important to initiate faster the growth of large cultures from a single colony. Do not extend the incubation time, as it may later lead to a lower amount of soluble biosensors and an increased occurrence of inclusion bodies.
      4. Transfer the culture to 20 °C and grow for 48 h at 220 rpm in the dark to avoid any photo-bleaching of FPs (Figure 2).
      5. Centrifuge the culture by using high-speed 50 ml centrifuge tubes for 30 min at 20,000 x g at 4 °C, discard the supernatant and freeze the sedimented cells at -20 °C overnight (Figure 2). Do not skip this freezing step, as it helps the purification process. A yellowish sediment of cells expressing the sensor should be visible.
        Notes:
        1. The yield of purified proteins may be increased by freezing and thawing the pellet, which helps to disrupt the cells.
        2. High-speed centrifuge tubes for 20,000 x g should be used to avoid breaking of the tubes during centrifugation. Alternatively: Normal falcon tubes can be used by reducing the speed to 17,000 x g in a way to prevent any leakage.


      Figure 2. Expression of GO-Matryoshka biosensors in E. coli. Workflow of the experiment starting from the screening of the colonies until the harvesting of the expressed biosensor as a bright yellow sediment.

    3. Biosensor purification
      1. Thaw the pellet on ice.
      2. Immediately resuspend the cells by pipetting up and down in 5 ml of lysis buffer containing protease inhibitor cocktail.
      3. Sonicate the cell suspension into the tubes on ice (10-15 cycles, with each cycle consisting of 10 s of sonication and 10-15 s of rest). Be careful with this step since it is important to optimally break the cells while at the same time preventing overheating samples, which may lead to protein denaturation. This step is critical for the quality of the protein sample.
        Note: Do not heat the sample (e.g., during sonication), which could lead to undesired protein denaturation and aggregation.
      4. Centrifuge the lysate by using high-speed 15 ml centrifuge tubes (4 °C; 20,000 x g) for at least 30 min.
      5. During centrifugation, prepare the column by adding 1-2 ml final volume of Ni-NTA agarose bead material into a column (placed on its holder).
      6. At room temperature, wash the Ni-NTA beads three times by adding 5-10 ml wash buffer to the column, then let it flow through by gravity.
      7. Add the lysate to the column. Allow the lysate to flow through by gravity. The His-tagged biosensors will bind to the Ni-NTA beads.
      8. Wash three times by gravity flow with 5-10 ml wash buffer.
      9. Elute the sensors by gravity flow with up to 5 successive fractions of 300 µl of elution buffer and store in the dark at 4 °C for at least 24 h before analysis to allow the maturation of all purified biosensors. Alternatively: incubate the elution fraction for 3 h at 37 °C.
        Note: Due to the longer maturation time of the LSSmOrange compared to derivatives (Shcherbakova et al., 2012), incubation at 4 degrees is crucial to obtain fully matured FPs.
      10. To remove imidazole, salt contaminants and bound ligands, the eluate can be further purified with a ZebaTM Spin desalting column in a final volume of 130 µl according to the manufacturer’s instructions.
    4. Quality control of purified biosensor
      Perform a Bradford assay to determine protein concentration according to manufacturer’s protocol. It is recommended that the concentration is at least about 1 mg/ml otherwise it is recommended to repeat the purification with a reduced elution volume.
      Note: Biosensors can be stored at 4 °C in the dark for a couple of weeks.

  2. Load 10-20 µg of protein samples into the SDS-Polyacrylamide gel (SDS-PAGE) (Figure 3).
    1. Mix the samples with 1x SDS Sample buffer and add water to a final volume of 20-40 µl.
    2. Boil the sample at 90 °C during 5 min to speed up the process of denaturation.
      Note: Once denatured, the samples can be kept at room temperature until loading.
    3. Load the sample into the wells of SDS-PAGE gel.
    4. After electrophoresis, proceed to the staining of 6xHis affinity-tagged protein (Pierce 6xHis Stain Reagent Set) according to the manufacturer’s instructions and to Coomassie staining for total protein visualization.
    5. Perform protein detection by using visible light (Coomassie staining) and UV-light excitation at a wavelength 280-310 nm (6xHis affinity-tagged staining).
      Note: The staining of 6xHis affinity-tagged proteins requires 5.6 pmol of His-tagged proteins per band for detection with a CCD camera or 57 pmol of His-tagged proteins per band for detection with a UV transilluminator.


      Figure 3. Isolation of GO-MatryoshCaMP6s sensors expressed in E. coli. Timeline of the experiment starting from the sedimented cells until the elution of the biosensor by affinity chromatography. Samples loaded into the SDS-PAGE gel are: MW (prestained molecular mass marker), CE (crude extract), L (lysate), FT (flow-through), W (wash), and E1-E5 (eluates from fractions 1 to 5). Upper gel panel was stained for total protein visualization using Coomassie stain buffer and lower gel panel was stained with 6xHis Protein Tag Stain Reagent Set (Thermo Fisher Scientific). Detection was performed by using UV-light excitation at a wavelength 280-310 nm.

  3. In vitro binding assay with purified biosensors
    1. Optimization of the sample dilution in final buffer (i.e., 20 mM MOPS, pH 7.0, see Recipes) and fluorescence reading parameters
      In this section, protocol users will identify the proper dilutions required for each purified biosensor solution based on individual excitation of cpGFP and LSSmOrange using their respective maximum excitation and emission wavelengths. Users will also identify a single wavelength that provides the greatest simultaneous excitation of both cpGFP and LSSmOrange.
      1. Select the fraction with the highest protein content and make a predilution of about 40 times to obtain a final concentration of about 5-10 µM.
      2. From the pre-diluted sample, prepare a dilution series (1; 1/2; 1/3; 1/4; 1/5; 1/10; 1/100) in a final volume of 200 µl and load into a 96-well plate suitable for reading fluorescence intensities and spectra in a microplate reader (see note).
        Note: Although best results are obtained with black transparent bottom 96-well plates, their cost is comparatively high. The use of this black plastic helps preventing signal contamination between wells and is therefore favored when precise measurements are needed. However, for preliminary sensor characterization, we use regular non-sterile 96-well plates to minimize costs. Alternatively, to avoid light emission contamination between wells, empty wells can be placed systematically in-between samples.
      3. Measure cpGFP and LSSmOrange fluorescence intensities of dilution series in the plate reader using the SPARKControl software and the following parameters: (1) cpGFP: Excitation at 480 nm with a band width of 20 nm, emission at 515 nm with a band width of 15 nm and (2) LSSmOrange: Excitation at 440 nm with a band width of 10 nm, emission at 570 nm with a band width of 15 nm. Adjust the gain and z-position of the reading so that the measurements fall roughly within the middle of the detector range. The gain is expected between 50-90 and should never exceed 130 while the Z position, when using 200 µl, is expected at about 20,000 μm.
        Note: While it is possible to find a compromise by using a single intermediate wavelength to excite both FPs, their excitation maxima are distinct, thus for in vitro characterization in a fast fluorimeter, it is better to excite the two FPs separately.
      4. Perform an excitation scan with 2 nm intervals between 440 to 480 nm to verify which excitation wavelength provides the highest amplitude for both GFP and the LSSmOrange emission in your conditions (Figure 4). This is typically around 455 nm for GO-Matryoshka biosensors.


         Figure 4. Excitation parameters for the detection of GO-Matryoshka sensors. Emission spectra of MatryoshCaMP6 reveal that increasing the excitation wavelength from 440 nm to 480 nm results in a rise in fluorescence intensity of cpGFP emission but a decrease in the emission of the stable reference LSSmOrange. A.u.: arbitrary units.

      5. Select the optimal dilution factor based on emission intensities obtained in Steps C1c and C1d. The dilution factor depends on each instrument, here the targeted range of intensity is of about 20,000 arbitrary units (a.u.) to stay within the optimal range of the detectors without leading to saturation. Lower gain factors of the instrument will yield higher quality data (less noise).
    2. Binding assays
      1. Design the sample loading pattern of your plate by including technical triplicates and purifications of lysate from 3 biological replicates for each biosensor. Depending on the expected Kd, include suitable concentrations of the ligand (e.g., dilution series from 0 mM up to 5 times the Kd; use sufficient data points to obtain reliable apo and saturated states as well as linear range of binding isotherm). Make sure that the previously selected dilution factor is compatible with this design, and include control wells that only contain buffer to measure background.
        Note: The order of components added does not matter. However, we recommend to add the component with the highest volume first, the buffer and end with the lowest volume, the biosensors. Load the 96-well plate according to your loading pattern. Technical triplicates for each of the three colonies will be loaded from a single Master Mix (see notes). Make sure to scale Master Mix for 4 reactions for technical triplicates, to maintain a margin of 1 reaction when pipetting triplicates. In addition, make sure that the final volume in each well remains identical, even with different ligand concentrations. Bubbles in the wells should be avoided, as they might affect the reading.
        Notes:
        1. Since the analysis is quantitative, accurate pipetting is crucial. Use high precision, use best practices when pipetting and handling pipettes, ensure that pipettes are in good state, ensure proper calibration of pipettes, prepare master mix (i.e., to avoid pipetting low volumes) and pipet slowly in order to reduce pipetting errors. Do not snap plunger, never allow any liquid to enter the shaft.
        2. Incubation time is recommended to be minimum 5 min. It is necessary to ensure to have reached binding equilibrium before performing the reading. The preparation of the 96-well plate is ususally taking a couple of minutes which is sufficient.
        3. It is recommended to include buffer-only wells as blanks.
      2. Read fluorescence using the plate reader as described in Step C1 to obtain three sets of data for each well: Emission spectrum and point measurements for cpGFP and LSSmOrange of maximum intensities of the GO-Matryoshka sensor (Figure 5).


      Figure 5. Screenshot of an excel worksheet displaying an example of data and parameters from one binding assay experiments. Three set of data are obtained after performing point measurements and emission spectrum scan of GO-MatryoshCaMP6s in biosensor binding assay.

    3. Analysis of the data for each biological replicate
      1. Set a default Excel worksheet, which can be used as template for automated analysis of data obtained in the previous section (Figure 5). This should be done by entering the required formula for processing data and generating graphs from any experiment by simply pasting its raw values. This template has to include:
        1. Calculation of the average value for the technical triplicates.
        2. Subtraction of the background fluorescence of the buffer for each condition and each data type (cpGFP and LSSmOrange intensities as well as the emission spectrum).
        3. Plotting the data by graphically representing the fluorescence intensity as a function of wavelength and generate emission spectra recorded at different ligand concentrations to quantify intensity changes responding to analyte concentration (Bermejo et al., 2011) (Figure 6).


        Figure 6. Binding assay using purified Matryoshka (here GO-MatryoshCaMP6s) biosensors. A. 96-well plate with GO-MatryoshCaMP6s plus increasing calcium concentrations, color code from green (without calcium) to red (10 µM of calcium), and in triplicate. B. The spectrum of MatryoshCaMP6, with excitation at 455 nm. Increasing concentrations of calcium result in a rise in the fluorescence intensity of cpGFP emission while the reference LSSmOrange emission stay stable (slight increase due to bleedthrough). [a.u.]: arbitrary units. Data are presented as the average of three technical replicates.
        Note: It is observed that bleed-through occurs and leads to a slight but significant apparent increase in fluorescence intensity of the reference FP LSSmOrange. However, separate point measurements by using two excitation wavelengths should get rid of this effect.

      2. Measure the affinity by using three biological replicates:
        1. Calculate the ratio (R) of intensities at 520 nm/575 nm emission for each ligand concentration by using the following equation:
          R = FI520nm/FI575nm
        2. Calculate R0 as the value of R without substrate.
        3. Calculate ∆R the value of R at a specific calcium concentration subtracted by R0 as follows:
          ∆R = R - R0
        4. Calculate ∆R/R0.
        5. Plot the data against the concentration by using e.g., MyCurveFit.
        6. Use a logarithmic scale for the X axis.
        7. Apply a sigmoidal fit by using, e.g., MyCurveFit, and calculate the Kd (Figure 7).


      Figure 7. In vitro saturation curves of purified GO-Matryoshka. Calcium binding isotherms of MatryoshCaMP6s. Analyses of sensor output at different calcium concentrations was performed using purified sensor. Calcium binding was determined as previously described (Ast et al., 2017). Data are presented as the average of three biological replicates (mean ± S.E.M. from biological replicates with n = 3).

Recipes

  1. LB liquid medium with antibiotics
    1. Dissolve 10 g BactoTM tryptone, 10 g sodium chloride and 5 g BactoTM yeast extract in 800 ml MilliQ water
    2. Adjust to pH 7.0 with NaOH
    3. Adjust final volume to 1 L with MilliQ water
    4. Autoclave at 121 °C for 20 min
    5. Before use, add the antibiotics corresponding to the required antibiotic resistance used for selection
  2. LB solid medium (pH 7.0)
    1. Dissolve 10 g BactoTM tryptone, 10 g sodium chloride and 5 g BactoTM yeast extract and 15 g of agar in 800 ml MilliQ water
    2. Adjust to pH 7.0 with NaOH
    3. Adjust final volume to 1 L with MilliQ water
    4. Autoclave at 121 °C for 20 min
    5. Allow to cool down to about 50-60 °C, then add the antibiotics corresponding to the required antibiotic resistance used for selection
  3. Auto-induction medium (pH 7.0)
    LB liquid medium
    0.05% w/v D-glucose
    0.2% w/v lactose
  4. Lysis buffer (pH 7.0)
    20 mM MOPS
    1 cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail per 50 ml of solution
    Note: The buffer can be stored at 4 degrees for months. 1 tablet of cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail is dissolved by extensive vortexing.
  5. Wash buffer (pH 7.0)
    20 mM MOPS
    1 cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail per 50 ml of solution
    Note: The buffer can be stored at 4 degrees for months. 1 tablet of cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail is dissolved by extensive vortexing.
  6. Elution buffer (pH 7.0):
    20 mM MOPS buffer
    250 mM imidazole
    1 cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail per 50 ml of solution
    Note: The buffer can be stored at 4 degrees. for months 1 tablet of cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail is dissolved by extensive vortexing.
  7. Final buffer (pH 7.0)
    20 mM MOPS
    Note: The buffer can be stored at 4 degrees for months. 1 tablet of cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail is dissolved by extensive vortexing.
  8. Ligand binding assay buffer (pH 7.0)
    20 mM MOPS with 0 to 10-20 mM calcium chloride
    Note: The buffer can be stored at 4 degrees for months. 1 tablet of cOmpleteTM ULTRA Tablet Mini protease inhibitor cocktail is dissolved by extensive vortexing.
  9. Coomassie staining buffer
    Coomassie Blue R250 staining reagent
    10% v/v acetic acid
    50% v/v methanol
  10. Stock solution of carbenicillin:
    1. Dissolve 1 g of disodium carbenicillin in 9.5 ml of MilliQ water
    2. Adjust final volume to 10 ml
    3. Filter-sterilize by using a 0.22-μm filter
    Note: Stock solution of carbenicillin can be stored at -20 °C.
  11. Running Buffer
    25 mM Tris
    192 mM glycine
    0.1% (w/v) SDS

Acknowledgments

The authors gratefully acknowledge grant support from Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy–EXC-2048/1–project ID 390686111, and the Alexander von Humboldt Professorship to WBF. Support from the CEA-Enhanced Eurotalents program and ANR-19-CE13-0007 “PHLOWZ” is gratefully acknowledged (HJ).

Competing interests

W.B.F. is author of the patent filed for the Matryoshka technology (U.S. Appl. No. 15/438,078). The remaining authors have no conflict of interest or competing interest to declare.

References

  1. Ast, C., Foret, J., Oltrogge, L. M., De Michele, R., Kleist, T. J., Ho, C. H. and Frommer, W. B. (2017). Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins. Nat Commun 8(1): 431.
  2. Bermejo, C., Haerizadeh, F., Takanaga, H., Chermak, D. and Frommer, W. B. (2011). Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast. Nat Protoc 6(11): 1806-1817.
  3. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263(5148): 802-805.
  4. Chudakov, D. M., Matz, M. V., Lukyanov, S. and Lukyanov, K. A. (2010). Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90(3): 1103-1163.
  5. Deuschle, K., Chaudhuri, B., Okumoto, S., Lager, I., Lalonde, S. and Frommer, W. B. (2006). Rapid metabolism of glucose detected with FRET glucose nanosensors in epidermal cells and intact roots of Arabidopsis RNA-silencing mutants. Plant Cell 18(9): 2314-2325.
  6. Frommer, W. B., Davidson, M. W. and Campbell, R. E. (2009). Genetically encoded biosensors based on engineered fluorescent proteins. Chem Soc Rev 38(10): 2833-2841.
  7. Kaper, T., Lager, I., Looger, L. L., Chermak, D. and Frommer, W. B. (2008). Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria. Biotechnol Biofuels 1(1): 11.
  8. Kuner, T. and Augustine, G. J. (2000). A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27(3): 447-459.
  9. March, J. C., Rao, G. and Bentley, W. E. (2003). Biotechnological applications of green fluorescent protein. Appl Microbiol Biotechnol 62(4): 303-315.
  10. Mehta, S., Zhang, Y., Roth, R. H., Zhang, J. F., Mo, A., Tenner, B., Huganir, R. L. and Zhang, J. (2018). Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities. Nat Cell Biol 20(10): 1215-1225.
  11. Miesenbock, G., De Angelis, D. A. and Rothman, J. E. (1998). Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394(6689): 192-195.
  12. Misteli, T. and Spector, D. L. (1997). Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol 15(10): 961-964.
  13. Perez Koldenkova, V. and Nagai, T. (2013). Genetically encoded Ca(2+) indicators: properties and evaluation. Biochim Biophys Acta 1833(7): 1787-1797.
  14. Shimomura, O., Johnson, F. H. and Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59: 223-239.
  15. Valeur, B. and Berberan-Santos, M. N. (2012). Molecular fluorescence: principles and applications. John Wiley & Sons.
  16. Shcherbakova, D. M., Hink, M. A., Joosen, L., Gadella, T. W. and Verkhusha, V. V. (2012). An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J Am Chem Soc 134(18): 7913-7923.

简介

[摘要]遗传编码的生物传感器是强大的工具为离子和代谢物的定量可视化在体内。设计和优化此类生物传感器通常需要分析大量变体。体外确定的传感器特性,例如底物特异性,亲和力,响应范围,动态范围和信噪比,对于评估体内数据很重要。该协议为新设计的传感器的体外结合测定提供了可靠的方法。这里我们提出了一个详细的协议用于纯化和体外表征的遗传编码的传感器,例示的His亲和标记的GO-(绿橙色)MatryoshCaMP6s钙传感器。GO-Matryoshka传感器基于在感兴趣的结合蛋白内一步插入一个包含两个嵌套荧光蛋白,圆形排列的荧光绿色FP(cpGFP )和Large Stoke Shift LSSmOrange的盒的方法,从而产生了利用被分析物触发的比例式传感器cpGFP的荧光变化。


[背景技术]将绿色荧光蛋白(GFP)在1962年被鉴定在水母水母维多利亚(下村等人,1962) 。30年后,描述了其首次用作报道基因(Chalfie等,1994)。自从发现以来,GFP变体和其他荧光蛋白为生物科学的主要进步做出了巨大贡献,并且现在已成为生物医学研究中的常用工具(Frommer等,2009)。

各种荧光蛋白(FP)和FP变异体已被用作报道分子或与所有生命王国的生物体中的蛋白融合(Chudakov等,2010 ;Valeur和Berberan- Santos,2012 )。不断地,已经鉴定出或正在设计具有增强特性的新型荧光蛋白,从而进一步改进和扩展了用于可视化体内过程的工具包。这些新颖的荧光团使我们能够监控各种各样的实时过程,从细胞的结构组织到活生物体的动态过程。可光活化的FP已经成功地用于追踪时空中的分子和细胞(Misteli和Spector,1997 ;March等,2003 )。最近,FP启用了生物传感器的设计。通常使用不同类别的基因编码生物传感器,包括基于单个荧光团强度的传感器和基于Förster共振能量转移(FRET)的两个荧光团传感器(Frommer等,2009)。在同一个单元格中使用了多种方法来执行多重传感器分析(Mehta et al。,2018)。

传感器操作 的机制可以依赖于FP自身的修饰,以响应其与配体的相互作用(如分别在pHluorin或Clomeleon中用于质子和氯化物)(Miesenbock等,1998;Kuner和Augustine,2000)。或者,可将FPs移植到配体结合域上。配体结合触发构象重排,其影响荧光性质或荧光团的相对位置(Deuschle等,2006;Kaper等,2008)。如今,遗传编码的生物传感器广泛用于体内监测离子和代谢物的水平和动态,转运蛋白的活性或张力(Frommer等,2009)。尽管成功使用了依赖于一对供体和受体FP相对位置变化而导致的荧光比率变化的生物传感器,但许多第一代传感器都受到其相对较低的动态范围和信噪比的限制[SNR;有关动态范围和SNR的定义,请参见Perez Koldenkova和Nagai(2013)。某些涉及使用构象敏感FP(csFP )的先进的单个荧光团强度生物传感器,可以具有令人印象深刻的高动态范围和高SNR。然而,生物传感器的表达水平的变化将影响读数,引起对体内使用的可靠性和伪像的担忧。体内测量的实际适用性需要比较目标组织中不同传感器的变体(Perez Koldenkova和Nagai,2013)。然而,这种传感器的体外特性是重要的信息,包括关于亲和力以及在诸如动作电位的快速过程中的动力学的定量信息。为了避免伪像,可以通过将csFP与参考FP耦合将强度传感器转换为比率传感器。

最近,一种名为Matryoshka的新技术提供了一个通用平台,以创建双FP生物传感器,该传感器具有大的动态范围,在广泛的pH和缓冲条件下都具有良好的稳定性,并且由于使用了大Stokes移位而获得了单波长激发(LSS)FP (Ast等人,2017)。将这种参考FP(例如,LSSmOrange )嵌套在报道分子FP(例如,圆形排列的绿色FP)内,允许在单个激发波长下激发两个FP。该技术已成功应用于分别生成胞质钙和铵转运传感器,GO- MatryoshCaMP和AmTryoshka变体(Ast等人,2017)。

在这里,我们提出了从大肠杆菌K12的遗传编码His标记的Matryoshka生物传感器的体外结合测定纯化和表征的详细协议。这里介绍的协议是专门为GO-MatryoshCaMP6的纯化和表征而开发的,但可以更广泛地用于其他具有轻微修饰的荧光生物传感器。

关键字:生物传感器, GO-Matryoshka, 体外结合测定, 蛋白质分离, His-标签, 钙, 比率测量, 荧光分析

材料和试剂

中号icropipette提示(吉尔森TM移液器经典TM ,目录号:吉尔森F12360x和,STARLAB ,TipOne ® ,目录号:1812至40年,一一一〇年至1840年,1110- 3800)
注意:移液精度对于获取可靠的原始数据至关重要。使用电动多通道移液器(100 µl)可能会有所帮助(例如,Sartorius AG)

将200μl TipOne ®移液器吸头(无菌),黄色,圆锥形,机架(STARLAB,目录号:S1111-6701-C)
0.22-μm过滤器
一次性塑料血清移液管(Sarstedt ,目录号:86.1254.001)
用于细胞培养的1升和2升玻璃烧瓶(SciLabware ,目录号:1135 / 26D和1135 / 30D)
50 ml管(Sarstedt ,目录号:62.547.004)
15 ml管(Sarstedt ,目录号:62.554.002)
1.5 ml微管(Sarstedt ,目录号:72.690.001)
Stellar Scientific离心管,高速(20,000 x g ),50 ml,红色螺帽(Stellar Scientific,目录号:T15-701)
Stellar Scientific离心管,高速(20,000 x g ),15 ml,红色螺旋盖(Stellar Scientific,目录号:T15-701)
圆形92 x 16毫米培养皿(Sarstedt ,目录号:82.1473)
一次性塑料柱(Thermo Scientific,目录号:29922)
非灭菌96孔平底透明板(Corning,目录号:9017)
带透明底的非无菌96孔平底黑色板(Corning,目录号:3631)
Zeba TM Spin脱盐柱(Thermo Fisher Scientific,目录号:89882)
纯化的质粒含His-标记的生物传感器为乳糖/ IPTG诱导型表达(例如,钙传感MatryoshCaMP6(阿斯特等人在载体中,2017年)的pRSET -B)为镍-次氮基三乙酸(的Ni-NTA)亲和层析,以及作为空载体,或缺少在BL21 GOLD(DE3)细胞中表达的FPs (阴性对照)的生物传感器骨架蛋白。质粒和菌株在下面更详细地描述
菌株
大肠杆菌BL21金(DE3)[ fhuA2 [ lon ] ompT gal(λDE3)[ dcm ] ∆ hsdS ;λDE3 = λsBa mHIo ∆ EcoRI -B int::( lacI :: PlacUV5 :: T7 gene1)i21 ∆nin5 ](New England Biolabs,目录号:C2527I)

cOmple te TM ULTRA片剂,小型,不含EDTA的蛋白酶抑制剂混合物(默克公司,目录号:11836170001)
Ni-NTA琼脂糖珠(Qiagen,目录号:30210)
Bradford分析试剂盒(Bio-Rad,目录号:500-0006)
MOPS(3-(N-吗啉代)丙烷磺酸)缓冲液(Carl Roth,目录号:6979.3)
咪唑(AppliChem,目录号:A1073,0500; UN3263)
D-(+)一水合葡萄糖(Sigma - Aldrich,目录号:14431-43-7)
乳糖(梅克,目录号:7660.0250)
BSA(牛血清白蛋白; Carl Roth,目录号:0163.3)
Bacto TM胰蛋白((BD BioSciences ,目录号:211705)
Bacto TM酵母提取物(BD BioSciences ,目录号:212750)
氯化钠(Carl Roth,目录号:3957.1)
Bacto TM琼脂(BD Biosciences,目录号:214030)
质粒选择所需的抗生素。例如:氨苄青霉素矢量的pRSET -含B MatryoshCaMP6 (西格玛- Aldrich公司,目录号:69-53-4)
氢氧化钠(Sigma - Aldrich,目录号:1310-73-2)
氯化钙(Sigma - Aldrich,目录号:10043-52-4)
4 - 20%的Mini-PROTEAN ® TGX TM预制普罗特EIN凝胶,10孔,50微升(生物-R广告,目录号:4561094)
4个SDS样本缓冲区(MERK,目录号:70607-3)
PageRuler预染的蛋白梯(赛默飞世尔科技,目录号:26616)
Pierce TM 6xHis蛋白标签染色试剂组(Thermo Fisher Scientific,目录号:24570)
具有抗生素的溶源性肉汤(LB)液体培养基(请参阅食谱)
LB固体培养基(请参阅食谱)
自动诱导培养基(对于组合物和说明,请参见[R ecipes)
裂解缓冲液(请参见食谱)
洗涤缓冲液(请参见食谱)
洗脱缓冲液(请参见配方)
最终缓冲区(请参见食谱)
配体结合测定缓冲液(请参见食谱)
考马斯亮斑缓冲液(请参阅食谱)
羧苄青霉素的储备溶液(见配方)
运行缓冲区(请参见食谱)
 

设备

 

标准微量移液器或多通道(8或12通道)移液器(用于100 µl)(例如,Sartorius AG,目录号:725240)
pH计(InoLab ® pH值级别1,目录号:72.690.001)
声波发生器(Branson Sonifier细胞破坏器B15)
离心机(海蒂诗Rotanta 460 / 460R)
酶标仪具有可调节的带宽(火花® ,Tecan公司)
荧光体视显微镜用于大视野观察(蔡司,型号:Axio Zoom.V16)
的Eppendorf Thermomixer中® C(的Eppendorf,目录号:5382000015)
高压灭菌器(Systec G mbH,型号:Systec V-150)
迷你-PROTEAN ®利垂直电泳细胞(生物-R广告,目录号:1658004)
NanoDrop 2000(LabX ,目录号:LV40609601)
的Eppendorf Thermomixer中® C(Eppendor ˚F ,目录号:5382000015)
的Eppendorf热块SmartBlock TM 1.5米升(Eppendor ˚F ,目录号:5360000038)
Gel Doc XR +凝胶文档系统(Bio -R ad,目录号:1708195)
 

软件

 

Tecan公司相关的软件(星火® ,Tecan公司生命科学)
Excel(Microsoft,Microsoft Office Professional 2016)
MyCurveFit 2019(MyCurveFit ,MyAssays Limited)
 

程序

 

一种。       纯化的制备ð比例荧光生物传感器

大肠杆菌的转化
解冻的DNA(携带生物传感器和阴性对照质粒微量制备物。例如,MatryoshCaMP6(阿斯特等人,2017)和矢量的pRSET -B )上的冰和等分BL21 GOLD(DE3)感受态细胞的10分钟。
向细胞中加入10-50 ng质粒,并在冰上放置30分钟。
注意:可以使用NanoDrop设备进行DNA定量。

使用42°C的加热块进行45-60 s的热冲击。
放在冰上2分钟。
通过在37°C下以200 rpm摇动,用800 µl LB培养基恢复细胞1 h 。
注意:可以使用SOC(超级理想肉汤)培养基(一种富含营养的细菌生长培养基)代替LB培养基,以提高转化后的回收率。

以11,000 x g离心5-10 s。
除去大部分上清液,并保持约100-200 µl。
将沉淀的细胞在剩余的LB培养基中重悬,然后将平板稀释液稀释到含有相应抗生素(此处为羧苄青霉素100)的固态LB平板上(稀释取决于转化效率) μ克/毫升(见配方小号)。
注意:要优化菌落密度,可将不同体积(即30和170 µl)铺板或稀释。

使细菌在固体琼脂上于37°C过夜生长。
将板在4 °C下保存过夜以实现荧光团成熟。
克隆选择和细胞培养
可视化cpGFP结构荧光立体显微镜下的荧光,并挑选菌落明亮的(图1) 。
注意:在LB固体板上观察到泄漏的表达。菌落的亮度可以用作评估传感器表达水平的标准。

 



图1.用空载体和GO-MatryoshCaMP6s在固体LB平板上转化的大肠杆菌细胞。AB 。用空载体转化后获得的菌落。Ç - d 。用GO-MatryoshCaMP6s传感器转化的细胞。所述的荧光的大肠杆菌细胞中使用ZEISS记录Axio上Zoom.V16荧光立体显微镜放大和GFP过滤器设置(λ EX四十○分之四百七nm和λ EM 50分之525纳米,与分束器在λ EM 495纳米)。使用明场照明(A,C)和GFP激发进行荧光检测(B,D)可视化菌落。比例尺s = 1毫米。

 

对于这3个生物学复制品中的每一个,请向5 ml的LB液体培养基(含用于选择质粒的抗生素)中接种1个独立的亮菌落,并在LB加抗生素的LB中于37°C和200 rpm摇动下生长过夜(图2)。
对于每个生物复制品,在一个大烧瓶中用5 ml预培养物接种200 ml含抗生素的自动诱导培养基(请参阅食谱),并在37°C的黑暗中于220 rpm的摇床上生长2 h (图2)。 。使用剩余预培养物中的样品来产生甘油储备。
ñ OTES:

为了获得最佳的蛋白质产量并避免毒性, 应使用自动诱导培养基(在此处提供详细信息)代替IPTG,表达生物传感器的细胞应在20°C而不是37°C下生长。自动诱导培养基用于获得乳糖诱导型表达系统的高水平重组蛋白表达。它不需要添加异丙基β-D-1-硫代半乳糖吡喃糖苷(IPTG),并且避免了在确定IPTG补充时间时监测光密度(OD)的限制。培养基含有以不同速率代谢的葡萄糖和乳糖。葡萄糖最初是受欢迎的,并抑制启动子活性。一旦葡萄糖被消耗掉,抑制将被减轻,乳糖导致转录的诱导。
自动诱导培养基用于乳糖/ IPTG诱导表达系统,否则必须定制协议的这一部分。
甘油库存是通过将细胞培养物与含有50%w / v甘油的LB培养基1:1混合而产生的。
生长200 ml培养物时,培养瓶的容量应为1或2L 。烧瓶的装填量不要超过总体积的20%,以确保最佳的充氧效果。
在37°C的初始温育对于加快单个菌落中大型培养物的生长至关重要。不要延长孵育时间,因为稍后可能会导致可溶性生物传感器的数量减少和包涵体的出现增加。
转移培养至20℃和在黑暗中以220rpm生长48小时,以避免任何光漂白FP的小号(图2) 。
离心培养物,通过使用高速50ml离心管30分钟,以20,000 X克于4 ℃,弃去上清液并在-20℃下过夜(图2)冻结沉淀的细胞。不要跳过此冷冻步骤,因为它有助于纯化过程。表达传感器的细胞应呈淡黄色沉淀物。
ñ OTE小号:

可以通过冷冻和解冻沉淀物来增加纯化蛋白的产量,这有助于破坏细胞。
应使用20,000 xg的高速离心管,以避免离心过程中管子破裂。可替换地:正常falcon管可以通过减小速度至17中使用,000 X克的方式,以防止任何泄漏。




图2. GO-Matryoshka生物传感器在大肠杆菌中的表达。W¯¯从殖民地的筛选,直到expresse的收获在实验开始的orkflow d生物传感器,亮黄色沉淀物。

 

生物传感器纯化
在冰上解冻沉淀。
通过在5 ml含蛋白酶抑制剂混合物的裂解缓冲液中上下吸液,立即重悬细胞。
在冰上将细胞悬浮液超声处理到试管中(10-15个循环,每个循环包括10秒钟的超声处理和10-15秒钟的休息)。在此步骤上要小心,因为重要的是最佳破碎细胞,同时防止样品过热,这可能导致蛋白质变性。此步骤对于蛋白质样品的质量至关重要。
注意:请勿加热样品(例如在超声处理过程中),这可能导致不希望的蛋白质变性和聚集。

离心升通过使用高速15ml离心管(4℃; 20000 ysate X克)为至少30分钟。
在离心过程中,通过将1-2 ml最终体积的Ni-NTA琼脂糖珠材料添加到色谱柱中(放置在其支架上)来准备色谱柱。
在室温下,通过向柱中添加5-10 ml洗涤缓冲液洗涤Ni-NTA珠子3次,然后使其在重力作用下流过。
将裂解液添加到该列中。允许裂解液通过重力流过。带His标签的生物传感器将与Ni-NTA磁珠结合。
用5-10 ml洗涤缓冲液在重力作用下洗涤3次。
通过重力流动用多达5个连续部分的300 µl洗脱缓冲液洗脱传感器,并在分析前于4°C的暗处保存至少24 h,然后进行分析,以使所有纯化的生物传感器成熟。或者:将洗脱液在37°C下孵育3小时。
注意:由于LSSmrange的成熟时间比衍生物长(Shcherbakova等人,2012),因此4度温育对于获得完全成熟的FP至关重要。

为了除去咪唑,盐污染物和结合的配体,可以使用Zeba TM Spin脱盐柱进一步纯化洗脱液,最终体积为130 µl(根据制造商的说明)。
纯化生物传感器的质量控制
根据制造商的方案进行Bradford分析以确定蛋白浓度。建议浓度至少约为1 mg / ml,否则建议以减少的洗脱体积重复纯化。

注意:生物传感器可以在黑暗中于4°C下存储几周。

 

B.将10-20 µg蛋白质样品装入SDS-聚丙烯酰胺凝胶(SDS-PAGE)(图3)。        

将样品与1x SDS样品缓冲液混合,加水至最终体积20-40 µl。
将样品在90°C下煮沸5分钟,以加快变性过程。
注意:变性后,样品可以保存在室温下直至上样。

将样品上样至SDS-PAGE凝胶孔中。
电泳后,根据制造商的说明进行6xHis亲和标签蛋白(Pierce 6xHis染色试剂组)的染色,并进行考马斯染色以进行总蛋白可视化。
通过使用可见光(库马西染色)和在280-310 nm波长的紫外光激发下进行蛋白质检测(6xHis亲和标记染色)。
注意:对6xHis亲和标记的蛋白进行染色需要每条带5.6 pmol His标记的蛋白才能用CCD相机检测,或者每条带57 pmol的His标记蛋白才能用UV透射仪检测。

 



图3.在大肠杆菌中表达的GO-MatryoshCaMP6s传感器的分离。实验的时间表是从沉淀的细胞开始,直到通过亲和色谱法洗脱生物传感器。样品负载编入SDS-PAGE凝胶是:MW(预染分子量标志物),CE(粗提取物),L (升ysate ),FT(˚F低通),W(洗涤),和E1-E5(洗脱液从分数1到5)。上凝胶板使用考马斯蓝染色缓冲液染色以进行总蛋白显像,下凝胶板用6xHis Protein Tag Stain Reagent Set(Thermo Fisher Scientific)染色。通过使用UV- ligt激发在280-310nm的波长下进行检测。

 

C.使用纯化的生物传感器进行体外结合测定        

该优化在最终缓冲液(样品稀释即,20毫MOPS,pH 7.0的,见配方)和荧光读取参数
在本节中,协议用户将根据cpGFP和LSSmOrange各自使用各自的最大激发和发射波长的激发,确定每种纯化的生物传感器溶液所需的适当稀释度。用户还将识别出一个单一波长,该波长可同时最大程度地激发cpGFP和LSSmOrange 。

选择蛋白质含量最高的级分,并进行约40倍的预稀释,以获得约5-10 µM的最终浓度。
从预稀释的样品中,制备最终体积为200 µl的稀释系列(1; 1/2; 1/3; 1/4; 1/5; 1/10; 1/100),然后装入96 -Well板适用于读取荧光强度和光谱在酶标仪(见ñ OTE) 。
注意:尽管使用黑色透明底部96孔板可获得最佳结果,但其成本相对较高。黑色塑料的使用有助于防止孔之间的信号污染,因此在需要精确测量时受到青睐。但是,为了进行初步的传感器表征,我们使用常规的非无菌96孔板将成本降至最低。或者,为了避免孔之间的发光污染,可以将空孔系统地放置在样品之间。

使用SPARKControl软件和以下参数,在酶标仪中测量cpGFP和LSSm稀释系列的橙色荧光强度:(1)cpGFP :在480 nm激发,带宽为20 nm,在515 nm激发,带宽为15 nm。 (2)LSSmOrange :在440nm处以10nm的带宽激发,在570nm处以15nm的带宽激发。调整读数的增益和z位置,以使测量值大致落在检测器范围的中间。增益预计50-90之间的,而在Z位置时,使用200μl的情况下,在约预期d不应超过130 20000 μ米。
注意:虽然可以通过使用单个中间波长来激发两个FP来找到折衷方案,但是它们的激发最大值是截然不同的,因此对于在快速荧光计中进行体外表征,最好分别激发两个FP。

以440至480 nm之间的2 nm间隔执行激发扫描,以验证哪种激发波长在您的条件下为GFP和LSSmOrange发射提供最大的振幅(图4)。对于GO-Matryoshka生物传感器,通常约为455 nm。
 



图4.用于检测GO-Matryoshka传感器的激励参数。MatryoshCaMP6的发射光谱表明,将激发波长从440 nm增加到480 nm导致cpGFP发射的荧光强度增加,但稳定的参考LSSmOrange的发射减少。Au .:任意单位。

 

根据在S teps C1 c和C1 d中获得的发射强度选择最佳稀释系数。稀释系数取决于每台仪器,此处目标强度范围约为20,000个任意单位(au 。),以保持在检测器的最佳范围内而不会导致饱和。仪器的增益系数越低,产生的数据质量越高(噪声越小)。
结合测定
通过对每个生物传感器进行3次生物重复,包括技术重复和裂解物纯化,设计平板的样品加载模式。取决于预期的K d ,包括合适的配体浓度(例如,从0 mM到K d的5倍的稀释系列;使用足够的数据点以获得可靠的载脂蛋白和饱和态以及结合等温线的线性范围) 。确保先前选择的稀释因子与此设计兼容,并包括仅包含用于测量背景的缓冲液的控制孔。
注意:添加的组件的顺序无关紧要。但是,我们建议首先添加体积最大的组件,即缓冲液,然后添加体积最小的组件,即生物传感器。根据您的加载方式加载96孔板。为三个殖民地的技术将一式三份从单一的预混(见加载ñ OTE小号)。确保按技术标准份量对4种反应的预混液进行缩放,以确保在移液一式三份时保持1次反应的余量。此外,即使配体浓度不同,也要确保每个孔的最终体积保持相同。应避免孔中有气泡,因为它们可能会影响读数。

注意小号:

由于分析是定量的,因此准确的移液至关重要。使用高精度,在移液和处理移液器时采用最佳实践,确保移液器处于良好状态,确保移液器的正确校准,准备预混液(即避免移取小体积),并缓慢移液以减少移液错误。请勿卡扣柱塞,切勿让任何液体进入轴。
建议孵育时间最少为5分钟。在进行读取之前,必须确保已达到结合平衡。通常需要花费几分钟的时间来准备96孔板。
建议包括纯缓冲孔和空白。
使用平板读数器如所描述的读出荧光在步骤C1 ,以获得三组数据的各孔:发射光谱和点测量为cpGFP结构和LSSmOrange的GO-俄罗斯套的最大强度的传感器(图5) 。
 



图5. excel工作表的屏幕快照,显示来自一个结合测定实验的数据和参数的示例。三组数据被进行点测量和发射光谱后获得的扫描的GO- MatryoshCaMP6s在生物传感器结合测定。

 

分析每个生物重复的数据
设置默认ë Xcel公司工作表,它可被用作模板用于自动分析的前面部分中所获得的数据(图5) 。这可以通过输入处理数据所需的公式并通过简单粘贴其原始值来从任何实验中生成图形来完成。该模板必须包括:
计算三次重复的平均值。
对于每种条件和每种数据类型(cpGFP和LSSmOrange强度以及发射光谱)减去缓冲液的背景荧光。
通过以图形方式将荧光强度表示为波长来绘制数据,并生成记录在不同配体浓度下的发射光谱,以量化响应分析物浓度的强度变化(Bermejo等人,2011)(图6)。
 



FIGUR Ë6.纯化,使用俄罗斯套(结合测定此处GO- MatryoshCaMP6s)生物传感器。A. 96孔板,带有GO- MatryoshCaMP6s和增加的钙浓度,颜色代码从绿色(无钙)到红色(钙为10 µM),一式三份。B. MatryoshCaMP6的光谱,在455 nm处激发。钙浓度的增加导致cpGFP发射的荧光强度增加,而参考LSSmOrange发射保持稳定(由于渗漏而略有增加)。[ au 。]:任意单位。数据表示为三个技术重复的平均值。

注意:观察到渗漏发生,并导致参考FP LSSmOrange的荧光强度轻微但明显的增加。^ h H但是,通过使用两个激发波长不同的点测量应该摆脱这种影响。

 

通过使用三个生物学重复来测量亲和力:
使用以下公式计算每种配体浓度在520 nm / 57 5 nm发射时的强度比(R):
R = FI 520nm / FI 57 5 nm

计算的R 0是无底物的R的值。
计算 R的值[R在由R中减去特定的钙浓度0为如下:
R = R-R 0

计算R / R 0 。
通过使用暗算浓度数据例如,MyCurveFit 。
X轴使用对数刻度。
通过使用,应用的S形拟合例如,MyCurveFit ,并计算ķ d (图7)。
 



图7.纯化的GO-Matryoshka的体外饱和曲线。MatryoshCaMP6s的钙结合等温线。使用纯化的传感器对不同钙浓度下的传感器输出进行分析。如先前所述确定钙结合(Ast等人,2017)。数据表示为三个生物学重复的平均值(平均值± SEM ,来自n = 3的生物学重复)。

 

菜谱

 

LB含抗生素的液体培养基
将10 g Bacto TM胰蛋白,、 10 g氯化钠和5 g Bacto TM酵母提取物溶于800 m l M illiQ水中
用NaOH调节至pH 7.0
调整最终体积至1L中号illiQ水
在121°C下高压灭菌20分钟
使用前,添加与选择所需的抗生素抗性相对应的抗生素
LB固体培养基(pH 7.0)
将10 g Bacto TM胰蛋白,、 10 g氯化钠和5 g Bacto TM酵母提取物和15 g琼脂溶解在800 ml M illiQ水中
用NaOH调节至pH 7.0
调整最终体积至1L中号illiQ水
在121°C下高压灭菌20分钟
让其冷却至约50-60°C,然后添加与选择所需的抗生素抗性相对应的抗生素
自动感应培养基(pH 7.0)
LB液体培养基

0.05%w / v D-葡萄糖

0.2%w / v乳糖

裂解缓冲液(pH 7.0)
20毫米MOPS

每50 ml溶液1个cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物

注意:缓冲区可以4度保存几个月。通过广泛涡旋溶解1片cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物。

洗涤缓冲液(pH 7.0)
20毫米MOPS

每50 ml溶液1个cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物

注意:缓冲区可以4度保存几个月。通过广泛涡旋溶解1片cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物。

洗脱缓冲液(pH 7.0):
20 mM MOPS缓冲区

250 mM咪唑

每50 ml溶液1个cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物

注意:缓冲区可以4度存储。连续1个月的cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂鸡尾酒通过广泛涡旋溶解。

最终缓冲液(pH 7.0)
20毫米MOPS

注意:缓冲区可以4度保存几个月。通过广泛涡旋溶解1片cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物。

配体结合测定缓冲液(pH 7.0)
20 mM MOPS和0至10-20 mM氯化钙

注意:缓冲区可以4度保存几个月。通过广泛涡旋溶解1片cOmplete TM ULTRA Tablet Mini蛋白酶抑制剂混合物。

考马斯染色缓冲液
考马斯亮蓝R250染色剂

10%v / v乙酸

50%v / v甲醇

羧苄青霉素原液:
D在9.5 ml M illiQ水中溶解1 g羧苄青霉素二钠
调整最终体积至10毫升
˚F ILTER-除菌通过使用0.22微米的过滤器
注意:小号羧苄青霉素的滴答溶液可以储存在- 20 ℃下。

运行缓冲区
25 mM Tris

192 mM甘氨酸

0.1%(w / v)的SDS

 

致谢

 

作者非常感谢来自德国赠款支持研究联合会在德国(DFG,德国研究基金会)“的精妙战略-EXC-2048 / 1-项目ID 390686111,和亚历山大·冯·洪堡教授到WBF。非常感谢CEA增强的欧洲人才计划和ANR-19-CE13-0007“ PHLOWZ”的支持(HJ)。

 

利益争夺

 

WBF是Matryoshka技术的专利(美国申请号15 / 438,078)的作者。在剩余的作者有没有CON佛罗里达州的利益ICT或竞争的利益申报。

 

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引用:Sadoine, M., Castro-Rodríguez, V., Poloczek, T., Javot, H., Sunal, E., Wudick, M. M. and Frommer, W. B. (2020). Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses. Bio-protocol 10(19): e3773. DOI: 10.21769/BioProtoc.3773.
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