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Dec 2018

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Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins
用于细胞蛋白VIPER标记的CoilR肽探针的制备   

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Abstract

Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag (Doh et al., 2018), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., CoilR). These two peptides deliver contrast to a protein of interest by forming a specific, high-affinity heterodimer. The probe peptide was designed with a single cysteine residue for site-specific modification via thiol-maleimide chemistry. This feature can be used to attach a variety of biophysical reporters to the peptide, including bright fluorophores for fluorescence microscopy or electron-dense nanoparticles for electron microscopy. In this Bio-Protocol, we describe our methods for expressing and purifying recombinant CoilR. Additionally, we describe protocols for making fluorescent or biotinylated probe peptides for labeling CoilE-tagged cellular proteins. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).

Keywords: Peptide (肽), Bioconjugation (生物偶联), Genetic tag (遗传标记), Microscopy (显微镜), Chemical biology (化学生物学), Fluorescent (荧光)

Background

Fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) enable investigations into the multi-protein complexes and macromolecular interactions that mediate normal and disease-associated cellular functions. However, multiscale microscopy is restricted by the shortage of methods for attaching FM-, EM-, and CLEM-compatible reporter chemistries to target proteins. Additionally, there are few methods for protein labeling that facilitate switching between imaging systems. As a result, most multiscale imaging studies obtain protein-specific contrast with immunolabeling. However, there are known drawbacks to immunolabeling. The large size of antibodies reduces localization precision, and labeling protocols can disrupt cellular ultra-structure (Schnell et al., 2012). Scarce proteins and rare interactions can elude detection unless immunolabeling is efficient (Griffiths and Hoppeler, 1986; Schnell et al., 2012; Griffiths and Lucocq, 2014). Many antibodies have poor target specificity and cross-reactivity (Berglund et al., 2008; Bordeaux et al., 2010; Baker, 2015; Bradbury and Pluckthun, 2015), which can result in misleading observations. 

The central obstacle that has limited progress in multiscale microscopy is the shortage of genetic tags for labeling proteins. Most tags were developed for FM (Liu et al., 2015), with the most commonly used tags being fluorescent proteins [e.g., GFP] (Tsien, 1998; Cranfill et al., 2016; Rodriguez et al., 2017). By comparison, there are few genetic tags for EM or CLEM (Ellisman et al., 2012). We saw this as an opportunity to create a new class of genetically-encoded peptide tags for multiscale microscopy (Zane et al., 2017; Doh et al., 2018). We named this technology Versatile Interacting Peptide (VIP) tags (Figure 1). VIP tags consist of a heterodimeric coiled-coil between a genetically-encoded peptide tag and a reporter-conjugated peptide (“probe peptide”). Binding is driven by a hydrophobic interface and inter-strand salt bridges between the two coils. Initially we reported VIP Y/Z, which was used to label cellular proteins with fluorophores and Qdots (Zane et al., 2017). This pair consists of a heterodimeric CoilY-CoilZ pair with a reported dissociation constant (KD) of less than 15 nM (Reinke et al., 2010). Either CoilY or CoilZ could serve as the genetically-encoded tag. In 2018, we reported the VIPER tag, which enables high-affinity labeling of proteins for imaging by FM and CLEM (Doh et al., 2018). Binding between the CoilE tag and the CoilR probe peptide to form VIPER is specific and nearly irreversible [KD ~10-11 M] (Moll et al., 2001).


Figure 1. Versatile interacting peptide (VIP) tags are a new technology for imaging proteins by FM, EM or CLEM. VIPER labeling of transferrin receptor 1 (TfR1) is mediated by heterodimer formation between the CoilE tag and a fluorescent CoilR probe peptide. Fluorescent micrograph: VIPER-tagged TfR1 labeled with CoilR-Cy5 (magenta) and colocalized with fluorescent transferrin (Tf-AF488; green) at the cell surface of transfected CHO TRVb cells (63x magnification). Magenta-green signal overlap appears white and nuclei are blue.


Figure 2. VIP tags are a versatile technology for multi-scale microscopy. After a target protein is tagged, it can be labeled using a variety of probe peptides selected for the particular application.

For VIP tags, the versatility is imparted by the customizable probe peptide. After introduction of the CoilE tag onto a target protein, the protein can be labeled with one of many different reporters attached to CoilR (Figure 2). For example, we imaged the transmembrane receptor, TfR1-CoilE, with CoilR-BODIPY, CoilR-Cy5 (see Figure 1), and CoilR-biotin (Doh et al., 2018). In other words, the probe peptide can be customized for different studies or imaging systems without changing the genetic tag. This is possible because CoilR encodes a single cysteine residue for site-specific modification via thiol-maleimide chemistry. The CoilR probe peptide can be bioconjugated to a variety of probes, including fluorophores, small molecules (e.g., biotin), or nanoparticles. Many companies sell thiol-reactive probes, which makes this conjugation reaction accessible to labs without synthetic chemistry expertise. For more information on bioconjugation reactions, we recommend reading Hermanson’s Bioconjugate Techniques (Hermanson, 2013).

In this Bio-Protocol, we provide methods for making CoilR probe peptides that can be used for VIPER-labeling of cellular proteins for imaging by FM or EM. The CoilR peptide and the CoilE tag sequences are provided in Table 1. As described in prior work (Doh et al., 2018), we used gene assembly PCR to enable the recombinant expression of probe peptides in E. coli. The method for peptide expression is described in Procedure A. CoilR was designed to interact with CoilE via an optimized alpha-helical coil-coil, as originally described by Vinson and coworkers (Moll et al., 2001). We included a hexahistidine tag at the C-terminus of CoilR for purification by immobilized metal affinity chromatography (IMAC) (Hochuli et al., 1987); this is described in Procedure B. 

Table 1. Sequences of CoilR and CoilE

§Italics: Linker sequence; Bold: Peptide coil; C: Cysteine (conjugation site).
Heptad position: Residues a and d form a hydrophobic interface, residues at e and g form inter-strand salt bridges. 

Procedures C and D describe thiol-maleimide reactions to label CoilR with a small molecule reporter. In Procedure C, we describe the method that we used to generate probe peptides in our prior work (Doh et al., 2018). In Procedure D, we adapted a method described by Weiss and coworkers for solid state-based labeling of peptides (Kim et al., 2008). Lastly, we include methods for purifying fluorophore-labeled (Procedure E) or biotinylated (Procedure F) probe peptide. This Bio-Protocol is accompanied by two companion articles, which include detailed methods for imaging VIPER-labeled cellular proteins by FM (Doh et al., 2019a) and CLEM (Doh et al., 2019b) (Figure 3). 


Figure 3. A decision tree for implementing VIPER. Procedures are color-coded by the publication in which they appear. Methods in this publication are color-coded purple. Methods in Doh et al., 2019a are yellow and methods in Doh et al., 2019b are orange. Publication 1: this article; Publication 2: Doh et al., 2019a; Publication 3: Doh et al., 2019b).

Materials and Reagents

Note: “*” indicates a brand that is critical to the success of the experiment.
Materials

  1. Universal pipette tips (USA Scientific TipOneTM, catalog numbers: 1112-1770, 1163-1730, and 1121-3812)
  2. Microcentrifuge tubes, 1.5 ml (Thermo Scientific, catalog number: 02-682-002)
  3. Sterile serological pipettes (Thermo Scientific, catalog number: 13-678-11D + E)
  4. Sterile 14 ml culture tubes (Corning, FalconTM, catalog number: 352059)
  5. Disposable polystyrene spectrophotometer cuvettes (Thermo Scientific, catalog number: 14-955-127)
  6. Conical 50 ml tubes (Thermo Scientific, NuncTM, catalog number: 12-565-270)
  7. Chromatography column (Bio-Rad, Econo-PacTM, catalog number: 7321010)
  8. Ring stand (Fisher, catalog number: 11-474-207)
  9. Adjustable ring stand clamps (United Scientific Supplies, catalog number: CLHD03)
  10. Molecular weight cut off (MWCO) 3 kDa filters (Sigma-Aldrich, Amicon UltraTM, catalog number: UFC900324)
  11. Quartz 10.00 mm cuvette (Hellma Analytics, Ultra-Micro Cell, catalog number: 105-250-15-40)
  12. Pipettes (e.g., Rainin Pipet-LiteTM XLS, catalog numbers: 17014407, 17014411, 17014412, and 17014413)
  13. Glass 2 L Erlenmeyer flask (Corning, PyrexTM, catalog number: 49802L)

Reagents
  1. Anti-biotin HRP antibody (Jackson Immunoresearch, catalog number: 200-032-211) 
  2. Streptavidin-HRP (Thermo Scientific, catalog number: ENN100)
  3. pET28b(+)_CoilR [Available by MTA from OHSU or made as published (Doh et al., 2018)]
  4. BL21 (DE3) E. coli (New England Biolabs, catalog number: C2527I)
  5. Glycine (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP381-500)
  6. SOC outgrowth media (New England Biolabs, catalog number: C2527I)
  7. Miller Luria-Bertani (LB) agar (BD DifcoTM, catalog number: 244520)
  8. Miller LB broth (BD DifcoTM, catalog number: BD 244610)
  9. 2X YT (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP9743500)
  10. Kanamycin sulfate (Thermo Scientific, Fisher Chemical, catalog number: BP906-5)
  11. IPTG (GoldBio, catalog number: I2481C5)
  12. *Ni-NTA agarose (Qiagen, catalog number: 30230)
  13. *Pierce Monomeric Avidin Agarose (Thermo Scientific PierceTM, catalog number: 20228)
  14. Sodium Phosphate Monobasic Anhydrous (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP329-500)
  15. Urea (Thermo Scientific, Fisher BioReagentsTM, catalog number: U15 3)
  16. Tris Base (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP152 5)
  17. Tris HCl (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP153 1)
  18. NaCl (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP358-1)
  19. Glycerol (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP229-1)
  20. Imidazole (ACROS Organics, catalog number: AC39674-1000)
  21. Coomassie Brilliant Blue R-250 (Thermo Scientific, catalog number: 20278)
  22. Methanol (Thermo Scientific, Fisher Chemical, catalog number: A412)
  23. Acetone (Thermo Scientific, Fisher Chemical, catalog number: A18)
  24. Nitrogen gas
  25. Ammonium sulfate (EMD Millipore, catalog number: AX1385-1)
  26. TCEP-HCl (GoldBio, catalog number: TCEP10)
  27. Dithiothreitol (DTT) (Thermo Scientific, Molecular ProbesTM, catalog number: D1532)
  28. TC-grade DMSO (Sigma-Aldrich, catalog number: D2650-5X10ML)
  29. *Sulfo-Cy5-maleimide (Lumiprobe, catalog number: 23380)
  30. *Biotin-PEG2-maleimide (Thermo Scientific, catalog number: 21901BID)
  31. D-Biotin (Ark Pharma, catalog number: AK-44010)
  32. Pierce BCA assay kit (Thermo Fisher Scientific, catalog number: 23227)
  33. 12% Bis-Tris polyacrylamide protein gels (Bio-Rad CriterionTM XT, catalog number: 3450119)
  34. MES (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP300-100)
  35. NaH2PO4 (Sigma-Aldrich, catalog number: S3139-250G)
  36. Ponceau Red (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP103-10)
  37. NaOH (Thermo Scientific, Fisher BioReagentsTM, catalog number: BP359-500)
  38. Buffer B (Ni-NTA peptide purification) (see Recipes)
  39. Buffer C (Ni-NTA peptide purification) (see Recipes)
  40. Buffer E (Ni-NTA peptide purification) (see Recipes)
  41. MES running buffer (see Recipes)
  42. TCEP/SDS Loading Dye (5x) (see Recipes)
  43. Coomassie Stain (see Recipes)
  44. Destain Solution (see Recipes)
  45. Tris-Buffered Saline (TBS) (see Recipes)
  46. TBS Urea (see Recipes)
  47. 0.5 M TCEP (see Recipes)
  48. TBS Urea Binding Buffer (see Recipes)
  49. Solid state-based labeling (SSL) Buffer, pH 7.5 (see Recipes)
  50. 1 M DTT (see Recipes)
  51. TBS Urea Imidazole (see Recipes)
  52. Biotin Buffer (see Recipes)
  53. Regeneration buffer (see Recipes)

Equipment

  1. Electronic pipettor (Eppendorf EasypetTM, catalog number: 4430000018)
  2. -20 °C freezer (Thermo Scientific, RevcoTM, catalog number: 13 990 206)
  3. Incubator and shaker (New Brunswick ExcellaTM E24, catalog number: M1352-0010)
  4. Spectrophotometer (Eppendorf, Biophotometer Plus, catalog number: 6132)
  5. Rotisserie (Thermo Scientific, catalog number: 400110Q)
  6. Sonifier (Branson UltrasonicsTM, catalog number: 101063198R)
  7. Sonifier 1/8 inch micro-tip (Branson UltrasonicsTM, catalog number: 22-309796)
  8. Refrigerated centrifuge (Thermo Scientific, Sorvall Legend XTR Centrifuge, catalog number: 75211731)
  9. Microcentrifuge (Eppendorf, catalog number: 022620304)
  10. Heat block (Fisher, IsotempTM, catalog number: 88-860-022)
  11. Electrophoresis cell (Bio-Rad CriterionTM, catalog number: 165-6001)
  12. Power supply (Bio-Rad PowerPacTM HC, catalog number: 1645052)
  13. Plate reader (Tecan Infinite M200 Pro, catalog number: 30050303)
  14. (Optional) Fluorescence and western blot imager (i.e., GE Healthcare AmershamTM Typhoon 5 multimode scanner, catalog number: 29187191 or Protein Simple, FluorChem Q)

Procedure

  1. Expression of recombinant CoilR
    CoilR is generated by recombinant expression in E. coli. The growth and purification of CoilR follows standard protocols for making and purifying histidine-tagged peptides under inducible expression. For detailed background, protocols, and troubleshooting, we recommend referring to the Qiaexpressionist handbook (Qiagen) (Morimoto-Tomita et al., 2003).
    1. Obtain or generate a plasmid encoding the CoilR peptide (i.e., pET28b(+)_CoilR) (Doh et al., 2018). The amino acid sequence of CoilR expressed from pET28b(+)_CoilR is provided in Table 1.
      Note: The pET28b(+)_CoilR plasmid encodes kanamycin resistance.
    2. Transform the plasmid into E. coli BL21 (DE3) competent cells, following NEB’s instructions for product C2527. 
      1. Plate cells on LB/agar/kanamycin (50 μg/ml) and grow overnight at 37 °C. 
      2. Pick single colonies and inoculate 5 ml starter cultures (one colony per 5 ml culture) in LB supplemented with kanamycin (50 μg/ml) in sterile 14 ml culture tubes. 
      3. Grow overnight in a shaking incubator (225 rpm, 37 °C). We grow several starter cultures in case of variation in growth is observed (e.g., a culture grows slowly).
    3. Use one overnight culture to inoculate (2.5 ml, 1:200 dilution) 500 ml of 2X YT sterile media in a 2 L Erlenmeyer flask supplemented with kanamycin (50 μg/ml). Grow at 225 rpm, 37 °C until the OD600 reaches 0.8 to 1.0.
      1. Monitor growth by measuring OD600 of the culture in a disposable cuvette on a spectrophotometer.
      2. It will take approximately 2-4 h for the culture to reach this OD600.
      3. Prior to induction, take a 1 ml sample of the uninduced culture for peptide expression analysis by SDS-PAGE.
        1. For each sample: Pellet 1 ml of bacterial culture in a microcentrifuge tube (10,000 x g, 2 min).
        2. Resuspend the pellet in Buffer B (Recipe 1). Normalize the sample by adding Buffer B to the pellet. Use the equation: volume = OD600 x 100 μl Buffer B.
        3. Freeze at -20 °C.
    4. Lower the temperature of the incubator/shaker to 25 °C and induce peptide expression for 2-4 h by addition of 0.1 mM IPTG.
      Note: The peptide will degrade if the induction is done at 37 °C, reducing the overall yield.
      1. Monitor expression by taking 1 ml samples every hour during the induction. Normalize as described in Step A3c.
    5. Harvest cells by centrifugation in a refrigerated centrifuge (5,000 x g, 15 min, 4 °C).
      1. Discard the supernatant.
      2. Transfer the pelleted bacteria to a tared 50 ml conical tube to obtain the weight of the wet pellet.
      3. Store the pellet frozen (-20 °C). The pellet can be stored for several months at -20 °C.
      Note: If desired, cell lysis (Step B4) can be performed on the same day as the peptide expression. However, we typically freeze the pellet before proceeding to purification the next day.
    6. Analyze peptide expression by SDS-PAGE. See Figure 4 for a representative SDS-PAGE analysis of CoilR expression and purification.
      1. Thaw protein samples from time-points collected during induction. Freezing and thawing in the presence of Buffer B will partially lyse the cells.
        Note: If lysis is incomplete, freeze-thaw the samples several times to break open cells.
      2. Pellet insoluble debris (10 min, 10,000 x g). 
      3. Transfer the clarified lysate to a new microcentrifuge tube.
      4. Add 5x TCEP-SDS loading dye (Recipe 5). Boil samples for 5 min, pellet by centrifugation, and then load 10-15 μl sample/well onto the protein gel.
      5. Analyze peptide expression by SDS-PAGE.
        1. We recommend analyzing on a BioRad Criterion Bis-Tris Gel (12%; 26-well) run in MES running buffer (Recipe 4) at constant voltage (180 V). 
        2. Run until the loading dye reaches the bottom of the gel, approximately 35 min.
        3. After electrophoresis, stain the protein gel with Coomassie stain (Recipe 6) and then destain (Destain solution, Recipe 7) before imaging.
        4. CoilR will migrate on the gel as a monomer (7.5 kDa) and a dimer (15 kDa).


      Figure 4. Peptide expression in E. coli and denaturing purification of CoilR. Samples were collected from uninduced (UI) and induced (2 h and 4 h) cells, lysed, and resolved by SDS-PAGE. CoilR was purified from clarified lysate by IMAC (i.e., on Ni-NTA resin) under denaturing conditions. CL = clarified lysate; FT = flow-through (unbound fraction). Wash 1: Buffer B (pH 8) with 10 mM imidazole. Washes 2-4: Buffer C (pH 6.8) with 10 mM imidazole. Elutions 1-3: Buffer E (pH 4.3). The CoilR peptide (MW = 7502.35 Da) migrates as a monomer at ~6 kDa and an apparent dimer at ~14 kDa (gray arrowheads).

  2. Purification of recombinant CoilR by IMAC
    1. Prior to peptide purification, measure and adjust the pH of all purification buffers.
      1. Buffer B: 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 8 (Recipe 1).
      2. Buffer C: 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 6.5 (Recipe 2).
      3. Buffer E: 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 4.5 (Recipe 3).
    Note: Unless otherwise noted, all steps should be done on ice with pre-chilled buffers.
    1. Thaw the E. coli pellet (from Step A5) on ice.
    2. Resuspend the pellet in Buffer B. Use 5 ml Buffer B per gram of wet weight.
    3. Lyse by sonication on ice.
      1. We use a Branson sonifier fitted with a 1/8” Branson microtip to lyse bacteria. We lysed cells at 40% duty cycle, output: 4. The sample was pulsed for 30 s and then left to rest for 1 min on ice for 8 cycles.
      2. Avoid foaming of the sample, which will cause protein loss.
      3. Alternatively, cells can be lysed by other methods (e.g., freeze-thaw or French press).
    4. Clarify the lysate by centrifugation in a refrigerated centrifuge (10,000 x g, 30 min, 4 °C).
      Note: Keep a sample of the clarified lysate for analysis by SDS-PAGE.
    5. Incubate the clarified lysate with Ni-NTA agarose resin (Qiagen) for 1 h at 4 °C on a rotisserie.
      1. Buffer B should be supplemented with 10-20 mM imidazole to reduce non-specific protein binding to the resin.
      2. Use 1 ml of resin per 1 gram of pellet (wet weight).
      3. Alternatively, bind at 4 °C overnight.
      Note: If you observe CoilR peptide in the flow-through and initial washes, then the resin was overloaded. Use more resin in the binding step.
    6. Load the lysate-resin mixture onto a clean, fritted chromatography column.
    7. Collect the flow-through. Save a sample for analysis by SDS-PAGE.
    8. Wash the resin with 5 column volumes (CV) of Buffer B.
    9. Wash the resin with 10-50 CV of Buffer C.
      1. The CoilR peptide will elute at ~pH 6. The wash buffer should be between pH 6.3 and pH 7.0.
      2. Save a sample from each wash step to analyze by SDS-PAGE.
      3. Wash until no further impurities elute.
    10. Elute the CoilR peptide in Buffer E. Elute in 5 fractions of 2 CV each. 
      1. Most of the peptide will elute in the first 3 elutions.
      2. Elute in additional volume if you detect incomplete elution (see Figure 4).
    11. Monitor the purification by SDS-PAGE. Analyze samples from the clarified lysate, flow-through, washes, and elution. 
      1. Gel running conditions are the same as from Step A6d-A6e.
    12. Combine fractions containing the purified peptide based on results from Step B12.
    13. Concentrate and exchange the purified CoilR into the desired buffer using a 3 kDa MWCO filter.
      1. We recommend exchanging into TBS Urea (Recipe 9) using the MWCO filter. This buffer is compatible with the thiol-maleimide conjugation reaction (Procedure C).
      2. Keep the peptide concentration between 0.5 mg/ml and 2 mg/ml to avoid issues with solubility. If a white precipitate is observed in the peptide solution, the peptide is too concentrated.
    14. Quantify the peptide concentration using the Pierce BCA assay kit (Thermo Fisher Scientific).
      1. The protein concentration can also be estimated by measuring the absorbance at 280 nm. However, the extinction coefficient of CoilR is low (2,980 L·mol-1·cm-1); so the concentration will be more accurate if determined by BCA assay.
      2. Include replicates and dilutions to obtain an accurate concentration.
    15. Add 5-10% glycerol to the concentrated peptide, aliquot (200 μl/tube), and freeze (-20 °C). CoilR peptide can be stored frozen for several months.

  3. Generation of CoilR probe peptides by thiol-maleimide conjugation in solution
    VIPER-labeling is specific and efficient in living and fixed cells expressing CoilE-tagged protein. However, the quality of labeling is directly related to the quality of the probe peptide. This is because unlabeled CoilR and labeled CoilR will both dimerize with CoilE-tagged cellular proteins. We recommend using peptides that are > 50% labeled with the reporter chemistry.
      We have found that the efficiency of the thiol-maleimide bioconjugation reaction is variable. Therefore we have included two approaches for modifying CoilR: Procedure C and Procedure D. We have used both successfully to label CoilR with reporters, with the preferred protocol being dependent on the researcher. Procedure C describes a conventional thiol-maleimide conjugation reaction in solution; this is the method used to generate probe peptides described in our 2018 publication (Doh et al., 2018). This method can be used to attach a fluorescent probe, such as Sulfo-Cyanine5 (Cy5)-maleimide, or to biotinylate CoilR. 
    1. Prepare buffers, TCEP, and a stock solution of the reactive maleimide. If these stocks are already made, then proceed to Step C2.
      1. We recommend labeling in TBS Urea (Recipe 9). The reaction should be done in a thiol-free buffer between pH 7 and pH 7.5.
      2. Degas the buffer before using. This can be done by bubbling a stream of nitrogen gas through the buffer or by vacuum degassing.
      3. Prepare 0.5 M TCEP (Recipe 10).
    2. Prepare a concentrated stock solution of the maleimide probe at 20-100 mg/ml in anhydrous DMSO. For Cy5-maleimide or other fluorophores, protect the solution from light. 
      1. More concentrated stocks are preferable to limit the amount of DMSO in the reaction.
      2. Stocks can be stored at -20 °C.
      3. The maleimide will hydrolyze in water, so storage in DMSO is recommended.
    3. Thaw the purified CoilR peptide on ice. The concentrated peptide stock should be in TBS Urea and degassed by nitrogen.
      1. The reaction will proceed better if the peptide is concentrated. We recommend using a stock that is 2 mg/ml (~270 µM).
      2. A typical labeling reaction will include 50 to 200 nmoles of CoilR peptide.
      3. If the peptide is not in an appropriate buffer, transfer into a different buffer at this point using a 3 kDa MWCO filter and degas before proceeding to Step C4.
    4. Reduce the peptide by the addition of a 10-fold molar excess of TCEP. Incubate for 30 min at 50 °C.
    5. Initiate the conjugation reaction by adding at least 20-fold molar excess of the maleimide probe. Mix well. Incubate for 2 h at room temperature or at 4 °C overnight on a rotisserie.
      1. For fluorophore-labeling, protect the reaction from light.
    6. After labeling, add TBS Urea Binding Buffer (Recipe 11) to a total volume of 15 ml.
      1. Save a sample of the crude reaction mixture for analysis by SDS-PAGE.
    7. Concentrate and buffer exchange the crude reaction on a 3 kDa MWCO filter to remove unreacted probe. Buffer exchange into TBS Urea Binding Buffer which is compatible with Ni-NTA purification. Save a sample for analysis by SDS-PAGE.
      1. For CoilR-fluorophores, continue the buffer exchange until the filtrate becomes colorless or stops changing color with subsequent buffer exchanges. Then proceed to Procedure E.
      2. For biotinylated CoilR, a 40 ml wash is sufficient to remove most of the free biotin moieties. Then proceed to Procedure F.

  4. Generation of CoilR probe peptides by thiol-maleimide chemistry using solid state-based labeling (SSL)
    In 2008, Weiss and coworkers described a new method for modifying proteins using thiol-maleimide chemistry (Kim et al., 2008). In that work, the protein was first precipitated with ammonium sulfate and reduced with DTT before fluorophore conjugation. They named this method solid state-based labeling (SSL). The advantage of this approach is that it is easy to do, efficient (70-90% labeled) and thiol-specific (Kim et al., 2008). We currently use both SSL and solution-based labeling to generate probe peptides. Procedure D is adapted from Weiss and coworkers published method (Kim et al., 2008).
    1. Prepare SSL Buffer (Recipe 12) and the reducing agents (1 M DTT [Recipe 13] and 0.5 M TCEP). If these stocks are already made, then proceed to Step D2.
    2. Thaw the purified CoilR peptide on ice.
      1. A typical labeling reaction will include 50 to 200 nmoles of CoilR peptide.
      2. The volume should be less than 1 ml, but the peptide will become concentrated by precipitation in Step D4.
    3. Reduce the peptide by the addition of 10 mM DTT, from a 1 M stock. Incubate for 30 min at 4 °C on a rotisserie.
    4. Precipitate the reduced peptide by slow addition of ammonium sulfate powder to a final concentration of 70-75%. 
      1. For an overview of protein precipitation, see the 1998 publication by Wingfield (Wingfield, 2001).
      2. Encor Biotechnology has a useful online tool for calculating the amount of ammonium sulfate to add; see: http://www.encorbio.com/protocols/AM-SO4.htm.
        Example: For a 500 µl peptide solution at 4 °C, add 0.23 g of ammonium sulfate to get a 70% saturated solution.
    5. After a precipitate forms, add 10 mM DTT and reduce for 2 h at 4 °C on a rotisserie.
    6. Wash the reduced peptide slurry with ice-cold SSL Buffer to remove DTT.
      1. Pellet the slurry by centrifugation (4 min, 14,000 x g, 4 °C). Discard the supernatant.
      2. Add 1 ml SSL Buffer and invert the sample several times.
      3. Repeat Steps D6a and D6b 3-5 times to remove all excess DTT.
      4. After the last centrifugation step, resuspend the pellet in 100 µl SSL Buffer.
      Note: Any residual DTT will react with the maleimide probe so it is critical to wash the peptide pellet several times.
    7. Perform the thiol-maleimide conjugation on reduced peptide in the solid state.
      1. Add 10- to 30-fold molar excess probe to the reduced peptide. Mix by inverting the tube several times.
        1. Use a concentrated stock (20-100 mg/ml) of maleimide probe (e.g., Cy5-maleimide) in anhydrous DMSO.
      2. Mix the reaction on a rotisserie for 15 min at 4 °C.
      3. Add 5- to 10-fold molar excess TCEP. Mix and continue to incubate for 45 min at 4 °C on a rotisserie.
        1. For fluorophore labeling, protect the tube from light.
        2. Keep the amount of the maleimide higher than the amount of TCEP in the reaction because the maleimide probe can undergo a side-reaction with TCEP (Kim et al., 2008).
        3. The reaction can be incubated overnight.
    8. After labeling, we recommend washing the reaction mixture with SSL Buffer to remove excess maleimide.
      1. Pellet the reaction by centrifugation (4 min, 14,000 x g, 4 °C). Discard the supernatant.
      2. Resuspend in SSL Buffer (1 ml).
      3. Pellet by centrifugation (4 min, 14,000 x g, 4 °C). Discard the supernatant.
    9. Resuspend the pellet from Step D8 in Buffer B.
      Notes: 
      1. High concentrations of EDTA will strip nickel from the Ni-NTA agarose used in Procedure E. Add enough Buffer B to ensure that the final concentration of EDTA is less than 1 mM.
      2. Save a sample of the crude reaction mixture for analysis by SDS-PAGE.
    10. Proceed to purification, following Procedure E (for fluorescent peptides) or Procedure F (for biotinylated peptides).

  5. Purification of CoilR-Fluorophore probe peptide
    This procedure removes excess unreacted free dye from fluorophore-labeled CoilR (i.e., CoilR-Cy5) while also purifying the peptide. This section additionally describes the method used to quantify the fluorophore labeling of the peptide.
    Note: Protect the peptide from light and keep the peptide on ice, unless otherwise noted.
    1. Bind the labeled CoilR peptide to Ni-NTA agarose resin for 1 h at 4 °C.
      1. For a typical labeling reaction (50-200 nmol CoilR), we recommend using 0.5 ml Ni-NTA resin and binding in a large volume (20-40 ml) of TBS Urea Binding Buffer.
    2. Load the lysate-resin mixture onto a clean, fritted chromatography column.
    3. Collect the flow-through and save a sample for analysis by SDS-PAGE.
    4. Wash the resin with 20 column volumes (CV) of TBS Urea Binding Buffer.
      1. Continue washing until fractions are colorless.
      2. Save washes for analysis by SDS-PAGE.
    5. Optional step: Wash the resin with 10 CV of TBS Urea Binding Buffer supplemented with 20% ethanol. The addition of ethanol can help remove free fluorophore.
    6. Elute the CoilR peptide with 5-10 CV of TBS Urea Imidazole (Recipe 14).
      1. Fractions should be dark blue for Cy5-labeled peptide.
      2. Continue to elute until the fractions are nearly colorless before proceeding to the next step.
      3. Alternatively, elute in a low pH buffer (e.g., Buffer E).
    7. Analyze the purification by SDS-PAGE, following Steps A6d-A6e. For a representative analysis see Figure 5.
      1. Analyze the crude reaction, samples from the purification (flow-through, washes, elutions), and the concentrated elution.
      2. Image the gel on a fluorescence scanner to detect labeled peptide. A representative 2-color scan acquired using a Protein Simple imaging system is provided in Figure 5A. Alternatively, we recommend imaging on a GE AmershamTM Typhoon multimode scanner using the appropriate detection settings (i.e., Cy5: ex: 635 nm, em: 670/30 nm).
      3. After fluorescence imaging, stain the protein gel with Coomassie stain, destain, and image to detect total protein.
    8. Concentrate and buffer exchange the elutions containing labeled peptide into a storage buffer of choice using a 3 kDa MWCO filter.
      Note: We recommend storing the peptide in TBS urea.
    9. Determine the degree of labeling (moles of fluorophore per mole of protein). We recommend following the protocol published by Thermo Scientific [Tech Tip #31: Calculate dye:protein (F/P) molar ratios] (Reference 22).
      1. Determine the amount of fluorophore in the solution by measuring the absorption at the fluorophore’s absorbance maximum (Absmax) and using the published extinction coefficient (εFL) (Table 2).
      2. Determine the amount of peptide in the solution by measuring the absorbance at 280 nm.
        1. The fluorophore will also absorb at 280 nm and a correction factor (CF) must be used (e.g., CF for Cy5 = 0.04).
        2. The extinction coefficient (ε) of the CoilR peptide is 2,980 L·mol-1·cm-1 (www.expasy.org).
      3. Calculate the molarity of the peptide and the degree of labeling using the following equations:



      4. We offer the following recommendations:
        1. Absorbance readings are only accurate in the linear range of the spectrophotometer (between 0.1 and 1.0).
        2. We suggest preparing several dilutions of the peptide and replicates to obtain more accurate results.
        3. We measured absorbance in a quartz cuvette on a Tecan Infinite M200 Pro with a cuvette port.


          Figure 5. Analysis of CoilR probe peptides by SDS-PAGE. CoilR was labeled with sulfo-Cyanine3 (CoilR-Cy3; 60% labeled), AlexaFluor-488 (CoilR-AF488; 45% labeled), or BODIPY-FL (CoilR-BDPY-FL: 40% labeled). The crude reaction (CR) was purified on Ni-NTA resin to remove free dye. Samples were resolved by SDS-PAGE and the gel was scanned for green (ex: 488 nm, em: 525/50 nm) and red (ex: 532 nm, em: 570/20 nm) fluorescence (A). The same gel was subsequently stained for total protein with Coomassie (B). CR = diluted crude reaction (pre-column), FT = flow-through (unbound protein/fluorophore), W = wash (TBS Urea Binding Buffer), E = elution (TBS Urea Imidazole). Unreacted CoilR peptide (15 and 30 µg) was included for reference and CoilR is indicated by a gray arrowhead.

          Table 2. Values for quantifying CoilR labeling with Cy5-maleimide

          Values provided on the Lumiprobe website: www.lumiprobe.com.

    10. Store the fluorophore-labeled peptide in 5-10% glycerol.
      1. Aliquot (100 μl/tube) and freeze (-20 °C). The peptide can be stored (frozen and protected from light) for several months.
      2. Final stocks should be between 1-50 μM for experimental convenience.
      3. To minimize freeze-thaw cycles, a thawed aliquot can be divided into smaller single-use volumes (e.g., 10 μl) and re-frozen.

  6. Purification of biotinylated probe peptide (CoilR-biotin)
    This procedure is intended for purifying CoilR peptide that was biotinylated using Procedure C or D. For an overview of avidin-based affinity chromatography and a troubleshooting guide, refer to the Pierce® Monomeric Avidin Agarose instructions, available online (Reference 18). After purification and elution from the monomeric avidin resin, the CoilR-biotin peptide is assumed to be 100% biotinylated.
    1. Prepare the buffers and equilibrate them to room temperature.
      1. TBS: 20 mM Tris, 150 mM NaCl pH 7 (Recipe 8).
      2. Biotin Buffer: 2 mM biotin in DPBS pH 7.4 (Recipe 15).
      3. Regeneration Buffer: 0.1 M glycine, pH 2.8 (Recipe 16).
    2. Add the Pierce Monomeric Avidin Agarose to a clean, fritted chromatography column and drain. 
      1. For 100 nmoles of CoilR peptide, use 1 ml of resin.
    3. Block non-reversible biotin binding sites on the resin:
      1. Wash with 5 column volumes (CV) of TBS.
      2. Wash with 5 CV of Biotin Buffer to block any non-reversible biotin binding sites.
      3. Wash with 5 CV of Regeneration Buffer to remove biotin bound to reversible biotin-binding sites on the resin.
      4. Wash with 5 CV of TBS to re-equilibrate the column.
      5. Plug the column to prevent flow; the resin is now ready to be used.
    4. Dilute the biotinylated peptide sample to approximately 5 ml in TBS. Apply to the column.
    5. Incubate the sample with the resin for 30 min at room temperature.
    6. Unplug the column and collect the flow-through. Save a sample of the flow-through and all subsequent wash and elution steps for analysis by SDS-PAGE.
    7. Wash the resin twice with 5 CV of TBS.
    8. Elute the biotinylated protein in 5 CV of Biotin Buffer. Collect 1 ml fractions.
    9. Elute in 5 CV of Regeneration Buffer. Collect 1 ml fractions. This elution step is included because some peptides do not elute with excess biotin.
    10. Regenerate the resin. Wash with 5 CV of Regeneration Buffer. Collect and analyze to ensure that this wash does not contain biotinylated peptide.
    11. Analyze all fractions by SDS-PAGE (see Steps A6d-A6e).
      Note: In our experience, CoilR-biotin elutes in both the Biotin Buffer and the Regeneration Buffer, with more eluting in the Biotin Buffer.
    12. Analyze all fractions by Western blot (using your preferred method) to detect biotinylated proteins. For example, we detect biotinylated proteins using either an anti-biotin HRP antibody (Jackson Immunoresearch) or using a streptavidin-HRP (Thermo Scientific).
    13. Combine fractions containing biotinylated peptide based on the analysis in Steps F11-F12.
    14. Concentrate and exchange the biotinylated peptide into desired buffer using a 3 kDa MWCO filter.
      1. We recommend exchanging into TBS Urea.
      2. Keep the peptide concentration between 0.5 mg/ml and 2 mg/ml.
    15. Quantify the protein yield of the purification using the Pierce BCA assay kit.
      1. The crude reaction will contain unmodified and biotinylated peptide. The amount of CoilR-biotin retrieved after monoavidin-based purification is thus expected to be less than the amount of CoilR used in the reaction.
      2. The biotinylation (%) of the peptide in the crude reaction mixture can be estimated by dividing the nmoles of CoilR-biotin obtained from the monoavidin purification by the nmoles of CoilR in the labeling reaction.
      3. The CoilR-biotin obtained from this procedure is presumed to be 100% biotinylated once it is eluted from the monoavidin column because unreacted peptide (i.e., CoilR) will not bind to the resin.
    16. Store the biotin-labeled peptide in 5-10% glycerol.
      1. Aliquot (100 μl/tube) and freeze (-20 °C). The peptide can be stored for several months.
      2. Final stocks should be between 1 μM and 50 μM for experimental convenience.
      3. To minimize freeze-thaw cycles, a thawed aliquot can be divided into smaller single-use volumes (e.g., 10 μl) and re-frozen.

Recipes

Notes:

  1. Buffers are made in autoclaved DI water unless otherwise stated.
  2. The pH of Tris buffers changes with temperature.
  3. The pH of urea-containing buffers (Buffer B, Buffer C, and Buffer E) should be checked and adjusted immediately prior to use.
  4. The pH of DPBS is 7.0.

  1. Buffer B (Ni-NTA peptide purification)
    8 M Urea
    100 mM NaH2PO4
    10 mM Tris-Cl pH 8.0
  2. Buffer C (Ni-NTA peptide purification)
    8 M Urea
    100 mM NaH2PO4
    10 mM Tris-Cl pH 6.5
  3. Buffer E (Ni-NTA peptide purification)
    8 M Urea
    100 mM NaH2PO4
    10 mM Tris-Cl pH 4.5
  4. MES running buffer
    50 mM MES
    50 mM Tris pH 7.3
    1 mM EDTA
    0.1% (w/v) SDS
  5. TCEP/SDS Loading Dye (5x)
    300 mM Tris pH 6.8
    50 mM TCEP
    10% (w/v) SDS
    65% (v/v) glycerol
    0.025% (v/v) Ponceau Red
  6. Coomassie Stain
    45% (v/v) methanol
    0.3% (w/v) Coomassie Brilliant Blue R-250
    10% v/v acetic acid
  7. Destain Solution
    20% (v/v) methanol
    10% (v/v) acetic acid
  8. Tris-Buffered Saline (TBS)
    20 mM Tris pH 7.4
    150 mM NaCl 
  9. TBS Urea
    20 mM Tris pH 7.4
    150 mM NaCl
    2 M Urea 
  10. 0.5 M TCEP
    Dissolve the TCEP and then adjust the pH to 7 by the addition of 10 M NaOH
    Note: Single-use aliquots of TCEP can be stored at -20 °C.
  11. TBS Urea Binding Buffer
    20 mM Tris pH 8.0
    150 mM NaCl
    2 M Urea 
  12. Solid state-based labeling (SSL) Buffer, pH 7.5
    125 mM NaH2PO4
    200 mM NaCl
    1.25 mM EDTA
    4.6 M Ammonium Sulfate (75% saturated solution)
  13. 1 M DTT
    Dissolve the DTT in autoclaved DI water
    Note: Single-use aliquots can be prepared and stored at -20 °C.
  14. TBS Urea Imidazole
    20 mM Tris pH 7.4
    150 mM NaCl
    2 M Urea
    500 mM imidazole
  15. Biotin Buffer
    2 mM biotin in DPBS 
  16. Regeneration buffer
    0.1 M glycine
    pH 2.8

Acknowledgments

KEB is grateful for support from the OHSU School of Medicine and the National Institutes of Health (R01 GM122854). JKD was partially funded by the Portland Chapter of Achievement Rewards for College Scientists (ARCS). The protocols described herein were originally described in two prior publications (Zane et al., 2017; Doh et al., 2018). We are grateful to our colleagues at OHSU, particularly Drs. Hannah Zane and Jonathan White, for their contributions to the development of the VIP tags.

Competing interests

The authors declare no financial or non-financial competing interests. A patent application is pending on the VIP technology (PCT/US17/60609).

References

  1. Baker, M. (2015). Reproducibility crisis: Blame it on the antibodies. Nature 521(7552): 274-276.
  2. Berglund, L., Bjorling, E., Oksvold, P., Fagerberg, L., Asplund, A., Szigyarto, C. A., Persson, A., Ottosson, J., Wernerus, H., Nilsson, P., Lundberg, E., Sivertsson, A., Navani, S., Wester, K., Kampf, C., Hober, S., Ponten, F. and Uhlen, M. (2008). A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol Cell Proteomics 7(10): 2019-2027.
  3. Bordeaux, J., Welsh, A., Agarwal, S., Killiam, E., Baquero, M., Hanna, J., Anagnostou, V. and Rimm, D. (2010). Antibody validation. Biotechniques 48(3): 197-209.
  4. Bradbury, A. and Pluckthun, A.(2015). Reproducibility: Standardize antibodies used in research. Nature 518(7537):27-29.
  5. Cranfill, P. J., Sell, B. R., Baird, M. A., Allen, J. R., Lavagnino, Z., de Gruiter, H. M., Kremers, G. J., Davidson, M. W., Ustione, A. and Piston, D. W. (2016). Quantitative assessment of fluorescent proteins. Nat Methods 13(7): 557-562.
  6. Doh, J. K., Enns, C. A. and Beatty, K. E. (2019a). Implementing VIPER for imaging cellular proteins by fluorescence microscopy. Bio-protocol 9(21): e3413.
  7. Doh, J. K., Chang, Y. H., Enns, C. A., Lopes, C. S. and Beatty, K. E. (2019b). Imaging VIPER-labeled cellular proteins by correlative light and electron microscopy. Bio-protocol 9(21): e3414.
  8. Doh, J. K., White, J. D., Zane, H. K., Chang, Y. H., López, C. S., Enns, C. A. and Beatty, K. E. (2018). VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy. Proc Natl Acad Sci U S A 115(51): 12961-12966.
  9. Ellisman, M. H., Deerinck, T. J., Shu, X. and Sosinsky, G. E. (2012) Chapter 8 - Picking faces out of a crowd: Genetic labels for identification of proteins in correlated light and electron microscopy imaging. In Methods in cell biology. Academic Press 111: 139-155.
  10. Griffiths, G. and Hoppeler, H. (1986). Quantitation in immunocytochemistry: correlation of immunogold labeling to absolute number of membrane antigens. J Histochem Cytochem 34(11): 1389-1398.
  11. Griffiths, G. and Lucocq, J. M. (2014). Antibodies for immunolabeling by light and electron microscopy: not for the faint hearted. Histochem Cell Biol 142(4): 347-360.
  12. Hermanson, G. T. (2013). Bioconjugate Techniques. 3rd ed. Academic Press. ISBN: 978-0-12-382239-0.
  13. Hochuli, E., Dobeli, H. and Schacher, A. (1987). New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411: 177-184.
  14. Kim, Y., Ho, S. O., Gassman, N. R., Korlann, Y., Landorf, E. V., Collart, F. R. and Weiss, S. (2008). Efficient site-specific labeling of proteins via cysteines. Bioconjug Chem 19(3): 786-791.
  15. Liu, Z., Lavis, L. D. and Betzig, E. (2015). Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell 58(4): 644-659.
  16. Moll, J. R., Ruvinov, S. B., Pastan, I. and Vinson, C. (2001). Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M. Protein Sci 10(3): 649-655.
  17. Morimoto-Tomita, M., Uchimura, K. and Rosen, S. D. (2003). Novel extracellular sulfatases: Potential roles in cancer. Trends Glycosci Glycotechnol 15(83):159-164.
  18. Pierce® Monomeric Avidin Agarose. Thermo Scientific Inc., 2011. (Accessed 12-Oct, 2019, at https://www.thermofisher.com/order/catalog/product/20228.)
  19. Reinke, A. W., Grant, R. A. and Keating, A. E. (2010). A synthetic coiled-coil interactome provides heterospecific modules for molecular engineering. J Am Chem Soc 132(17): 6025-6031.
  20. Rodriguez, E. A., Campbell, R. E., Lin, J. Y., Lin, M. Z., Miyawaki, A., Palmer, A. E., Shu, X., Zhang, J. and Tsien, R. Y. (2017). The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem Sci 42(2): 111-129.
  21. Schnell, U., Dijk, F., Sjollema, K. A. and Giepmans, B. N. (2012). Immunolabeling artifacts and the need for live-cell imaging. Nat Methods 9(2): 152-158.
  22. TECH TIP #31 Calculate dye:protein (F/P) molar ratios. Thermo Scientific Inc., 2011. (Accessed 12-Oct, 2019, at https://assets.thermofisher.com/TFS-Assets/LSG/brochures/TR0031-Calc-FP-ratios.pdf.)
  23. Tsien, R. Y. (1998). The green fluorescent protein. Annu Rev Biochem 67: 509-544.
  24. Wingfield, P.(2001). Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci Appendix 3:Appendix 3F.
  25. Zane, H. K., Doh, J. K., Enns, C. A. and Beatty, K. E. (2017). Versatile Interacting Peptide (VIP) tags for labeling proteins with bright chemical reporters. Chembiochem 18(5): 470-474.

简介

多功能相互作用肽(VIP)标签是一类新的遗传编码标签,旨在通过荧光和电子显微镜对细胞蛋白进行成像。在2018年,我们报道了VIPER标签(Doh et al。,2018),它包含两个元素:基因编码的肽标签( ie ,CoilE)和一个探针肽段( ie ,CoilR)。这两种肽通过形成特异性的高亲和力异二聚体而与目的蛋白形成对比。探针肽经设计具有单个半胱氨酸残基,可通过硫醇-马来酰亚胺化学进行位点特异性修饰。此功能可用于将各种生物物理报告分子连接到肽上,包括用于荧光显微镜的明亮荧光团或用于电子显微镜的电子致密纳米颗粒。在此生物协议中,我们描述了表达和纯化重组CoilR的方法。此外,我们描述了用于制作荧光或生物素化探针肽以标记CoilE标签的细胞蛋白的协议。该协议由另外两个概述了VIPER使用的生物协议补充(Doh等,2019a和2019b)。
【背景】荧光显微镜(FM),电子显微镜(EM)以及相关光和EM(CLEM)使人们能够研究介导正常和与疾病相关的细胞功能的多蛋白复合物和大分子相互作用。但是,由于缺乏将FM-,EM-和CLEM兼容的报告分子化学连接至靶蛋白的方法的限制,因此多尺度显微镜受到了限制。另外,很少有用于蛋白质标记的方法可以促进成像系统之间的切换。结果,大多数多尺度成像研究都通过免疫标记获得了蛋白质特异性对比。然而,免疫标记存在已知的缺点。较大的抗体会降低定位精度,标记方案会破坏细胞的超结构(Schnell et al。,2012)。除非免疫标记有效,否则稀有蛋白和稀有相互作用可能无法检测(Griffiths和Hoppeler,1986; Schnell et al。,2012; Griffiths和Lucocq,2014)。许多抗体具有较差的靶标特异性和交叉反应性(Berglund等,2008; Bordeaux等,2010; Baker,2015; Bradbury和Pluckthun,2015),这可能会导致误导性的观察。
在多尺度显微镜技术方面取得进展的主要障碍是缺乏用于标记蛋白质的遗传标签。大多数标签是针对FM开发的(Liu et al。,2015),最常用的标签是荧光蛋白[ eg ,GFP](Tsien,1998; Cranfill <等人,,2016;罗德里格斯等人,,2017)。相比之下,EM或CLEM的遗传标签很少(Ellisman et al。,2012)。我们认为这是为多尺度显微镜创建一类新的遗传编码肽标签的机会(Zane et al。,2017; Doh et al。,2018)。我们将该技术命名为多功能相互作用肽(VIP)标签(图1)。 VIP标签由基因编码的肽标签和报道分子偶联的肽(“探针肽”)之间的异二聚体卷曲螺旋组成。结合是由两个线圈之间的疏水界面和链间盐桥驱动的。最初我们报道了VIP Y / Z,它用于用荧光团和Qdot标记细胞蛋白(Zane et al。,2017)。该对由异二聚体CoilY-CoilZ对组成,据报道其解离常数(K D )小于15 nM(Reinke等,2010)。 CoilY或CoilZ都可以用作基因编码标签。在2018年,我们报道了VIPER标签,该标签可对蛋白进行高亲和力标记,以通过FM和CLEM进行成像(Doh et al。,2018)。 CoilE标签和CoilR探针肽之间形成VIPER的结合是特异性的,几乎是不可逆的[K D 〜10 -11 M](Moll等,,2001)。
“”
图1.多功能相互作用肽(VIP)标签是通过FM,EM或CLEM成像蛋白质的新技术。转铁蛋白受体1(TfR1)的VIPER标记是由CoilE标签和荧光CoilR探针肽。荧光显微照片:VIPER标签的TfR1标记有CoilR-Cy5(品红色),并与荧光转铁蛋白(Tf-AF488;绿色)共定位在转染的CHO TRVb细胞的细胞表面(放大63倍)。品红色-绿色信号重叠显示为白色,原子核为蓝色。

“”
图2. VIP标签是用于多尺度显微镜的通用技术。标记目标蛋白后,可以使用为特定应用选择的多种探针肽对其进行标记。

对于VIP标签,通用性是由可定制的探针肽赋予的。将CoilE标签引入目标蛋白后,可以用连接到CoilR的许多不同报告基因之一标记该蛋白(图2)。例如,我们对跨膜受体TfR1-CoilE和CoilR-BODIPY,CoilR-Cy5(参见图1)和CoilR-生物素进行了成像(Doh et al。,2018)。换句话说,探针肽可以被定制用于不同的研究或成像系统而无需改变遗传标签。这是可能的,因为CoilR编码单个半胱氨酸残基,可通过硫醇-马来酰亚胺化学进行位点特异性修饰。 CoilR探针肽可以与多种探针生物偶联,包括荧光团,小分子(例如, ,生物素)或纳米颗粒。许多公司出售硫醇反应性探针,这使得这种偶联反应对于没有合成化学专业知识的实验室来说是可行的。有关生物偶联反应的更多信息,我们建议阅读Hermanson的 Bioconjugate Techniques (Hermanson,2013年)。
在此生物协议中,我们提供了制备CoilR探针肽的方法,该肽可用于VIPER标记细胞蛋白以通过FM或EM成像。表1中提供了CoilR肽和CoilE标签序列。如先前工作所述(Doh et al。,2018),我们使用基因组装PCR使探针肽在em> E。大肠杆菌。肽表达的方法在步骤A中进行了描述。如Vinson和同事最初所描述的那样,将CoilR设计为通过优化的α-螺旋线圈与CoilE相互作用(Moll等,2001)。 。我们在CoilR的C末端加入了一个六组氨酸标签,用于通过固定金属亲和色谱法(IMAC)进行纯化(Hochuli等人,1987);这在程序B中进行了描述。

表1. CoilR和CoilE的顺序
“”
§ 斜体:链接器序列; 粗体:肽线圈; C :半胱氨酸(结合位点)。
‡臀部位置:残基 a 和 d 疏水界面, e 和 g 处的残基形成链间盐桥。
程序C和D描述了用小分子报道分子标记CoilR的硫醇-马来酰亚胺反应。在程序C中,我们描述了先前工作中用于生成探针肽的方法(Doh et al。,2018)。在程序D中,我们采用了Weiss和他的同事描述的基于固态标记肽的方法(Kim et al。,2008)。最后,我们提供了纯化荧光团标记的(程序E)或生物素化的(程序F)探针肽的方法。该生物协议随附两篇附带的文章,其中包括通过FM(Doh等人,2019a)和CLEM(Doh等人,2019)对VIPER标记的细胞蛋白进行成像的详细方法。 / em>,2019b)(图3)。

“”
图3.用于实施VIPER的决策树。过程由出现的出版物以颜色进行编码。本出版物中的方法为颜色编码的紫色。 Doh等人,2019a中的方法为黄色,而Doh等人,2019b中的方法为橙色。出版物1:这篇文章;出版物2:Doh等人,2019a;出版物3:Doh等人,2019b)。

关键字:肽, 生物偶联, 遗传标记, 显微镜, 化学生物学, 荧光

材料和试剂

注意:“ *”表示对实验成功至关重要的品牌。
材料

  1. 通用移液器吸头(USA Scientific TipOne TM ,目录号:1112-1770、1163-1730和1121-3812)
  2. 1.5 ml微量离心管(Thermo Scientific,目录号:02-682-002)
  3. 无菌血清移液器(Thermo Scientific,目录号:13-678-11D + E)
  4. 无菌14 ml培养管(Corning,Falcon TM ,货号:352059)
  5. 一次性聚苯乙烯分光光度计比色杯(Thermo Scientific,目录号:14-955-127)
  6. 锥形50毫升试管(Thermo Scientific,Nunc TM ,货号:12-565-270)
  7. 色谱柱(Bio-Rad,Econo-Pac TM ,目录号:7321010)
  8. 环架(Fisher,目录号:11-474-207)
  9. 可调式环形支架夹具(United Scientific Supplies,目录号:CLHD03)
  10. 截留分子量(MWCO)3 kDa过滤器(Sigma-Aldrich,Amicon Ultra TM ,目录号:UFC900324)
  11. 石英10.00毫米比色皿(Hellma Analytics,超微池,目录号105-250-15-40)
  12. 移液器(例如,Rainin Pipet-Lite TM XLS,目录号:17014407、17014411、17014412和17014413)
  13. 2 L玻璃锥形烧瓶(Corning,Pyrex TM ,货号:49802L)

试剂
  1. 抗生物素HRP抗体(杰克逊免疫研究,目录号:200-032-211)
  2. 链霉亲和素-HRP(Thermo Scientific,目录号:ENN100)
  3. pET28b(+)_ CoilR [由MSU从OHSU提供,或由公开发行(Doh 等人,2018)]
  4. BL21(DE3) E。大肠杆菌(新英格兰生物实验室,目录号:C2527I)
  5. 甘氨酸(Thermo Scientific,Fisher BioReagents TM ,货号:BP381-500)
  6. SOC增长媒体(新英格兰生物实验室,目录号:C2527I)
  7. Miller Luria-Bertani(LB)琼脂(BD Difco TM ,目录号:244520)
  8. Miller LB汤汁(BD Difco TM ,目录号:BD 244610)
  9. 2X YT(Thermo Scientific,Fisher BioReagents TM ,目录号:BP9743500)
  10. 硫酸卡那霉素(Thermo Scientific,Fisher Chemical,目录号:BP906-5)
  11. IPTG(GoldBio,目录号:I2481C5)
  12. * Ni-NTA琼脂糖(Qiagen,目录号:30230)
  13. * Pierce单体抗生物素蛋白琼脂糖(Thermo Scientific Pierce TM ,目录号:20228)
  14. 磷酸二氢钠无水(Thermo Scientific,Fisher BioReagents TM ,货号:BP329-500)
  15. 尿素(Thermo Scientific,Fisher BioReagents TM ,货号:U15 3)
  16. Tris Base(Thermo Scientific,Fisher BioReagents TM ,目录号:BP152 5)
  17. Tris HCl(Thermo Scientific,Fisher BioReagents TM ,目录号:BP153 1)
  18. NaCl(Thermo Scientific,Fisher BioReagents TM ,货号:BP358-1)
  19. 甘油(Thermo Scientific,Fisher BioReagents TM ,目录号:BP229-1)
  20. 咪唑(ACROS Organics,目录号:AC39674-1000)
  21. 考马斯亮蓝R-250(Thermo Scientific,目录号:20278)
  22. 甲醇(Thermo Scientific,Fisher Chemical,目录号:A412)
  23. 丙酮(Thermo Scientific,Fisher Chemical,目录号:A18)
  24. 氮气
  25. 硫酸铵(EMD Millipore,目录号:AX1385-1)
  26. TCEP-HCl(GoldBio,目录号:TCEP10)
  27. 二硫苏糖醇(DTT)(Thermo Scientific,Molecular Probes TM ,货号:D1532)
  28. TC级DMSO(Sigma-Aldrich,目录号:D2650-5X10ML)
  29. *磺基-Cy5-马来酰亚胺(Lumiprobe,目录号:23380)
  30. *生物素-PEG2-马来酰亚胺(Thermo Scientific,目录号:21901BID)
  31. D-生物素(方舟制药,目录号:AK-44010)
  32. Pierce BCA化验试剂盒(Thermo Fisher Scientific,目录号:23227)
  33. 12%Bis-Tris聚丙烯酰胺蛋白质凝胶(Bio-Rad Criterion TM XT,目录号:3450119)
  34. MES(Thermo Scientific,Fisher BioReagents TM ,目录号:BP300-100)
  35. NaH 2 PO 4 (Sigma-Aldrich,目录号:S3139-250G)
  36. 丽春红(Thermo Scientific,Fisher BioReagents TM ,货号:BP103-10)
  37. NaOH(Thermo Scientific,Fisher BioReagents TM ,货号:BP359-500)
  38. 缓冲液B(Ni-NTA肽纯化)(请参见食谱)
  39. 缓冲液C(Ni-NTA肽纯化)(请参阅食谱)
  40. 缓冲液E(Ni-NTA肽纯化)(请参阅食谱)
  41. MES运行缓冲区(请参阅食谱)
  42. TCEP / SDS上染染料(5x)(请参阅配方)
  43. 考马斯染色(见食谱)
  44. 脱色溶液(请参阅食谱)
  45. Tris缓冲盐水(TBS)(请参阅食谱)
  46. TBS尿素(请参阅食谱)
  47. 0.5 M TCEP(请参阅食谱)
  48. TBS尿素结合缓冲液(请参阅配方)
  49. 固态标记(SSL)缓冲液,pH 7.5(请参见食谱)
  50. 1 M DTT(请参阅食谱)
  51. TBS尿素咪唑(请参阅食谱)
  52. 生物素缓冲液(请参阅食谱)
  53. 再生缓冲液(请参见配方)

设备

  1. 电子移液器(Eppendorf Easypet TM ,目录号:4430000018)
  2. -20°C冷冻机(Thermo Scientific,Revco TM ,货号:13990206)
  3. 孵化器和振荡器(New Brunswick Excella TM E24,目录号:M1352-0010)
  4. 分光光度计(Eppendorf,Biophotometer Plus,目录号:6132)
  5. 烤肉店(Thermo Scientific,目录号:400110Q)
  6. Sonifier(Branson Ultrasonics TM ,目录号:101063198R)
  7. Sonifier 1/8英寸微尖端(Branson Ultrasonics TM ,目录号:22-309796)
  8. 冷冻离心机(Thermo Scientific,Sorvall Legend XTR离心机,目录号:75211731)
  9. 微量离心机(Eppendorf,目录号:022620304)
  10. 加热块(Fisher,Isotemp TM ,货号:88-860-022)
  11. 电泳池(Bio-Rad Criterion TM ,目录号:165-6001)
  12. 电源(Bio-Rad PowerPac TM HC,目录号:1645052)
  13. 酶标仪(Tecan Infinite M200 Pro,目录号:30050303)
  14. (可选)荧光和蛋白质印迹成像仪(即,GE Healthcare Amersham TM Typhoon 5多模式扫描仪,目录号:29187191或Protein Simple,FluorChem Q)

程序

  1. 重组CoilR的表达
    CoilR是通过在 E中重组表达而产生的。大肠杆菌。 CoilR的生长和纯化遵循在诱导表达下制备和纯化组氨酸标签肽的标准方案。有关详细的背景,协议和疑难解答,建议您参考 Qiaexpressionist 手册(Qiagen)(Morimoto-Tomita 等人,,2003年)。
    1. 获取或生成编码CoilR肽的质粒( i.e。,pET28b(+)_ CoilR)(Doh et al。,2018)。表1提供了由pET28b(+)_ CoilR表达的CoilR的氨基酸序列。
      注意:pET28b(+)_ CoilR质粒编码卡那霉素抗性。
    2. 将质粒转化为 E。请按照NEB关于产品C2527的说明进行大肠杆菌BL21(DE3)感受态细胞的处理。
      1. 将细胞置于LB /琼脂/卡那霉素(50μg/ ml)上,并在37°C下生长过夜。
      2. 挑选单个菌落,并在无菌的14 ml培养管中,在补充有卡那霉素(50μg/ ml)的LB中接种5 ml起始培养物(每5 ml培养物一个菌落)。
      3. 在振荡培养箱(225 rpm,37°C)中过夜生长。在观察到生长变化的情况下,我们会生长几种发酵剂文化(例如,一种文化生长缓慢)。
    3. 使用一夜培养液在补充有卡那霉素(50μg/ ml)的2 L锥形瓶中接种(2.5 ml,1:200稀释)500 ml 2X YT无菌培养基。在225 rpm和37°C下生长,直到OD 600 达到0.8至1.0。
      1. 通过在分光光度计上的一次性比色皿中测量培养物的OD 600 来监控生长。
      2. 培养物达到此OD 600 大约需要2-4小时。
      3. 诱导前,取1 ml未诱导培养物样品,用于通过SDS-PAGE进行肽表达分析。
        1. 对于每个样品:在微量离心管中沉淀1 ml细菌培养物(10,000 x g ,2分钟)。
        2. 将沉淀重悬在缓冲液B中(配方1)。通过向颗粒中加入缓冲液B来标准化样品。使用等式:volume = OD 600 x 100μl缓冲液B。
        3. 在-20°C下冷冻。
    4. 将培养箱/振荡器的温度降至25°C,并通过添加0.1 mM IPTG诱导2-4小时的肽表达。
      注意:如果在37°C下诱导,肽会降解,从而降低总产量。
      1. 在诱导过程中每小时采集1 ml样品,监控表达。按照步骤A3c中的描述进行归一化。
    5. 通过在冷冻离心机(5,000 x g ,15分钟,4°C)中离心收集细胞。
      1. 丢弃上清液。
      2. 将沉淀的细菌转移到去皮重的50 ml锥形管中,以获得湿沉淀的重量。
      3. 储存沉淀(-20°C)。沉淀可以在-20°C下保存几个月。
      注意:如果需要,可以在肽表达的同一天进行细胞裂解(步骤B4)。但是,我们通常先将沉淀物冷冻,然后再进行第二天的纯化。
    6. 通过SDS-PAGE分析肽表达。有关CoilR表达和纯化的代表性SDS-PAGE分析,请参见图4。
      1. 从诱导过程中收集的时间点解冻蛋白质样品。在缓冲液B存在下冻结和解冻将部分溶解细胞。
        注意:如果裂解不完全,则将样品冻融数次以破坏开孔细胞。
      2. 颗粒状不溶性碎片(10分钟,10,000 x g )。
      3. 将澄清的裂解液转移到新的微量离心管中。
      4. 添加5x TCEP-SDS加载染料(配方5)。煮沸样品5分钟,离心沉淀,然后将10-15μl样品/孔上样至蛋白质凝胶。
      5. 通过SDS-PAGE分析肽表达。
        1. 我们建议在恒定电压(180 V)的MES运行缓冲液(配方4)中运行的BioRad Criterion Bis-Tris凝胶(12%; 26孔)上进行分析。
        2. 运行约35分钟,直至负载染料到达凝胶底部。
        3. 电泳后,在成像前用考马斯蓝染色(配方6)对蛋白凝胶染色,然后脱色(去污溶液,配方7)。
        4. CoilR将作为单体(7.5 kDa)和二聚体(15 kDa)在凝胶上迁移。


      图4. E中的肽表达。大肠杆菌和CoilR的变性纯化。从未诱导(UI)和诱导(2 h和4 h)细胞中收集样品,裂解并通过SDS-PAGE分离。在变性条件下,通过IMAC( i.e。,在Ni-NTA树脂上)从澄清的裂解物中纯化CoilR。 CL =澄清的裂解物; FT =流通量(未结合分数)。洗涤1:用10 mM咪唑缓冲液B(pH 8)。洗涤2-4:用10 mM咪唑缓冲液C(pH 6.8)。洗脱1-3:缓冲液E(pH 4.3)。 CoilR肽(MW = 7502.35 Da)在〜6 kDa处以单体形式迁移,在〜14 kDa处以表观二聚体形式迁移(灰色箭头)。

  2. 通过IMAC纯化重组CoilR
    1. 在肽纯化之前,测量并调节所有纯化缓冲液的pH。
      1. 缓冲液B:8 M尿素,100 mM NaH 2 PO 4 ,10 mM Tris-Cl,pH 8(配方1)。
      2. 缓冲液C:8 M尿素,100 mM NaH 2 PO 4 ,10 mM Tris-Cl,pH 6.5(配方2)。
      3. 缓冲液E:8 M尿素,100 mM NaH 2 PO 4 ,10 mM Tris-Cl,pH 4.5(配方3)。
    注意:除非另有说明,否则所有步骤均应在冰上并使用预先冷却的缓冲液。
    1. 解冻 E。冰上的大肠杆菌沉淀(来自步骤A5)。
    2. 将沉淀重悬在缓冲液B中。每克湿重使用5 ml缓冲液B。
    3. 通过在冰上超声处理来裂解。
      1. 我们使用配有1/8英寸Branson microtip的Branson超声仪来裂解细菌。我们以40%的占空比裂解细胞,输出:4.将样品脉冲处理30 s,然后在冰上静置1分钟,进行8个循环。
      2. 避免样品起泡,否则会造成蛋白质损失。
      3. 或者,可以通过其他方法(例如,冻融或French press)裂解细胞。
    4. 通过在冷冻离心机(10,000 x g ,30分钟,4°C)中离心来澄清裂解物。
      注意:保留澄清的裂解液样品,以通过SDS-PAGE分析。
    5. 将澄清的裂解物与Ni-NTA琼脂糖树脂(Qiagen)在烤肉架上于4°C孵育1小时。
      1. 缓冲液B应补充10-20 mM咪唑,以减少非特异性蛋白质与树脂的结合。
      2. 每1克颗粒(湿重)使用1毫升树脂。
      3. 或者,在4°C下过夜。
      注意:如果在流通和初始洗涤过程中观察到CoilR肽,则表明树脂超载。在粘合步骤中使用更多的树脂。
    6. 将裂解物-树脂混合物上样到干净的多孔色谱柱上。
    7. 收集流通。保存样品以通过SDS-PAGE分析。
    8. 用5倍柱体积(CV)的缓冲液B洗涤树脂。
    9. 用10-50 CV的缓冲液C洗涤树脂。
      1. CoilR肽将在约pH 6时洗脱。洗涤缓冲液应在pH 6.3和pH 7.0之间。
      2. 保存每个洗涤步骤中的样品以通过SDS-PAGE分析。
      3. 洗涤直至没有其他杂质洗脱。
    10. 用缓冲液E洗脱CoilR肽。以5份2 CV洗脱。
      1. 大部分肽会在前3个洗脱液中洗脱。
      2. 如果检测到洗脱不完全,则以其他体积洗脱(请参见图4)。
    11. 通过SDS-PAGE监控纯化。从澄清的裂解液,流通液,洗涤液和洗脱液中分析样品。
      1. 凝胶运行条件与步骤A6d-A6e中的条件相同。
    12. 根据步骤B12的结果合并含有纯化肽的馏分。
    13. 浓缩并使用3 kDa MWCO过滤器将纯化的CoilR交换到所需的缓冲液中。
      1. 我们建议使用MWCO过滤器更换为TBS尿素(配方9)。该缓冲液与硫醇-马来酰亚胺偶联反应兼容(程序C)。
      2. 将肽浓度保持在0.5 mg / ml和2 mg / ml之间,以避免溶解性问题。如果在肽溶液中观察到白色沉淀,则肽浓度过高。
    14. 使用Pierce BCA分析试剂盒(Thermo Fisher Scientific)定量肽浓度。
      1. 也可以通过测量280 nm处的吸光度来估算蛋白质浓度。但是,CoilR的消光系数低(2,980 L·mol -1 ·cm -1 )。因此,如果用BCA分析法测定浓度,则浓度会更准确。
      2. 包括重复和稀释以获得准确的浓度。
    15. 将5-10%甘油添加到浓缩肽中,等分试样(200μl/管),然后冷冻(-20°C)。 CoilR肽可以冷冻保存几个月。

  3. 溶液中巯基-马来酰亚胺共轭产生CoilR探针肽
    VIPER标记在表达CoilE标签蛋白的活细胞和固定细胞中具有特异性和高效性。但是,标记的质量与探针肽的质量直接相关。这是因为未标记的CoilR和标记的CoilR都会与带有CoilE标签的细胞蛋白二聚。我们建议使用> 50%标记有报告分子化学物质。
    &nbsp;我们发现硫醇-马来酰亚胺生物缀合反应的效率是可变的。因此,我们包括了两种修改CoilR的方法:过程C和过程D。我们都成功地使用了两种标记报告基因的CoilR,首选的方案取决于研究者。方法C描述了溶液中的常规硫醇-马来酰亚胺共轭反应;这是我们2018年出版物中描述的用于生成探针肽的方法(Doh et al。,2018)。该方法可用于连接荧光探针,例如磺基-花菁5(Cy5)-马来酰亚胺,或连接生物素化的CoilR。
    1. 准备缓冲液,TCEP和反应性马来酰亚胺的储备溶液。如果已经准备好这些库存,请继续执行步骤C2。
      1. 我们建议在TBS尿素中贴标签(第9条)。该反应应在pH 7至pH 7.5之间的无硫醇缓冲液中进行。
      2. 使用前,请给缓冲液脱气。这可以通过使氮气流通过缓冲液鼓泡或通过真空脱气来完成。
      3. 准备0.5 M TCEP(配方10)。
    2. 在无水DMSO中以20-100 mg / ml制备马来酰亚胺探针的浓缩原液。对于Cy5-马来酰亚胺或其他荧光团,请保护溶液避光。
      1. 为了限制反应中DMSO的量,更浓缩的原料是优选的。
      2. 库存可以在-20°C下保存。
      3. 马来酰亚胺会在水中水解,因此建议在DMSO中储存。
    3. 在冰上解冻纯化的CoilR肽。浓缩的肽原液应放在TBS尿素中,并用氮气脱气。
      1. 如果肽被浓缩,反应将进行得更好。我们建议使用2 mg / ml(〜270 µM)的储备液。
      2. 典型的标记反应将包括50至200纳摩尔的CoilR肽。
      3. 如果该肽不在适当的缓冲液中,请在此时使用3 kDa MWCO过滤器转移至其他缓冲液中,然后脱气,然后继续进行步骤C4。
    4. 通过添加10倍摩尔过量的TCEP来减少肽段。在50°C下孵育30分钟。
    5. 通过加入至少20倍摩尔过量的马来酰亚胺探针来引发偶联反应。拌匀在烤肉店中于室温或4°C下孵育2小时。
      1. 对于荧光团标记,请保护反应避光。
    6. 标记后,添加TBS尿素结合缓冲液(配方11)至总体积为15 ml。
      1. 保存粗反应混合物的样品,以通过SDS-PAGE分析。
    7. 浓缩液和缓冲液在3 kDa MWCO过滤器上交换粗反应物,以除去未反应的探针。将缓冲液更换为与Ni-NTA纯化兼容的TBS尿素结合缓冲液。保存样品以通过SDS-PAGE分析。
      1. 对于CoilR-荧光团,请继续进行缓冲液更换,直到滤液变为无色或停止颜色变化,随后再进行缓冲液更换。然后继续执行过程E。
      2. 对于生物素化的CoilR,40 ml清洗液足以去除大部分游离的生物素部分。然后继续执行程序F。

  4. 使用基于固态的标记(SSL)通过硫醇-马来酰亚胺化学生成CoilR探针肽
    2008年,Weiss及其同事描述了一种使用硫醇-马来酰亚胺化学修饰蛋白质的新方法(Kim et al。,2008)。在这项工作中,在荧光团偶联之前,先用硫酸铵沉淀蛋白质,然后用DTT还原。他们将这种方法命名为基于固态的标签(SSL)。这种方法的优点是操作简便,效率高(标记为70-90%)且硫醇特异性(Kim et al。,2008)。我们目前同时使用SSL和基于解决方案的标记来生成探针肽。程序D改编自Weiss和同事发表的方法(Kim et al。,2008)。
    1. 准备SSL缓冲液(配方12)和还原剂(1 M DTT [配方13]和0.5 M TCEP)。如果已经准备好这些库存,请继续执行步骤D2。
    2. 用冰冷的SSL缓冲液洗涤还原的肽浆,以去除DTT。
      1. 通过离心(4分钟,14,000 x g ,4°C)制浆。丢弃上清液。
      2. 加入1 ml SSL缓冲液,并将样品倒置几次。
      3. 重复步骤D6a和D6b 3-5次,以清除所有多余的DTT。
      4. 最后一个离心步骤后,将沉淀重悬于100 µl SSL缓冲液中。
      注意:任何残留的DTT都会与马来酰亚胺探针发生反应,因此多次洗涤肽沉淀至关重要。
    3. 在固态还原肽上进行巯基-马来酰亚胺偶联。
      1. 向还原的肽中添加10至30倍摩尔过量的探针。颠倒管数次进行混合。
        1. 在无水DMSO中使用浓缩的马来酰亚胺探针(例如,Cy5-马来酰亚胺)(20-100 mg / ml)。
      2. 在烤肉架上于4°C混合反应15分钟。
      3. 加入5至10倍摩尔过量的TCEP。混合并继续在烤肉架上于4°C孵育45分钟。
        1. 对于荧光标记,请保护管避光。
        2. 反应中马来酰亚胺的量应高于TCEP的量,因为马来酰亚胺探针可能会与TCEP发生副反应(Kim et al。,2008)。
        3. 反应可以孵育过夜。
    4. 标记后,我们建议用SSL Buffer洗涤反应混合物以除去过量的马来酰亚胺。
      1. 通过离心(4分钟,14,000 x g ,4°C)沉淀反应。丢弃上清液。
      2. 重悬于SSL缓冲液(1 ml)中。
      3. 离心沉淀(4分钟,14,000 x g ,4°C)。丢弃上清液。
    5. 将步骤D8中的沉淀重悬在缓冲液B中。
      注意:
      1. 高浓度的EDTA将从程序E中使用的Ni-NTA琼脂糖中去除镍。添加足够的缓冲液B以确保EDTA的最终浓度小于1 mM。
      2. 保存粗反应混合物的样品,以进行SDS-PAGE分析。
    6. 按照步骤E(对于荧光肽)或步骤F(对于生物素化肽)进行纯化。

  5. CoilR-荧光团探针肽的纯化
    该程序从荧光团标记的CoilR(即,CoilR-Cy5)中去除了过量的未反应的游离染料,同时还纯化了肽。本节还介绍了用于定量肽段荧光团标记的方法。
    注意:除非另有说明,否则应保护肽避光并保持在冰上。
    1. 在4°C下将标记的CoilR肽与Ni-NTA琼脂糖树脂结合1小时。
      1. 对于典型的标记反应(50-200 nmol CoilR),我们建议使用0.5 ml Ni-NTA树脂并在大体积(20-40 ml)的TBS尿素结合缓冲液中结合。
    2. 将裂解物-树脂混合物上样到干净的多孔色谱柱上。
    3. 收集流通液并保存样品以通过SDS-PAGE分析。
    4. 用20柱体积(CV)的TBS尿素结合缓冲液洗涤树脂。
      1. 继续洗涤直至级分无色。
      2. 保存洗涤液以通过SDS-PAGE分析。
    5. 可选步骤:用补充有20%乙醇的10 CV TBS尿素结合缓冲液洗涤树脂。添加乙醇可以帮助去除游离的荧光团。
    6. 用5-10 CV的TBS尿素咪唑洗脱CoilR肽(方案14)。
      1. Cy5标记的肽级分应为深蓝色。
      2. 继续洗脱直到馏分几乎无色,然后再进行下一步。
      3. 或者,在低pH的缓冲液(例如,缓冲液E)中洗脱。
    7. 按照步骤A6d-A6e,通过SDS-PAGE分析纯化。有关代表性分析,请参见图5。
      1. 分析粗反应,纯化样品(流通,洗涤,洗脱)和浓缩洗脱。
      2. 将凝胶在荧光扫描仪上成像,以检测标记的肽。图5A提供了使用Protein Simple成像系统获得的代表性2色扫描图。另外,我们建议使用适当的检测设置(即,Cy5:例如:635 nm,em:670/30 nm)在GE Amersham TM Typhoon多模扫描仪上成像。
      3. 荧光成像后,用考马斯染色对蛋白凝胶染色,脱色并成像以检测总蛋白。
    8. 使用3 kDa MWCO过滤器浓缩和缓冲液将含有标记肽的洗脱液交换到所选的存储缓冲液中。
      注意:我们建议将肽储存在TBS尿素中。
    9. 确定标记的程度(每摩尔蛋白质的荧光团摩尔数)。我们建议遵循Thermo Scientific发布的协议[技术提示#31:计算染料:蛋白质(F / P)摩尔比](参考文献22)。
      1. 通过测量最大荧光团吸光度(Abs max )的吸收并使用公布的消光系数(ε FL )(表2),确定溶液中的荧光团数量。
      2. 通过测量280 nm的吸光度确定溶液中肽的量。
        1. 荧光团也将在280 nm处吸收,因此必须使用校正因子(CF)(例如,对于Cy5 = 0.04,CF)。
        2. CoilR肽的消光系数(ε)为2,980 L·mol -1 ·cm -1 ( www.expasy.org )。
      3. 使用以下公式计算肽的摩尔浓度和标记程度:



      4. 我们提供以下建议:
        1. 吸光度读数仅在分光光度计的线性范围内(0.1到1.0之间)是准确的。
        2. 我们建议准备几种肽的稀释液并复制以获得更准确的结果。
        3. 我们在带有比色皿端口的Tecan Infinite M200 Pro上的石英比色皿中测量了吸光度。


          图5.通过SDS-PAGE分析CoilR探针肽。将CoilR标记为磺基花菁3(CoilR-Cy3; 60%标记),AlexaFluor-488(CoilR-AF488; 45%标记),或BODIPY-FL(CoilR-BDPY-FL:标记为40%)。在Ni-NTA树脂上纯化粗反应物(CR)以除去游离染料。通过SDS-PAGE解析样品,并且扫描凝胶的绿色(例如:488nm,em:525 / 50nm)和红色(例如:532nm,em:570 / 20nm)荧光(A)。随后用考马斯(B)对同一凝胶进行总蛋白染色。 CR =稀释的粗反应(柱前),FT =流通液(未结合的蛋白质/荧光团),W =洗涤液(TBS尿素结合缓冲液),E =洗脱液(TBS尿素咪唑)。包括未反应的CoilR肽(15和30 µg)作为参考,并且CoilR用灰色箭头指示。

          表2.用Cy5-maleimide量化CoilR标记的值‡

          ‡在Lumiprobe网站上提供的值: www.lumiprobe.com 。

    10. 将荧光团标记的肽存储在5-10%的甘油中。
      1. 分装(100μl/管)并冷冻(-20°C)。该肽可以保存(冷冻并避光)几个月。
      2. 为了实验方便,最终库存应在1-50μM之间。
      3. 为了最大程度地减少冻融循环,可以将已融化的等分试样分成较小的单次使用体积(例如, ,10μl)并重新冷冻。

  6. 生物素化探针肽(CoilR-生物素)的纯化
    此过程旨在纯化使用过程C或D生物素化的CoilR肽。有关基于亲和素的亲和色谱法和故障排除指南的概述,请参阅Pierce ®亲和力Avidin琼脂糖单体说明,在线可得。 (参考文献18)。从单体亲和素树脂纯化和洗脱后,假定CoilR-生物素肽已100%被生物素化。
    1. 准备缓冲液并平衡至室温。
      1. TBS:20 mM Tris,150 mM NaCl pH 7(配方8)。
      2. 生物素缓冲液:pH 7.4的DPBS中2 mM生物素(配方15)。
      3. 再生缓冲液:0.1 M甘氨酸,pH 2.8(配方16)。
    2. 将Pierce Monomeric亲和素琼脂糖添加到干净的多孔色谱柱中并沥干。
      1. 对于100 nmole的CoilR肽,请使用1 ml树脂。
    3. 阻止树脂上不可逆的生物素结合位点:
      1. 用5倍柱体积(CV)的TBS洗涤。
      2. 用5 CV的生物素缓冲液洗涤,以阻断任何不可逆的生物素结合位点。
      3. 用5 CV的再生缓冲液洗涤,以去除与树脂上可逆生物素结合位点结合的生物素。
      4. 用5 CV的TBS洗涤以重新平衡色谱柱。
      5. 塞住色谱柱以防止流动;现在就可以使用树脂了。
    4. 在TBS中将生物素化的肽样品稀释至大约5 ml。应用于列。
    5. 将样品与树脂在室温下孵育30分钟。
    6. 拔下色谱柱并收集流出物。保存流通液以及所有后续洗涤和洗脱步骤的样品,以通过SDS-PAGE分析。
    7. 用5 CV的TBS洗涤树脂两次。
    8. 在5 CV的生物素缓冲液中洗脱生物素化的蛋白质。收集1毫升馏分。
    9. 在5 CV的再生缓冲液中洗脱。收集1毫升馏分。包括此洗脱步骤,因为某些肽不会被过量的生物素洗脱。
    10. 再生树脂。用5 CV的再生缓冲液洗涤。收集并分析以确保该洗涤液不含生物素化肽。
    11. 通过SDS-PAGE分析所有馏分(请参阅步骤A6d-A6e)。
      注意:根据我们的经验,CoilR-生物素在生物素缓冲液和再生缓冲液中均被洗脱,而在生物素缓冲液中则洗脱更多。
    12. 通过蛋白质印迹法(使用您的首选方法)分析所有级分,以检测生物素化的蛋白质。例如,我们使用抗生物素HRP抗体(杰克逊免疫研究)或链霉亲和素-HRP(Thermo Scientific)检测生物素化蛋白。
    13. 根据步骤F11-F12中的分析合并含有生物素化肽的馏分。
    14. 使用3 kDa MWCO过滤器浓缩生物素化肽并将其交换为所需的缓冲液。
      1. 我们建议换成TBS尿素。
      2. 保持肽浓度在0.5 mg / ml和2 mg / ml之间。
    15. 使用Pierce BCA分析试剂盒定量纯化的蛋白质产量。
      1. 粗反应将包含未修饰的生物素化肽。因此,在基于单抗生物素蛋白的纯化后回收的CoilR-生物素的量预计将小于反应中使用的CoilR的量。
      2. 粗反应混合物中肽的生物素化率(%)可以通过标记反应中用单亲和素纯化得到的CoilR-生物素的纳摩尔除以CoilR的纳摩尔来估算。
      3. 一旦未纯化的CoilR-生物素未反应的肽(即,CoilR)不会与树脂结合,则认为通过该方法获得的CoilR-生物素已100%被生物素化。
    16. 将生物素标记的肽储存在5-10%的甘油中。
      1. 分装(100μl/管)并冷冻(-20°C)。该肽可以保存几个月。
      2. 为了实验方便,最终库存应在1μM和50μM之间。
      3. 为了最大程度地减少冻融循环,可以将已融化的等分试样分成较小的单次使用体积(例如, ,10μl)并重新冷冻。

菜谱

注意:

  1. 除非另有说明,否则缓冲液是在高压灭菌的去离子水中制成的。
  2. Tris缓冲液的pH值随温度变化。
  3. 使用含尿素的缓冲液(缓冲液B,缓冲液C和缓冲液E)的pH值应在使用前立即进行检查和调整。
  4. DPBS的pH为7.0。

  1. 缓冲液B(Ni-NTA肽纯化)
    8 M尿素
    100 mM NaH 2 PO 4
    10毫米Tris-Cl pH 8.0
  2. 缓冲液C(Ni-NTA肽纯化)
    8 M尿素
    100 mM NaH 2 PO 4
    10毫米Tris-Cl pH 6.5
  3. 缓冲液E(Ni-NTA肽纯化)
    8 M尿素
    100 mM NaH 2 PO 4
    10 mM Tris-Cl pH 4.5
  4. MES运行缓冲区
    50 mM MES
    50 mM Tris pH 7.3
    1毫米EDTA
    0.1%(w / v)的SDS
  5. TCEP / SDS上染染料(5x)
    300 mM Tris pH 6.8
    50毫米TCEP
    10%(w / v)SDS
    65%(v / v)甘油
    0.025%(v / v)丽春红
  6. 考马斯染色
    45%(v / v)甲醇
    0.3%(w / v)考马斯亮蓝R-250
    10%v / v乙酸
  7. 脱色溶液
    20%(v / v)甲醇
    10%(v / v)乙酸
  8. Tris缓冲盐水(TBS)
    20 mM Tris pH 7.4
    150 mM氯化钠
  9. TBS尿素
    20 mM Tris pH 7.4
    150 mM氯化钠
    2 M尿素
  10. 0.5 M TCEP
    溶解TCEP,然后通过添加10 M NaOH将pH调节至7
    注意:TCEP的一次性使用等分试样可以在-20°C下保存。
  11. TBS尿素结合缓冲液
    20 mM Tris pH 8.0
    150 mM氯化钠
    2 M尿素
  12. 固态标记(SSL)缓冲液,pH 7.5
    125 mM NaH 2 PO 4
    200 mM氯化钠
    1.25 mM EDTA
    4.6 M硫酸铵(75%饱和溶液)
  13. 1 M DTT
    将DTT溶于高压灭菌的去离子水中
    注意:可以准备一次性使用的等分试样,并在-20°C下保存。
  14. TBS尿素咪唑
    20 mM Tris pH 7.4
    150 mM氯化钠
    2 M尿素
    500 mM咪唑
  15. 生物素缓冲液
    DPBS中2 mM生物素
  16. 再生缓冲液
    0.1 M甘氨酸
    pH值2.8

致谢

KEB非常感谢OHSU医学院和国立卫生研究院(R01 GM122854)的支持。 JKD由波特兰大学科学家成就奖章(ARCS)资助。本文描述的协议最初在两个现有出版物中进行了描述(Zane 等人,,2017; Doh 等人,,2018)。我们感谢OHSU的同事,特别是Drs。 Hannah Zane和Jonathan White,为他们对VIP标签的开发做出了贡献。

利益争夺

作者声明没有任何金融或非金融竞争利益。 VIP技术正在申请专利(PCT / US17 / 60609)。

参考文献

  1. Baker,M.(2015年)。 再现性危机:怪罪于抗体。 自然 521(7552):274-276。
  2. 贝尔格伦德·L·比约林·E·奥克斯沃尔德·P·法格贝格·L·阿斯普伦德·A·西吉亚托·CA,佩尔森·A·奥托森J. ,E.,Sivertsson,A.,Navani,S.,Wester,K.,Kamppf,C.,Hober,S.,Ponten,F。和Uhlen,M。(2008)。 基于基因的人类蛋白质图谱,用于基于抗体的表达谱。 分子细胞蛋白质组学 7(10):2019-2027。
  3. Bordeaux,J.,Welsh,A.,Agarwal,S.,Killiam,E.,Baquero,M.,Hanna,J.,Anagnostou,V.和Rimm,D.(2010)。 抗体验证。 生物技术 48(3):197 -209。
  4. Bradbury,A.和Pluckthun,A.(2015年)。 可重复性:标准化研究中使用的抗体。 自然 518 (7537):27-29。
  5. Cranfill,P.J.,Sell,B.R.,Baird,M.A.,Allen,J.R.,Lavagnino,Z.,de Gruiter,H.M.,Kremers,G.J.,Davidson,M.W.,Ustione,A.和Piston,D.W.(2016)。 荧光蛋白的定量评估。 自然方法 13( 7):557-562。
  6. Doh,J.K.,Enns,C.A.和Beatty,K.E.(2019a)。 通过荧光显微镜对VIPER进行细胞蛋白成像。 生物协议 9(21 ):e3413。 DOI:10.21769 / BioProtoc.3413。
  7. Doh,J.K.,Chang,Y.H.,Enns,C.A.,Lopes,C.S.和Beatty,K.E.(2019b)。 通过相关的光学和电子显微镜对VIPER标记的细胞蛋白进行成像。 生物协议 9(21):E3414。 DOI:10.21769 / BioProtoc.3414。
  8. Doh,J.K.,White,J.D.,Zane,H.K.,Chang,Y.H.,López,C.S.,Enns,C.A.和Beatty,K.E.(2018)。 VIPER是用于荧光和电子显微镜的遗传编码肽标签。 Proc。 Natl。学院科学美国 115(51):12961-12966。
  9. Ellisman,MH,Deerinck,TJ,Shu,X.和Sosinsky,GE(2012)第8章-从人群:在相关的光学和电子显微镜成像中用于鉴定蛋白质的遗传标记。在《细胞生物学方法》中。 Academic Press 111:139-155。
  10. Griffiths,G.和Hoppeler,H.(1986)。 免疫细胞化学中的定量:免疫金标记与膜抗原绝对数量的关联。 J Histochem Cytochem 34(11):1389-1398。
  11. Griffiths,G.和Lucocq,J.M.(2014)。 用于通过光学和电子显微镜进行免疫标记的抗体:不适用于胆小的人。 Histochem Cell Biol 142(4):347-360。
  12. Hermanson,G.T.(2013)。 Bioconjugate Techniques 。第三版。学术出版社。 ISBN:978-0-12-382239-0。
  13. Hochuli,E.,Dobeli,H。和Schacher,A。(1987)。 新型的金属螯合吸附剂,对含有相邻组氨酸残基的蛋白质和肽具有选择性。 Chromatogr 411:177-184。
  14. Kim,Y.,Ho,S.O.,Gassman,N.R.,Korlann,Y.,Landorf,E.V.,Collart,F.R. and Weiss,S.(2008年)。 通过半胱氨酸对蛋白质进行高效的位点特异性标记。 Bioconjug Chem 19(3):786-791。
  15. Liu,Z.,Lavis,L.D. and Betzig,E.(2015)。 在单分子水平上成像活细胞动力学和结构。 大声笑细胞。 58(4):644-659。
  16. Moll,J。R.,Ruvinov,S。B.,Pastan,I。和Vinson,C.(2001)。 设计了异二聚化亮氨酸拉链,其pI值范围高达10(-15)M。 / a> Protein Sci 10(3):649-655。
  17. Morimoto-Tomita,M.,Uchimura,K。和Rosen,S.D。(2003)。 新颖的细胞外硫酸酯酶:在癌症中的潜在作用。 趋势糖精。乙二醇技术。 15(83):159-164。
  18. Pierce®单体亲和素琼脂糖。 Thermo Scientific Inc.,2011年。(于2019年10月12日访问,网址为 https:// www.thermofisher.com/order/catalog/product/20228。)
  19. Reinke,A. W.,Grant,R. A.和Keating,A. E.(2010)。 合成的盘绕线圈相互作用基因组为分子工程提供了异源模块。 J Am Chem Soc 132(17):6025-6031。
  20. Rodriguez,E.A.,Campbell,R.E.,Lin,J.Y.,Lin,M.Z.,Miyawaki,A.,Palmer,A.E.,Shu,X.,Zhang,J.和Tsien,R.Y.(2017)。 荧光和光敏蛋白的生长和发光工具箱。 趋势生物化学科学 42(2):111-129。
  21. Schnell,U.,Dijk,F.,Sjollema,K.A.和Giepmans,B.N.(2012)。 免疫标记伪影以及对活细胞成像的需求。 自然方法 9(2):152-158。
  22. TECH TIP#31计算染料:蛋白质(F / P)的摩尔比。 Thermo Scientific Inc.,2011年。(访问时间:2019年10月12日,位于 https://assets.thermofisher.com/TFS-Assets/LSG/brochures/TR0031-Calc-FP-ratios.pdf。)
  23. Tsien,R.Y.(1998)。 绿色荧光蛋白。 Annu Rev Biochem 67: 509-544。
  24. Wingfield,P.(2001年)。 使用硫酸铵沉淀蛋白质。 Curr Protoc Protein Sci 附录3:附录3F。
  25. Zane,H.K.,Doh,J.K.,Enns,C.A.和Beatty,K.E.(2017)。 通用的相互作用肽(VIP)标签,用于使用明亮的化学报告分子标记蛋白质。 Chembiochem 18(5):470-474。
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引用:Doh, J. K., Tobin, S. J. and Beatty, K. E. (2019). Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins. Bio-protocol 9(21): e3412. DOI: 10.21769/BioProtoc.3412.
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