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Feb 2018
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Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
双标签亲和纯化法从细菌中纯化人可溶重组Tau蛋白   

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Abstract

Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies.

Keywords: Tau (Tau蛋白), MAPT (MAPT), Protein purification (蛋白纯化), Soluble Tau (可溶性Tau), Recombinant Tau (重组Tau), Alzheimer’s (阿尔茨海默病)

Background

Tau is traditionally defined as a microtubule binding protein; however, in human diseases Tau can dissociate from axonal microtubules and mislocalize to other neuronal compartments including the soma, dendrites, and synapses, where interactions with non-microtubule proteins and structures drive neuronal dysfunction (Iqbal et al., 2016; Wang and Mandelkow, 2016; Zhou et al., 2017; McInnes et al., 2018). Although Tau aggregates in the form of neurofibrillary tangles are commonly found in post-mortem diseased brain tissue, studies suggest that soluble Tau, not aggregated Tau, is largely responsible for neuronal dysfunction (Crimins et al., 2012; Polydoro et al., 2014; Koss et al., 2016). As such, investigating the soluble functions of Tau in disease, such as identifying protein-protein interaction partners, is therefore of critical importance to target Tau dysfunction.

Purifying soluble Tau protein has been challenging since many ectopically expressed recombinant proteins aggregate into insoluble inclusion bodies in bacteria. Tau furthermore contains aggregation-prone motifs, making purifying recombinant Tau even more difficult. We have therefore developed a protocol that overcomes these obstacles by optimizing two key aspects of this process: first, we express Tau as a fusion protein with an N-terminal Glutathione S-transferase (GST) tag to enhance solubility of the protein, and utilize a mild induction paradigm to minimize the formation of insoluble Tau aggregates. We chose GST over other fusion proteins because it is relatively small, robustly enhances protein solubility and stability, can be easily cleaved, and is relatively inexpensive to purify. Second, our purification protocol utilizes dual affinity tag purification of both an N-terminal GST tag and a C-terminal 8xHis tag (Figure 1); because this purification protocol requires both N- and C-terminal tags to be available to bind affinity resin, the protocol therefore specifically enriches for soluble, non-aggregated protein (in the event of aggregation, at least one of these epitopes is hidden within the aggregate). Following the first purification step against the N-terminal GST tag, the GST fusion protein is cleaved using PreScission protease, resulting in a final product of purified Tau protein that contains only an additional eight histidine residues (8xHis tag) at its C-terminal domain that can also be used for downstream applications including pull-down experiments or detection using anti-His antibodies. This method is versatile and can be easily modified to purify different isoforms of Tau, Tau carrying point mutations, or Tau truncations by altering the pGEX_GST-Tau0N4R-8xHis plasmid using site-directed mutagenesis or traditional cloning methods. We have successfully utilized this protocol to produce various isoforms of Tau, including with point mutations or domain truncations, for use in in vitro binding assays with synaptic vesicles (Zhou et al., 2017) and as bait in protein-protein interaction studies (McInnes et al., 2018).


Figure 1. Schematic overview of the recombinant GST-Tau-8xHis fusion protein and purification steps

Materials and Reagents

  1. 1 ml micropipette tips
  2. Microcentrifuge tubes
  3. Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (Merck, catalog number: UFC501024 )
  4. Plasmid pGEX_GST-Tau0N4R-8xHis
    Note: This plasmid can be generated by cloning a cDNA encoding human MAPT into a pGEX-6P-1 vector backbone (such as the BamHI/EcoRI restriction sites of Addgene plasmid number 46408). The cDNA should be at downstream of N-terminal GST fusion with PreScission Protease cleavage site. The 8xHis tag can be inserted into the 3’ primer used to amplify the hTau cDNA. In this protocol, we use human MAPT cDNA corresponding to the 0N4R isoform.
  5. RosettaTM bacteria cells (Merck, catalog number: 70953 )
  6. LB Broth (Sigma-Aldrich, catalog number: L3522 )
  7. Ampicillin (Sigma-Aldrich, catalog number: A9393 )
  8. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  9. Anti-His antibody (such as Thermo Fisher Scientific, catalog number: 37-2900 ) or anti-Tau antibody (such as Agilent Technologies, DAKO, catalog number: A0024 )
  10. Isopropyl β-D-thiogalactoside (IPTG) (Sigma-Aldrich, catalog number: I6758 )
    Note: Prepare 100 mM stock in sterile H2O and freeze aliquots at -20 °C. Do not re-freeze after thawing.
  11. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  12. Complete protease inhibitor tablets, EDTA-free (Roche Diagnostics, catalog number: 11873580001 )
  13. Phenylmethanesulfonyl fluoride (PMSF), 100 mM solution (Sigma-Aldrich, catalog number: 93482 )
  14. Benzonase Nuclease, 250 units/μl (Sigma-Aldrich, catalog number: E1014 )
  15. PreScission Protease, 2,000 units/ml (GE Healthcare, catalog number: 27084301 )
    Note: Upon receiving stock, thaw on ice and prepare ~10 μl aliquots and freeze at -20 °C. Do not re-freeze aliquots after thawing.
  16. Ni-NTA ProfinityTM IMAC Resin, Ni-charged (Bio-Rad Laboratories, catalog number: 1560131 )
  17. Quick StartTM Bradford Protein Assay (Bio-Rad Laboratories, catalog number: 5000201 )
  18. PageBlueTM Protein Staining Solution (Thermo Fisher Scientific, catalog number: 24620 )
  19. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  20. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  21. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
  22. Sodium phosphate dibasic anhydrous (Na2HPO4) (Sigma-Aldrich, catalog number: 795410 )
  23. DL-Dithiothreitol (DTT), 1 M stock solution in H2O (Sigma-Aldrich, catalog number: 646563 )
  24. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  26. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  27. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  28. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  29. Imidazole (Sigma-Aldrich, catalog number: 792527 )
  30. Bacteria lysis buffer (see Recipes)
  31. Phosphate buffered saline (PBS) (see Recipes)
  32. PBS + 250 mM NaCl (see Recipes)
  33. PreScission protease cleavage buffer (see Recipes)
  34. Ni-NTA wash buffer (see Recipes)
  35. Ni-NTA elution buffer (see Recipes)

Equipment

  1. Pipettes
  2. Table-top microcentrifuge capable of speeds up to 16,000 x g
  3. Microplate reader or spectrophotometer capable of absorbance readings at 600 nm for bacterial density measurements and at 595 nm for Bradford protein assay

Procedure

Notes:

  1. The following procedure is given for purifying Tau protein from a 50 ml culture volume of bacteria, which yields approximately 4 μg pure protein. This protocol can easily be scaled up if more protein is required.
  2. To guarantee the solubility of Tau, it is essential that the protein is purified fresh and is used for experiment immediately upon finishing purification. Freezing purified Tau protein nucleates aggregation and results in loss of solubility and function.
  3. Perform all purification procedures at 4 °C and/or on ice, and pre-chill all tubes on ice.


  1. Bacterial cell induction and harvest
    1. Transform a suitable bacterial strain for protein production with the pGEX_GST-Tau0N4R-8xHis plasmid (ampicillin-resistant). We recommend using Rosetta bacteria that carry a chloramphenicol-resistant tRNA plasmid to enhance expression of mammalian proteins.
    2. Starter culture: In the afternoon, inoculate 5 ml LB media containing appropriate antibiotics (100 μg/ml ampicillin for pGEX_GST-Tau0N4R-8xHis plasmid; additionally, 25 µg/ml chloramphenicol if using Rosetta cells, highly recommended) with a single colony of bacteria (or 10 μl from a glycerol stock) and let grow overnight at 37 °C with shaking at 220-250 rpm.
    3. Main culture: The following morning, dilute the saturated overnight 5 ml culture (OD600 ~2.0) into 45 ml fresh LB medium containing appropriate antibiotics (1:10 dilution, to OD600 ~0.2), and incubate for 1 h at 37 °C with shaking at 220-250 rpm to allow bacteria to recover to exponential growth phase.
    4. After 1 h, add IPTG to a final concentration of 0.4 mM and let bacteria grow at 37 °C for 2 h.
      Optional: Confirm bacteria have re-entered exponential growth phase by measuring the density of the culture at OD600. Bacteria should have recovered from OD600 ~0.2 to at least OD600 ~0.4 before induction.
    5. Two hours following induction, pellet bacteria by centrifugation at 5,000 x g for 15 min at 4 °C. Freeze bacterial cell pellet at -80 °C until use.
      Note: Bacterial cell pellets are stable for at least 3 months at -80 °C. We routinely prepare 0.5-2 L batches of bacteria, freeze 50 ml cell culture volume pellets in aliquots, and take single aliquots for purification on experiment days.

  2. Bacterial cell lysis
    1. Take a single bacterial cell pellet from a 50 ml culture and thaw on ice. Thoroughly resuspend pellet in 1.5 ml ice-cold bacteria lysis buffer by pipetting up and down using 1 ml micropipette tip that has been cut to make it wide-bore and transfer the lysate to a microcentrifuge tube on ice.
      Note: Be sure to freshly prepare lysis buffer (see Recipes).
    2. Incubate lysate for 30 min at 4 °C on a rotating wheel to allow for complete lysis.
    3. Centrifuge lysate at 16,000 x g for 20 min at 4 °C to pellet insoluble material and debris, and transfer the supernatant (cleared lysate) to a new microcentrifuge tube on ice. Complete lysis will be evident as the lysate becomes light brown in color.

  3. Purification against N-terminal GST tag
    1. Preparation of glutathione sepharose: Using a cut pipette tip, transfer 75 μl of Glutathione Sepharose 4B slurry into a microcentrifuge tube and add 1 ml ice-cold PBS to wash.
      Note: Make sure glutathione sepharose slurry is thoroughly mixed by gentle shaking for 30 sec before using.
    2. Pellet sepharose by centrifugation at 3,500 x g for 3 min. 
    3. Discard supernatant. Wash sepharose again with 1 ml ice-cold PBS, centrifuge and discard supernatant.
    4. Transfer the total volume of the cleared bacterial lysate (approximately 1.5 ml lysate from 50 ml bacterial culture) into the tube containing washed glutathione sepharose beads.
    5. Incubate lysate with glutathione sepharose for 2 h at 4 °C with rotation to allow GST tag to bind glutathione.
    6. After binding, pellet sepharose (3,500 x g for 3 min at 4 °C) and wash with 1 ml of PBS + 250 mM NaCl. Pellet again and discard supernatant. Repeat washing 2 more times.

  4. Cleavage of the N-terminal GST tag
    1. Prepare PreScission protease cleavage buffer by adding fresh DTT to a final concentration of 1 mM immediately before use.
    2. Equilibrate beads (GST-Tau-8xHis bound sepharose) by washing twice each with 1 ml PreScission protease cleavage buffer with DTT (same washing procedure as in Procedure C).
    3. After the second wash, discard supernatant and resuspend bead slurry in 240 μl PreScission protease cleavage buffer with DTT. 
    4. Transfer the suspension to a smaller tube (e.g., 0.5 ml tube) and add 10 μl (20 U) PreScission protease.
    5. Incubate the cleavage mixture overnight at 4 °C with rotation. Ensure tube is of appropriate volume to allow for sufficient mixing of the reaction.
      Note: The minimum cleavage time recommended by GE Healthcare is 4 h at 4 °C, but we recommend overnight incubation.

  5. Purification against the C-terminal His tag
    1. The next morning, pellet the sepharose slurry by centrifugation at 3,500 x g for 5 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube and discard the sepharose bead slurry. This supernatant contains the cleaved Tau-His as well as residual PreScission protease. 
    2. Prepare fresh glutathione sepharose slurry (50 μl), wash twice in cleavage buffer containing DTT, then incubate supernatant from Step E1 wish fresh glutathione sepharose for 1 h at 4 °C with mixing.
      Note: This step will remove the residual GST-tagged PreScission protease as well as any residual uncleaved GST-Tau-His protein in the mixture. 
    3. After 1 h, pellet the sepharose by centrifugation at 3,500 x g for 5 min at 4 °C, and transfer the supernatant, which contains the free Tau-His protein, to a new microcentrifuge tube on ice.
    4. Prepare Ni-NTA resin by pipetting 25 μl of Ni-NTA slurry into a microcentrifuge tube and wash twice in ice-cold PreScission protease cleavage buffer to equilibrate the resin.
    5. Apply the supernatant containing Tau-His protein to the Ni-NTA resin and allow to bind for 45 min at 4 °C with mixing.
    6. After 45 min, pellet Ni-NTA slurry at 3,500 x g for 3 min at 4 °C and discard supernatant. Wash beads three times with ice-cold 1 ml Ni-NTA wash buffer.
    7. Elute the bound Tau-His protein by resuspending washed beads in ice-cold 500 μl Ni-NTA elution buffer and incubating for 10 min at 4 °C with rotation.
    8. Pellet Ni-NTA beads by centrifugation at 10,000 x g for 2 min at 4 °C. Collect the supernatant, being careful not to transfer any sedimented Ni-NTA beads, and transfer to a new microcentrifuge tube on ice.
    9. Concentrate the 500 μl protein eluate down to a volume of approximately 100 μl by applying the eluate to an Amicon Ultra-0.5 ml Centrifugal Filter Unit (30 kDa molecular weight cutoff) and centrifuging at 14,000 x g for approximately 8 min at 4 °C, or longer until the volume has reached 100 μl. Recover solution by inverting the filter unit into a clean collection tube and centrifuging at 1,000 x g for 2 min at 4 °C.
    10. Measure protein concentration using the Bradford Protein Assay, or other appropriate protein quantification method. Confirm protein purity by SDS-PAGE and staining with PageBlue colloidal Coomassie reagent.
      Note: Expect the protein concentration to be approximately 20-60 μg/ml, which requires putting up to 10 μl of protein sample into a 200 μl Bradford reaction for detection.

Data analysis

  1. To evaluate the purity of the final product, run 1 μg protein on an SDS-PAGE gel and stain with colloidal Coomassie. We routinely observe greater than 90% protein purity using this protocol (Figure 2).
  2. The identity of the purified protein product can be confirmed by immunoblotting using an anti-His antibody or anti-Tau antibody. For examples of immunoblots detecting full-length and truncated Tau proteins purified using this protocol see Zhou et al. (2017) and McInnes et al. (2018).
  3. The aggregation status of purified Tau protein can be assessed by ultracentrifugation of the sample at 100,000 x g for 1 h at 4 °C, followed by analyzing the presence of Tau aggregates in the pellet by SDS-PAGE. Analyzing freshly purified protein using this protocol, we observe minimal Tau protein in the insoluble fraction. In contrast, freezing and thawing the purified protein results in abundant Tau protein in the pellet upon centrifugation at 100,000 x g, indicating aggregation.


    Figure 2. Purity of Tau-His protein. One microgram purified protein was mixed with 1x NuPAGE LDS sample buffer (InvitrogenTM) containing 1% β-mercaptoethanol, heated at 70 °C for 10 min, and separated on a NuPAGE 4-12% Bis-Tris Protein Gel (InvitrogenTM) with MOPS running buffer. The gel was stained with PageBlue colloidal Coomassie solution.

Recipes

  1. Bacteria lysis buffer
    1x PBS (from 10x stock solution)
    1% Triton X-100 (from 10% stock solution)
    10% glycerol (from 50% stock solution)
    1 mg/ml lysozyme (dissolve fresh from powder)
    1x Complete Protease Inhibitor cocktail, EDTA-free (Roche Diagnostics)
    1 mM PMSF (from 100 mM stock solution)
    250 U/ml Benzonase (1:1,000 from enzyme stock)
    Note: Add Complete protease inhibitor cocktail, PMSF, lysozyme and Benzonase fresh immediately before use .
  2. Phosphate buffered saline (PBS)
    10 mM Na2HPO4
    2.7 mM KCl
    137 mM NaCl
    Adjust pH to 7.4
  3. PBS + 250 mM NaCl
    Dilute a 5 M stock of NaCl into PBS to give a final concentration of 250 mM NaCl
  4. PreScission protease cleavage buffer
    20 mM Tris-HCl
    50 mM NaCl
    0.5 mM EDTA
    0.01% Tween-20
    1 mM DTT
    Adjust pH to 7.0 with NaOH
    Notes:
    1. Buffer (without DTT) can be kept at 4 °C for up to 1 month.
    2. Immediately before use, take an aliquot of buffer and supplement with DTT.
  5. Ni-NTA wash buffer
    50 mM NaH2PO4
    300 mM NaCl
    20 mM imidazole
    pH 8.0
    Store at 4 °C
  6. Ni-NTA elution buffer
    50 mM NaH2PO4
    300 mM NaCl
    250 mM imidazole
    pH 8.0
    Store at 4 °C

Acknowledgments

This work was supported by a European Research Council consolidator grant (646671), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO), and an academic-industrial collaboration between the VIB-KU Leuven Center for Brain and Disease Research and Janssen Pharmaceutical Companies of Johnson & Johnson.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Crimins, J. L., Rocher, A. B. and Luebke, J. I. (2012). Electrophysiological changes precede morphological changes to frontal cortical pyramidal neurons in the rTg4510 mouse model of progressive tauopathy. Acta Neuropathol 124(6): 777-795.
  2. Iqbal, K., Liu, F. and Gong, C. X. (2016). Tau and neurodegenerative disease: the story so far. Nat Rev Neurol 12(1): 15-27.
  3. Koss, D. J., Jones, G., Cranston, A., Gardner, H., Kanaan, N. M. and Platt, B. (2016). Soluble pre-fibrillar tau and beta-amyloid species emerge in early human Alzheimer's disease and track disease progression and cognitive decline. Acta Neuropathol 132(6): 875-895.
  4. McInnes, J., Wierda, K., Snellinx, A., Bounti, L., Wang, Y. C., Stancu, I. C., Apostolo, N., Gevaert, K., Dewachter, I., Spires-Jones, T. L., De Strooper, B., De Wit, J., Zhou, L. and Verstreken, P. (2018). Synaptogyrin-3 mediates presynaptic dysfunction induced by Tau. Neuron 97(4): 823-835 e8.
  5. Polydoro, M., Dzhala, V. I., Pooler, A. M., Nicholls, S. B., McKinney, A. P., Sanchez, L., Pitstick, R., Carlson, G. A., Staley, K. J., Spires-Jones, T. L. and Hyman, B. T. (2014). Soluble pathological tau in the entorhinal cortex leads to presynaptic deficits in an early Alzheimer's disease model. Acta Neuropathol 127(2): 257-270.
  6. Wang, Y. and Mandelkow, E. (2016). Tau in physiology and pathology. Nat Rev Neurosci 17(1): 5-21.
  7. Zhou, L., McInnes, J., Wierda, K., Holt, M., Herrmann, A. G., Jackson, R. J., Wang, Y. C., Swerts, J., Beyens, J., Miskiewicz, K., Vilain, S., Dewachter, I., Moechars, D., De Strooper, B., Spires-Jones, T. L., De Wit, J. and Verstreken, P. (2017). Tau association with synaptic vesicles causes presynaptic dysfunction. Nat Commun 8: 15295.

简介

微管相关蛋白Tau(由 MAPT 基因编码)的功能障碍已经涉及20多种神经退行性疾病,包括阿尔茨海默病。 因此,Tau的生理和疾病相关功能引起了研究界的极大兴趣。 妨碍对Tau功能的研究和产生靶向Tau的药理学试剂的一个障碍是难以获得纯化形式的可溶性Tau蛋白。 在这里,我们描述了一种方案,该方案使用双亲和标签纯化从细菌中选择性纯化可溶性重组Tau蛋白,所述细菌对于下游应用具有功能活性,包括免疫,微管结合测定和蛋白质 - 蛋白质相互作用研究。
【背景】Tau传统上被定义为微管结合蛋白;然而,在人类疾病中,Tau可以与轴突微管分离并错误定位到其他神经元区室,包括体细胞,树突和突触,其中与非微管蛋白和结构的相互作用驱动神经元功能障碍(Iqbal et al。 ,2016; Wang和Mandelkow,2016; Zhou et al。,2017; McInnes et al。,2018)。尽管神经原纤维缠结形式的Tau聚集体通常存在于死后患病的脑组织中,但研究表明,可溶性Tau,而不是聚集的Tau,是神经元功能障碍的主要原因(Crimins et al。,2012 ; Polydoro et al。,2014; Koss et al。,2016)。因此,研究Tau在疾病中的可溶性功能,例如鉴定蛋白质 - 蛋白质相互作用配偶体,因此对于靶向Tau功能障碍是至关重要的。

纯化可溶性Tau蛋白一直是具有挑战性的,因为许多异位表达的重组蛋白聚集成细菌中的不溶性包涵体。 Tau还含有易聚集的基序,使得纯化重组Tau更加困难。因此,我们通过优化该过程的两个关键方面开发了一种克服这些障碍的方案:首先,我们将Tau表达为具有N-末端谷胱甘肽S-转移酶(GST)标签的融合蛋白,以增强蛋白质的溶解性,并利用温和的诱导范例,以尽量减少不溶性Tau聚集体的形成。我们选择GST优于其他融合蛋白,因为它相对较小,强有力地增强蛋白质溶解度和稳定性,可以容易地裂解,并且相对便宜地纯化。其次,我们的纯化方案利用N端GST标签和C端8xHis标签的双亲和标签纯化(图1);因为这种纯化方案需要N-和C-末端标签可用于结合亲和树脂,因此该方案特异性地富集可溶性,非聚集蛋白质(在聚集的情况下,这些表位中的至少一个隐藏在骨料)。在针对N-末端GST标签的第一个纯化步骤后,使用PreScission蛋白酶切割GST融合蛋白,产生纯化的Tau蛋白的最终产物,其在C-末端结构域仅含有另外8个组氨酸残基(8xHis标签)。也可用于下游应用,包括下拉实验或使用抗His抗体检测。该方法是通用的,并且可以通过使用定点诱变或传统克隆方法改变pGEX_GST-Tau0N4R-8xHis质粒,可以容易地修饰以纯化Tau,Tau携带点突变或Tau截短的不同同种型。我们已成功利用该方案产生各种Tau同种型,包括点突变或结构域截短,用于与突触小泡的体外结合试验(Zhou et al。 ,2017)和蛋白质 - 蛋白质相互作用研究中的诱饵(McInnes et al。,2018)。


图1.重组GST-Tau-8xHis融合蛋白的示意图和纯化步骤

关键字:Tau蛋白, MAPT, 蛋白纯化, 可溶性Tau, 重组Tau, 阿尔茨海默病

材料和试剂

  1. 1毫升微量吸管尖端
  2. 微量离心管
  3. 带Ultracel-10膜的Amicon Ultra-0.5离心过滤装置(默克,产品目录号:UFC501024)
  4. 质粒pGEX_GST-Tau0N4R-8xHis(Addgene,目录编号:XXX)
  5. Rosetta T M  细菌细胞(默克,目录号:70953)
  6. LB肉汤(Sigma-Aldrich,目录号:L3522)
  7. 氨苄西林(Sigma-Aldrich,目录号:A9393)
  8. 氯霉素(Sigma-Aldrich,目录号:C0378)
  9. 抗His抗体(如Thermo Fisher Scientific,目录号:37-2900)或抗Tau抗体(如Agilent Technologies,DAKO,目录号:A0024)
  10. 异丙基β-D-硫代半乳糖苷(IPTG)(Sigma-Aldrich,目录号:I6758)
    注意:在无菌H 2 O中制备100 mM原液,并在-20°C下冷冻等分试样。解冻后不要再冻结。
  11. Glutathione Sepharose 4B(GE Healthcare,目录号:17075601)
  12. 完整的蛋白酶抑制剂片剂,不含EDTA(Roche Diagnostics,目录号:11873580001)
  13. Phenylmethanesulfonyl fluoride(PMSF),100 mM溶液(Sigma-Aldrich,目录号:93482)
  14. Benzonase核酸酶,250单位/μl(西格玛奥德里奇,目录号:E1014)
  15. PreScission Protease,2,000单位/ ml(GE Healthcare,目录号:27084301)
    注意:收到原种后,在冰上解冻并准备约10μl等分试样并在-20°C下冷冻。解冻后不要重新冷冻等分试样。
  16. Ni-NTA Profinity TM IMAC树脂,Ni-charged(Bio-Rad Laboratories,目录号:1560131)
  17. 快速入门 TM Bradford蛋白质分析(Bio-Rad Laboratories,目录号:5000201)
  18. PageBlue TM 蛋白质染色液(Thermo Fisher Scientific,目录号:24620)
  19. Triton X-100(Sigma-Aldrich,目录号:X100)
  20. 甘油(Sigma-Aldrich,目录号:G5516)
  21. 溶菌酶(Sigma-Aldrich,目录号:L6876)
  22. 无水磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:795410)
  23. DL-二硫苏糖醇(DTT),在H 2 O中的1M储备溶液(Sigma-Aldrich,目录号:646563)
  24. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  25. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  26. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  27. Tween-20(西格玛奥德里奇,目录号:P1379)
  28. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  29. 咪唑(Sigma-Aldrich,目录号:792527)
  30. 细菌裂解缓冲液(见食谱)
  31. 磷酸盐缓冲盐水(PBS)(见食谱)
  32. PBS + 250 mM NaCl(参见食谱)
  33. PreScission蛋白酶切割缓冲液(见食谱)
  34. Ni-NTA洗涤缓冲液(见食谱)
  35. Ni-NTA洗脱缓冲液(见食谱)

设备

  1. 移液器
  2. 台式微量离心机,速度可达16,000 x g
  3. 微孔板读数器或分光光度计能够在600 nm处进行细菌密度测量的吸光度读数,在595 nm处用于Bradford蛋白质测定的吸光度读数

程序

注意:

  1. 以下步骤用于从50ml培养体积的细菌中纯化Tau蛋白,其产生约4μg纯蛋白质。如果需要更多的蛋白质,这个协议可以很容易地扩大规模。
  2. 为了保证Tau的溶解性,必须将蛋白质新鲜纯化,并在完成纯化后立即用于实验。冷冻纯化的Tau蛋白会使聚集成核并导致溶解度和功能的丧失。
  3. 在4°C和/或冰上进行所有纯化步骤,并在冰上预冷所有管。


  1. 细菌细胞诱导和收获
    1. 用pGEX_GST-Tau0N4R-8xHis质粒(氨苄青霉素抗性)转化合适的细菌菌株用于蛋白质生产。我们建议使用携带氯霉素抗性tRNA质粒的Rosetta细菌来增强哺乳动物蛋白的表达。
    2. 初级培养:下午,接种含有适当抗生素的5 ml LB培养基(100μg/ ml氨苄青霉素用于pGEX_GST-Tau0N4R-8xHis质粒;另外,25μg/ ml氯霉素,如果使用Rosetta细胞,强烈推荐),单个细菌菌落(或从甘油原液中10μl)并在37℃下以220-250rpm振荡生长过夜。
    3. 主培养:第二天早上,将饱和的5ml过夜培养液(OD 600 ~2.0)稀释到45ml含有适当抗生素的新鲜LB培养基中(1:10稀释,OD 600 ~0.2),并在37℃下以220-250rpm振荡孵育1小时,以使细菌恢复到指数生长期。
    4. 1小时后,加入IPTG至终浓度为0.4 mM,让细菌在37°C生长2小时。
      可选:通过测量OD 600 处培养物的密度,确认细菌已重新进入指数生长期。诱导前,细菌应从OD 600 ~0.2恢复到至少OD 600 ~0.4。
    5. 诱导后2小时,通过在4℃下以5,000×g×g离心15分钟沉淀细菌。将细菌细胞沉淀在-80°C冷冻直至使用。
      注意:细菌细胞颗粒在-80°C下至少可稳定保存3个月。我们常规制备0.5-2L批次的细菌,以等分试样冷冻50ml细胞培养体积颗粒,并在实验日取单个等分试样进行纯化。

  2. 细菌细胞裂解
    1. 从50ml培养物中取出一个细菌细胞沉淀并在冰上融化。通过用1ml微量移液管尖端上下移液,将沉淀物彻底重悬于1.5ml冰冷的细菌裂解缓冲液中,所述微量移液管尖端已被切割以使其宽孔并将裂解物转移至冰上的微量离心管中。
      注意:确保新鲜制备裂解缓冲液(参见食谱)。
    2. 将裂解物在4°C下在旋转轮上孵育30分钟以允许完全裂解。
    3. 在4℃下以16,000 x g 离心裂解物20分钟以沉淀不溶性物质和碎片,并将上清液(澄清的裂解物)转移到冰上的新微量离心管中。当裂解物变成浅棕色时,完全裂解将是明显的。

  3. 针对N端GST标签的纯化
    1. 谷胱甘肽琼脂糖凝胶的制备:使用切割的移液管尖端,将75μl谷胱甘肽Sepharose 4B浆液转移到微量离心管中,加入1ml冰冷的PBS进行洗涤。
      注意:确保谷胱甘肽琼脂糖浆液在使用前轻轻摇动30秒彻底混合。
    2. 通过在3,500 x g 离心3分钟沉淀琼脂糖凝胶。 
    3. 丢弃上清液。用1ml冰冷的PBS再次洗净琼脂糖凝胶,离心并弃去上清液。
    4. 将澄清的细菌裂解物的总体积(来自50ml细菌培养物的约1.5ml裂解物)转移到含有洗涤的谷胱甘肽琼脂糖珠的管中。
    5. 将裂解物与谷胱甘肽琼脂糖凝胶在4℃下孵育2小时,旋转以使GST标签结合谷胱甘肽。
    6. 结合后,沉淀琼脂糖凝胶(3,500 x g 在4℃下3分钟)并用1ml PBS + 250mM NaCl洗涤。再次沉淀并弃去上清液。重复洗涤2次。

  4. N末端GST标签的切割
    1. 在使用前立即加入新鲜的DTT至终浓度为1mM,制备PreScission蛋白酶切割缓冲液。
    2. 通过用含有DTT的1ml PreScission蛋白酶裂解缓冲液洗涤两次(GST-Tau-8xHis结合的琼脂糖凝胶)平衡珠子(与步骤C中相同的洗涤程序)。
    3. 在第二次洗涤后,弃去上清液并将珠浆液重悬于240μl含有DTT的PreScission蛋白酶裂解缓冲液中。 
    4. 将悬浮液转移到较小的管(例如,0.5ml管)中并加入10μl(20U)PreScission蛋白酶。
    5. 将裂解混合物在4℃下旋转孵育过夜。确保管具有适当的体积,以使反应充分混合。
      注意:GE Healthcare推荐的最短切割时间为4小时4小时,但我们建议过夜孵育。

  5. 针对C末端His标签的纯化
    1. 第二天早上,通过在4,500℃下在3℃下离心5分钟来沉淀琼脂糖凝胶浆液。将上清液转移到新的微量离心管中并丢弃琼脂糖凝胶珠浆液。该上清液含有裂解的Tau-His以及残留的PreScission蛋白酶。 
    2. 制备新鲜的谷胱甘肽琼脂糖凝胶浆液(50μl),在含有DTT的裂解缓冲液中洗涤两次,然后将来自步骤E1的上清液在4℃下在4℃下搅拌混合使用新鲜谷胱甘肽琼脂糖凝胶1小时。
      注意:此步骤将去除残留的GST标记的PreScission蛋白酶以及混合物中任何残留的未切割的GST-Tau-His蛋白。 
    3. 1小时后,通过在4℃下在3,500×g离心5分钟沉淀琼脂糖凝胶,并将含有游离Tau-His蛋白的上清液转移到冰上的新微量离心管中。
    4. 通过将25μlNi-NTA浆液移液到微量离心管中制备Ni-NTA树脂,并在冰冷的PreScission蛋白酶裂解缓冲液中洗涤两次以平衡树脂。
    5. 将含有Tau-His蛋白的上清液应用于Ni-NTA树脂,并在4℃下混合45分钟。
    6. 45分钟后,在4℃下在3,500×gg /小时下沉淀Ni-NTA浆液3分钟并弃去上清液。用冰冷的1ml Ni-NTA洗涤缓冲液洗涤珠子三次。
    7. 通过在冰冷的500μlNi-NTA洗脱缓冲液中重悬浮洗涤的珠子并在4℃下旋转孵育10分钟来洗脱结合的Tau-His蛋白质。
    8. 通过在4℃下以10,000 x g 离心2分钟来沉淀Ni-NTA珠粒。收集上清液,注意不要转移任何沉淀的Ni-NTA珠子,并转移到冰上的新微量离心管中。
    9. 通过将洗脱液应用于Amicon Ultra-0.5 ml离心过滤装置(30 kDa分子量截止值)并在14,000 xg 离心约8,将500μl蛋白质洗脱液浓缩至约100μl的体积。最低温度为4°C或更长时间,直至体积达到100μl。通过将过滤器单元倒置到干净的收集管中并在4℃下以1,000 x g 离心2分钟来回收溶液。
    10. 使用Bradford蛋白质测定法或其他适当的蛋白质定量方法测量蛋白质浓度。通过SDS-PAGE确认蛋白纯度,并用PageBlue胶体考马斯试剂染色。
      注意:预计蛋白质浓度约为20-60μg/ ml,这需要将最多10μl蛋白质样品加入200μlBradford反应中进行检测。

数据分析

  1. 为了评估最终产品的纯度,在SDS-PAGE凝胶上运行1μg蛋白质并用胶体考马斯染色。我们常规使用该方案观察到超过90%的蛋白质纯度(图2)。
  2. 纯化的蛋白质产物的特性可以通过使用抗His抗体或抗-Tau抗体的免疫印迹来确认。对于检测使用该方案纯化的全长和截短的Tau蛋白的免疫印迹的实例,参见Zhou 等人(2017)和McInnes 等人(2018)。
  3. 纯化的Tau蛋白的聚集状态可以通过在4℃下以100,000×gg 超速离心样品1小时来评估,然后通过SDS-PAGE分析沉淀中Tau聚集体的存在。使用该方案分析新鲜纯化的蛋白质,我们在不溶性部分中观察到最小的Tau蛋白质。相反,冷冻和解冻纯化的蛋白质在100,000 x g 离心后导致沉淀中的大量Tau蛋白,表明聚集。


    图2. Tau-His蛋白的纯度。将1微克纯化的蛋白质与含有1%β-巯基乙醇的1x NuPAGE LDS样品缓冲液(Invitrogen TM )混合,加热至70℃。 ℃保持10分钟,并在具有MOPS运行缓冲液的NuPAGE 4-12%Bis-Tris蛋白凝胶(Invitrogen TM )上分离。凝胶用PageBlue胶体考马斯溶液染色。

食谱

  1. 细菌裂解缓冲液
    1x PBS(来自10x原液)
    1%Triton X-100(来自10%原液)
    10%甘油(来自50%原液)
    1毫克/毫升溶菌酶(从粉末中溶解新鲜)
    1x完全蛋白酶抑制剂鸡尾酒,不含EDTA(罗氏诊断)
    1mM PMSF(来自100mM储备溶液)
    250 U / ml Benzonase(酶原1:1,000)
    注意:使用前立即加入完全蛋白酶抑制剂混合物,PMSF,溶菌酶和Benzonase。
  2. 磷酸盐缓冲盐水(PBS)
    10mM Na 2 HPO 4
    2.7 mM KCl
    137 mM NaCl
    将pH调节至7.4
  3. PBS + 250 mM NaCl
    将5 M NaCl的稀释液稀释到PBS中,使最终浓度为250 mM NaCl
  4. PreScission蛋白酶切割缓冲液
    20 mM Tris-HCl
    50 mM NaCl
    0.5 mM EDTA
    0.01%吐温-20
    1 mM DTT
    用NaOH调节pH至7.0
    注意:
    1. 缓冲液(不含DTT)可在4°C下保存长达1个月。
    2. 在使用前,立即取一份缓冲液并补充DTT。
  5. Ni-NTA洗涤缓冲液
    50mM NaH 2 PO 4
    300 mM NaCl
    20 mM咪唑
    pH 8.0
    储存在4°C
  6. Ni-NTA洗脱缓冲液
    50mM NaH 2 PO 4
    300 mM NaCl
    250 mM咪唑
    pH 8.0
    储存在4°C

致谢

这项工作得到了欧洲研究委员会合并补助金(646671),Fonds voor Wetenschappelijk Onderzoek Vlaanderen(FWO)的支持,以及VIB-KU鲁汶脑与疾病研究中心和Johnson& Janssen Pharmaceutical Companies之间的学术 - 工业合作。 ;约翰逊。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Crimins,J.L.,Rocher,A.B。和Luebke,J.I。(2012)。 电生理改变先于rTg4510小鼠进行性tau病变模型中额叶皮质锥体神经元的形态学改变。 Acta Neuropathol 124(6):777-795。
  2. Iqbal,K.,Liu,F。和Gong,C。X.(2016)。 Tau和神经退行性疾病:迄今为止的故事。 Nat Rev Neurol 12(1):15-27。
  3. Koss,D.J.,Jones,G.,Cranston,A.,Gardner,H.,Kanaan,N.M。和Platt,B。(2016)。 可溶性前纤维状tau蛋白和β-淀粉样蛋白物种在早期人类阿尔茨海默病中出现,并追踪疾病进展和认知能力下降。 Acta Neuropathol 132(6):875-895。
  4. McInnes,J.,Wierda,K.,Snellinx,A.,Bounti,L.,Wang,YC,Stancu,IC,Apostolo,N.,Gevaert,K.,Dewachter,I.,Spiers-Jones,TL,De Strooper,B.,De Wit,J.,Zhou,L。和Verstreken,P。(2018)。 Synaptogyrin-3介导由Tau诱导的突触前功能障碍。 Neuron 97(4):823-835 e8。
  5. Polydoro,M.,Dzhala,VI,Pooler,AM,Nicholls,SB,McKinney,AP,Sanchez,L.,Pitstick,R.,Carlson,GA,Staley,KJ,Spires-Jones,TL and Hyman,BT(2014 )。 内嗅皮质中可溶性病理性tau导致早期阿尔茨海默病模型中的突触前缺陷。 Acta Neuropathol 127(2):257-270。
  6. Wang,Y。和Mandelkow,E。(2016)。 Tau in physiology and pathology。 Nat Rev Neurosci 17 (1):5-21。
  7. Zhou,L.,McInnes,J.,Wierda,K.,Holt,M.,Herrmann,AG,Jackson,RJ,Wang,YC,Swerts,J.,Beyens,J.,Miskiewicz,K.,Vilain,S 。,Dewachter,I.,Moechars,D.,De Strooper,B.,Spiers-Jones,TL,De Wit,J。和Verstreken,P。(2017)。 Tau与突触小泡的关联导致突触前功能障碍。 Nat Commun 8:15295。
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引用:McInnes, J., Zhou, L. and Verstreken, P. (2018). Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification. Bio-protocol 8(22): e3043. DOI: 10.21769/BioProtoc.3043.
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Sandeep Rai
Sandeep Rai
Dear Author, During purification of tau protein, do you get truncated products (appear as smear on SDS gel) of tau as well? Generally, C-terminal tagged protein has advantage over other forms of protein as only those fragments will binds to the affinity resin, which is completely translated. But considering the truncation in tau (frequently encountered when tau protein is purified without tag), do you also encounter the similar observation in this method of protein preparation?. Looking forward to hearing from you. Thanks in advance. Regards Sandeep Rai
2020/12/15 1:34:09 回复