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In vitro Assay of the Glycosyltransferase Activity of a Heterologously Expressed Plant Protein
异源表达植物蛋白的糖基转移酶活性的体外检测   

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Abstract

Glycosyltransferases are carbohydrate active enzymes containing catalytic modules involved in catalysing the biosynthesis of glycosidic bonds in oligo- and polysaccharides and glycoconjugates. One of the most comprehensive collections of Carbohydrate Active enZYmes is the CAZy database (http://www.cazy.org) comprising 120,000 glycosyltransferases allocated to 96 families based mainly on sequence homologies of their conserved and catalytically active domains (Cantarel et al., 2009). Interestingly, the glycosyltransferase activities of only about 1.6% of these proteins have been experimentally characterized (Lombard et al., 2014). In recent years, membrane-bound glycosyltransferases of a number of families have been shown to play a key role in the biosynthesis of plant cell-wall polysaccharides (Doblin et al., 2010; Scheller and Ulvskov, 2010; Driouich et al., 2012). They catalyze the transfer of glycosyl residues from donor nucleotide sugars to acceptors, forming the glycosidic bonds between adjacent glycosyl residues. Family 34 contains glycosyltransferases that have been shown to be involved in the biosynthesis of xyloglucans and transfer xylosyl residues to (1→4)-β-glucan chains (Keegstra and Cavalier, 2011). Our previous work suggests that Pinus radiata protein PrGT34B is a xyloglucan (1→6)-α-xylosyltransferase (Ade et al., 2014). Here, we describe a procedure for determining the xylosyltransferase activity of PrGT34B in vitro. We measured the transfer of xylose from the donor substrate UDP-xylose to different cello-oligosaccharide acceptor substrates under controlled reaction conditions. The assays include quantification of radioactively labeled reaction products and their identification by mass spectrometry. We also describe the purification, identification and quantification of the heterologously expressed recombinant protein PrGT34B in preparation for its use in the assays. This procedure may be applied to a wide range of glycosyltransferases in many different plant species.

Materials and Reagents

  1. Spodoptera frugiperda cells (Sf9) (Life Technologies, InvitrogenTM, catalog number: 11496-015 )
  2. Vector pFastBacHTaTM (Life Technologies, InvitrogenTM, catalog number: 10584-027 )
  3. Escherichia coli (E. coli) strains DH10Bac (Life Technologies, InvitrogenTM, catalog number: 10359-016 )
  4. 20% ethanol
  5. SDS
  6. β-mercaptoethanol
  7. Glycerol
  8. Bromophenol blue
  9. HEPES-NaOH (pH 7.0)
  10. MgCl2
  11. MnCl2
  12. Triton X-100
  13. Benzonase® nuclease (Novagen, catalog number: 70746 )
  14. Protease inhibitors (complete EDTA free, protease inhibitor cocktail tablet) (Roche Diagnostics, catalog number: 0 4693159001 )
  15. Imidazole
  16. Cellotetraose, cellopentaose and cellohexaose (Associates of Cape Cod, Northstar BioProducts®, catalog numbers: 400402-1 , 400404-1 , 400406-1 )
  17. Cellobiose (Sigma-Aldrich, catalog number: C7252 )
  18. UDP-D-xylose (CarboSource Services, Complex Carbohydrate Research Center, product code: UDP-Xylose )
  19. UDP-[14C-U]xylose (151.8 mCi/mmol) (PerkinElmer, catalog number: NEC543005UC )
  20. TALON metal affinity resin (Takara Bio Company, Clontech, catalog number: 635501 )
  21. BenchMark His-tagged protein standard (Life Technologies, InvitrogenTM, catalog number: LC5606 )
  22. Precision Plus protein unstained standard (Bio-Rad Laboratories, catalog number: 161-0363 )
  23. Mouse anti-His6 monoclonal antibody (GE Healthcare, product code: 27-4710-01 )
  24. Horseradish peroxidase (HRP) conjugated secondary sheep anti-mouse antibody (GE Healthcare, product code: NA931-100UL )
  25. SYPRO® Ruby protein gel stain (Life Technologies, Molecular Probes®, catalog number: S-21900 )
  26. Ion exchange resin Dowex 1x8-200 (chloride form) (Sigma-Aldrich, catalog number: 217425 )
  27. 2,5-Dihydroxybenzoic acid (Sigma-Aldrich, catalog number: 85707 )
  28. Starscint high flash-point LSC-cocktail (PerkinElmer)
  29. PAGE loading dye (see Recipes)
  30. Hypotonic lysis buffer (see Recipes)
  31. Equilibration/wash buffer for IMAC (see Recipes)
  32. Sample preparation buffer for IMAC (see Recipes)
  33. IMAC elution buffer (see Recipes)
  34. Reaction buffer for enzymatic assays (with radioactively labeled UDP-xylose) (see Recipes)
  35. Reaction buffer for enzymatic assays (without radioactively labeled UDP-xylose) (see Recipes)

Equipment

  1. Empty gravity-flow chromatography columns (20 ml volume) (Bio-Rad Laboratories, catalog number: 732-1010 )
  2. Micro Bio-Spin chromatography columns (Bio-Rad Laboratories, catalog number: 732-6204 )
  3. Amersham ECL detection system (GE Healthcare, product code: RPN2105 )
  4. PVDF transfer membrane (Amersham Hybond-P) (GE Healthcare, product code: 10600023 )
  5. NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific)
  6. LAS-3000 Lite imager with software LAS-3000 Image Reader (Fujifilm Corporation)
  7. FLA 2000 phosphorimager with software FLA 2000G (Fujifilm Corporation)
  8. Rackbeta LKB Wallac (PerkinElmer, model: 1209 )
  9. Voyager DETM PRO MALDI-TOF Workstation (Life Technologies, Applied Biosystems®)
    Note: We believe that the reproducibility of the experiments does not depend on the precise equipment stated above but can be achieved using any other equivalent instrumentation.

Software

  1. Software Multi Gauge v2.2 (Fujifilm Corporation)
  2. Data Explorer (version 4.0.0.0) (Life Technologies, Applied Biosystems®)
  3. Software FLA 2000G (Fujifilm Corporation)
  4. Software LAS-3000 Image Reader (Fujifilm Corporation)

Procedure

  1. Heterologous expression of the glycosyltransferase
    Note: There are a vast number of protocols describing procedures for the heterologous expression of proteins. Successful expression of plant glycosyltransferase proteins for enzymatic analyses has been achieved in bacteria, yeast and various insect cell systems. We do not give a detailed description of the heterologous expression of protein PrGT34B, but only outline the procedure we used and give some information that may be helpful.
    1. In our study (Ade et al., 2014), we expressed the xylosyltransferase PrGT34B in Spodoptera frugiperda cells (Sf9) using an Autographa californica nuclear polyhydrosis virus based baculovirus expression vector system.
      1. The cell line Sf9 had previously been used to successfully express plant glycosyltransferases (Egelund et al., 2006). Furthermore, Sf9 cells are highly susceptible to infection with Autographa californica nuclear polyhydrosis virus and the corresponding baculovirus expression vector was shown to result in high expression levels of the desired gene product in insect cells (Condreay and Kost, 2007).
      2. The sequence of interest was cloned into vector pFastBacHTaTM, recombinant bacmid DNA was generated in E. coli strains DH10Bac or DH10MultiBac (Berger et al., 2004) and baculovirus was produced and amplified in Sf9 cells following the instructions in the manual “Bac-to-Bac Baculovirus Expression System”.
      3. Sf9 suspension cultures (200 ml), with a starting concentration of 2.0 x 106 cells ml-1, were inoculated with baculovirus (1:1,000 v/v of amplified virus stock to Sf9 cell suspension) and incubated with shaking (130 rpm, 72 h, 28 °C). Infected cells were harvested by centrifugation (500 x g, 10 min, 4 °C), and the cell pellets used for protein extraction (see below for details).
    2. For easy purification of the recombinant protein prior to its use in enzymatic assays, we strongly suggest that histidine tagged (6x histidine) fusion protein is expressed.
    3. In addition, many plant GTs, including xylosyltransferase PrGT34B, are type-II membrane bound proteins with a single transmembrane domain (TMD) close to the N-terminal end of the peptide, which acts as a signal anchor for the protein’s localization to the Golgi apparatus via the ER. For such proteins, we suggest expressing an N-terminus truncated version of the protein, lacking the N-terminal TMD but containing the complete functional domain. This may result in better solubility of the heterologously expressed protein and hence an easier extractability from the host cells (see below for details).
    4.  If the His6-tag is to be fused to the protein’s N-terminus, the position of the N-terminal truncation of the protein has to be chosen carefully. We truncated the GT upstream of the most prominent stretch of charged amino acids between the TMD and the conserved domain to allow for an optimal presentation of the histidine-tag [please refer to Ade et al. (2014) for details on the exact positions of protein truncation and His6 tagging].

  2. Extraction of functional heterologous protein from Sf9 insect cells
    1. Extract native recombinant protein PrGT34B from 8 x 108 freshly pelleted Sf9 cells using ice-cold hypotonic lysis buffer (20 ml). Lyse the cells on ice (40 min) and then mix the lysate on an orbital shaker (50 rpm, 2 h, 4 °C).
    2. Clarify the lysate by centrifugation (14,000 x g, 5 min, 4 °C) and transfer the supernatant containing the Triton X-100 solubilized protein to a fresh tube.
    3. Proceed to the purification of the His6-tagged recombinant protein PrGT34B from the total extracted protein as soon as possible.
      Note: We kept the protein extract on ice for no longer than 2 h before proceeding to protein purification to avoid loss of functional protein by protein degradation, protein precipitation or protein denaturation.
    4. In addition, extract total protein from 8 x 108 freshly pelleted Sf9 cells infected with non-recombinant baculovirus or with baculovirus recombinant for empty vector pFastBacHTaTM for negative control reactions required to calculate background GT activities (see below).

  3. Purification of the His6-tagged recombinant GT using immobilized metal affinity chromatography (IMAC)
    Note: The TALON resin consists of Sepharose beads with a tetradentate metal chelator that holds Co2+ ions in electronegative pockets. At neutral pH, the histidine side groups of the target protein’s polyhistidine tags are unprotonated and bind to the chelated cobalt ions. Under these conditions, increasing imidazole concentrations in the elution buffer during chromatography competitively elutes bound polyhistidine-tagged protein from the resin. Purification of the GT is required to precisely determine the quantity of specific protein to be used in the enzymatic assay. Only in this way can the specific enzymatic activity of the GT be calculated.
    1. Load an empty gravity-flow chromatography column with 2 ml of a 50% (w/v) TALON metal affinity resin slurry in 20% ethanol at room temperature.
    2. After the resin has settled, apply a further frit and wash the column with double distilled water (ddH2O) (20 ml) at room temperature.
    3. Equilibrate the TALON resin with equilibration/wash buffer (10 ml) at room temperature.
    4. Mix the total protein lysate (20 ml) (see above) with ice-cold sample preparation buffer (5 ml).
    5. Load the ice-cold 25 ml of soluble protein lysate, adjusted to 50 mM HEPES-NaOH (pH 7.0), 100 mM NaCl, 5 mM imidazole, 10% glycerol, 1.6% Triton X-100, 1.6 mM MgCl2, on to the column.
      Note: The increased ionic strength of the sample adjusted to 100 mM NaCl minimizes non-specific binding to the resin and stabilizes the soluble protein on the column during fractionation. The initial imidazole concentration of 5 mM increases the stringency for the affinity binding and decreases background protein binding.
    6. Wash the TALON resin with equilibration/wash buffer (20 ml) and then elute His6-tagged protein from the column with IMAC elution buffer (15 ml) at room temperature.
    7. Capture successive fractions (1 ml) of the eluted protein and directly store on ice. Analyze aliquots (10 µl) by standard SDS-PAGE to determine the fraction with the highest concentration and purity of the recombinant protein to be used in subsequent enzymatic assays.
    8. Keep eluted soluble protein at 4 °C for only a short time before determining its enzymatic activity.
      Note: We kept the purified protein on ice for no longer than 24 h before using it in GT assays to avoid loss of functional protein by protein degradation, protein precipitation or protein denaturation.

  4. Identification of heterologously expressed recombinant protein PrGT34B by immunoblot analysis and peptide mass fingerprinting (PMF)
    Note: Verification of the recombinant protein’s identity was achieved by a combination of immunoblot analysis of the His6-tagged protein and a mass spectrometric peptide analysis.
    1. Denature an aliquot (10 µl) of the total protein extract (the total protein extracted from 5.0 x 105 Sf9 cells) and of the IMAC purified and eluted protein by heating in PAGE loading dye (5 min at 95 °C).
    2. Separate the denatured protein on a 12% polyacrylamide gel together with a suitable marker such as the BenchMark His-tagged protein standard.
    3. Transfer the protein to a PVDF transfer membrane using any kind of suitable electrophoretic protein transfer method.
    4. Detect the His6-tagged protein with a mouse anti-His6 monoclonal antibody (3,000-fold diluted) in combination with a horseradish peroxidase (HRP) conjugated secondary sheep anti-mouse antibody (7,000-fold diluted) using any kind of suitable hybridization protocol.
    5. Visualize chemiluminescent signals on the membrane using the Amersham ECL detection system and any kind of suitable analysis system such as the LAS-3000 Lite with the software LAS-3000 Image Reader.
    6. Optional: In addition to identification of the His6-tagged protein in the total protein extract and the IMAC eluted fraction by immunoblot analysis, separate a second aliquot of the IMAC purified heterologously expressed protein by standard SDS-PAGE. Stain the polyacrylamide gel with Coomassie blue, excise the relevant protein band and subject to PMF, using any kind of suitable commercial provider or academic proteomics facility.
      Note: We recommend the analysis of the purified protein once by PMF to unambiguously verify its identity.

  5. On-gel quantification of the purified protein PrGT34B
    Note: The purity of the His6-tagged recombinant protein after IMAC purification is not 100%. Consequently, accurate quantification of one specific protein in a protein mixture is not possible by spectrophotometric methods such as the NanoDrop ND-1000 and requires alternative quantification methods.
    1. Load equal volumes (10 µl) of different dilutions (e.g. undiluted, 2, 5, 10 and 20-fold diluted) of the IMAC purified heterologously expressed protein together with equal volumes (20 µl) of different dilutions of a quantitative protein mass ladder such as the Precision Plus protein unstained standard on a 12% polyacrylamide gel.
    2. Separate the protein by any kind of standard SDS-PAGE protocol.
    3. Stain the gel with the quantitative luminescent SYPRO Ruby protein gel stain following the manufacturer’s instructions.
    4. Visualize the fluorescence signals with any kind of suitable analysis system such as the FLA 2000 phosphorimager with FLA 2000G software.
      Note: The emission maximum of the SYPRO Ruby protein stain is 610 nm.
    5. Quantify the protein bands of the previously identified specific protein species on the gel based on a calibration curve obtained from the known quantities of protein in the reference bands of the various dilutions of the quantitative protein ladder using the software Multi Gauge v2.2.
      Note: The 100, 50 and 20 kDa bands of undiluted Precision Plus protein standard (20 µl) correspond to 300, 1,500 and 300 ng of protein, respectively.


      (C)
      No
      Description
      LAU
      Area (mm2)
      BG
      LAU-BG
      Amount of protein on gel (loaded)
      21
      Background
      27.19
      6.83
      27.19
      0
      0 ng
      1
      Standard
      18.16
      2.72
      12.73
      5.44
      75.0 ng
      2
      Standard
      19.80
      2.72
      10.81
      8.98
      112.5 ng
      3
      Standard
      32.02
      4.23
      16.83
      15.19
      150.0 ng
      4
      Standard
      43.80
      4.24
      16.88
      26.92
      225.0 ng
      5
      Standard
      57.65
      4.54
      18.05
      39.60
      300.0 ng
      6
      Standard
      90.78
      5.11
      20.34
      70.44
      375.0 ng
      7
      Standard
      145.31
      6.87
      27.32
      117.99
      562.5 ng
      8
      Standard
      188.50
      8.61
      34.27
      154.23
      750.0 ng
      9
      Standard
      284.96
      11.20
      44.55
      240.40
      1125.0 ng
      10
      Standard
      385.81
      12.92
      51.40
      334.42
      1500.0 ng
      11
      Standard
      21.49
      3.97
      15.81
      5.68
      75.0 ng
      12
      Standard
      26.14
      3.74
      14.88
      11.25
      112.5 ng
      13
      Standard
      33.63
      4.98
      19.82
      13.81
      150.0 ng
      14
      Standard
      53.90
      6.13
      24.40
      29.50
      225.0 ng
      15
      Standard
      67.53
      6.66
      26.50
      41.04
      300.0 ng

      No
      Description
      LAU 
      Area (mm2)
      BG
      LAU-BG
      Amount of protein on gel (calculated)
      Protein conc. of undiluted sample
      17
      10 μl sample (20-fold dilution)
      221.24
      7.33
      29.18
      192.06
      918.07 ng
      1.8361 mg/ml
      18
      10 μl sample (10-fold dilution)
      474.50
      10.92
      43.44
      431.06
      2060.52 ng
      2.0605 mg/ml
      19
      10 μl sample (5-fold dilution)
      1031.81
      18.71
      74.45
      957.36
      4576.29 ng
      2.2881 mg/ml
      20
      10 μl sample (2-fold dilution)
      2241.51
      24.06
      95.75
      2145.75
      10256.93 ng
      2.0514 mg/ml






      Concentration mean value: 2.0590 mg/ml
      Figure 1. Quantification of IMAC purified heterologously expressed protein by fluorometric analysis of a SYPRO Ruby stained SDS-gel. Aliquots (10 μl) of four dilutions of the purified recombinant protein and five different volumes of the protein mass standard Precision Plus protein standard were separated on a 12% acrylamide gel. The gel was stained with SYPRO Ruby protein gel stain (Molecular Probes) and the fluorescent signals were visualised (A). Reference bands and bands to be quantified were manually selected and the signal intensities of the selected areas were detected as linear arbitrary units (LAU). The known protein quantities for each selected reference band and their background-corrected signal intensity values (LAU-BG) were used to generate a calibration curve (B). Protein amounts in the selected sample bands were calculated using the equation of the calibration curve. The final concentration of recombinant protein in the sample was determined by taking loaded volumes and dilution factors into account (C).

  6. Quantitative in vitro glycosyltransferase assay for the Pinus radiata xylosyltransferase PrGT34B
    General information:
    1. This protocol describes how to determine the in vitro GT activity of the xyloglucan xylosyltransferase PrGT34B by measuring, under constant reaction conditions, the transfer of the monosaccharide moiety from a nucleotide sugar such as UDP-xylose, used as the donor substrate, to an acceptor substrate such as a cello-oligosaccharide.
    2. An assay temperature of 25 °C and a pH of 6.5-7.0 were found to be optimal in a study of the activities of other GTs located in the Golgi apparatus in P. radiata (Mast et al., 2009). Furthermore, recent literature suggests that GT activities in the Golgi apparatus depend on a slightly acidic environment (Reyes and Orellana, 2008). However, successful enzymatic in vitro assays with enzymes closely related to the protein PrGT34B were done under neutral or slightly alkaline conditions in previous studies (Edwards et al., 1999; Faik et al., 2002; Cavalier and Keegstra, 2006). Consequently, we used a neutral pH for all assays described in the present protocol.
    3. For all GT activity assays described in this protocol, the protein PrGT34B was either in total Sf9 protein extract, adjusted to a total protein concentration of 10 mg/ml with hypotonic lysis buffer (see above), or in protein extract purified by IMAC and adjusted to a total protein concentration of 2.5 mg/ml with IMAC elution buffer (see above). In addition, dilutions of the extract purified by IMAC were prepared with IMAC elution buffer (see above) containing specific PrGT34B concentrations of 2,000, 100 and 40 µg/ml.
    4. Total protein extract contains HEPES-NaOH (pH 7.0) (50 mM), MgCl2 (2 mM), glycerol (10%) and Triton X-100 (2%) and the IMAC purified protein extract contains HEPES-NaOH (pH 7.0) (50 mM), NaCl (100 mM), imidazole (50 mM) and glycerol (10%).
      Note: Xyloglucan xylosyltransferase activity of the heterologously expressed protein PrGT34B was assayed with radioactively labeled UDP-[14C-U]xylose and cellobiose, cellotetraose, cellopentaose or cellohexaose.
    1. Mix protein solution (total protein extract or IMAC-purified extract) (10 µl) with reaction buffer (40 µl), containing UDP-xylose, UDP-[14C]xylose and cello-oligosaccharide as acceptor substrate.
    2. Incubate (25 °C for e.g. 1, 17 or 44 h) the resulting reaction mixture (50 µl) containing the extracted protein in HEPES-NaOH (pH 7.0) (50 mM), MnCl2 (5 mM), MgCl2 (5 mM), UDP-xylose (1 or 4 mM), UDP-[14C]xylose (9 kBq, 54,300 dpm, 150 pmol) and cello-oligosaccharides (1 mM).
      Notes:
      1. These reaction conditions correspond to 50 nmol acceptor substrate and 50 or 200 nmol unlabelled UDP-sugar donor substrate in the reaction mixture, resulting in molar ratios of acceptor and donor of 1:1 or 1:4. The amount of 150 pmol UDP-[14C] xylose added to the reaction mixture corresponds to a concentration in the reaction mixture of only 3 µM, changing the total concentration of added UDP-xylose only insignificantly.
      2. The assay can be done with any other molar ratio between acceptor and donor substrate. Other GT activities can be assayed using appropriate nucleotide sugars and donor substrates in the assay mixture such as UDP-galactose and UDP-[14C]galactose as donor substrates and manno-oligosaccharides as acceptor substrates to assay the galactosyltransferase activity of galactomannan galactosyltransferases.
    3. Stop the reaction by heating (5 min, 95 °C), remove precipitated protein by centrifugation (10,000 x g, 2 min, 4 °C) and transfer the supernatant with the reaction products to a fresh tube and place on ice.
    4. Remove unincorporated nucleotide sugars (UDP-xylose and UDP-[14C]xylose) from the reaction mixture by anion exchange chromatography (AEC) using the ion exchange resin Dowex 1x8-200 (chloride form).
      Note: The positively charged groups of the resin act as strong anion exchangers that stay charged over a wide pH range and bind negatively charged molecules such as nucleotide sugars in exchange for the counter ion chloride.
    5. Mix Dowex 1 x 8-200 resin (1 g) with water (10 ml), allow the resin to swell over-night, wash the resin twice with fresh water (10 ml), allow the resin to settle (10 min), discard the supernatant and add fresh water to give a 50% (w/v) resin-water slurry.
    6. Directly mix the cleared supernatant from the assay reaction (50 µl) with the 50% (w/v) resin-water slurry (400 µl) and incubate on an orbital shaker (50 rpm, 5 min, at room temperature).
    7. Transfer the mixture to an empty Micro Bio-Spin chromatography column fitted with a frit to retain the resin and centrifuge (700 x g, 1 min, at room-temperature).
      Note: The effluent may be collected in a fresh microcentrifuge tube. The Micro Bio-Spin column with the empty microcentrifuge tube may be placed into a 15 ml Falcon tube with lid, so the centrifugation can be done without the risk of contaminating the centrifuge.
    8. Mix the complete effluent (250-300 µl), containing the uncharged reaction products with the incorporated radioactivity, with scintillation counting fluid (Starscint high flash-point LSC-cocktail) (1 ml).
    9. Measure the incorporated radioactivity with a liquid scintillation counter for 5 min.
      Note: Conversion of measured “counts per minute” (cpm) into “disintegrations per minute” (dpm) is done automatically by the instrument using a calibration curve that is generated by plotting detected cpm values for external standards of known activity against their known spectral quench parameters [SQP(E)].
    10. Prepare control incubations for enzymatic assays identical to those described above but without protein and with protein extracts from Sf9 cells infected with non-recombinant virus to determine background radioactivity resulting from incomplete retention of unincorporated UDP-[14C]sugar during anion exchange chromatography, enzyme independent degradation of UDP-sugar and UDP-sugar hydrolyzing background activity. Treat these control incubations identically to the samples and measure the remaining unspecific background radioactivity in the AEC effluent.
    11. Calculate the xylosyltransferase activity as follows.
      Incorporation of xylose into reaction products is calculated from the total radioactivity retained in the sample according to the formula “amount of incorporated xylose into acceptor substrate per assay (in pmol) = (A/B)*C”; with A representing the radioactivity retained in the sample following anion exchange chromatography (in dpm), B representing the total radioactivity added to the assay (in dpm) and C representing the total amount of UDP-sugar (labeled + unlabeled) added to the assay (in pmol).
      1. GT activity can be shown as xylose incorporation per assay (in pmol/assay) or as xylose incorporation per time (in pmol/h) if the assays are incubated for a discrete time (e.g. 1, 17 and 44 h).
      2. For assays containing a known quantity of the heterologous protein PrGT34B, specific GT activity can be determined from the quantity (in pmol) of xylose incorporated into acceptor substrates per time and per quantity of specific enzyme (in pmol/h/µg).
      3. Background radioactivity detected in control assays (in dpm) may be used to calculate the unspecific activity in a sample or may be directly subtracted from the corresponding experimental sample to provide a background-corrected value for the calculation of enzymatic activity.

  7. Identification and characterization of PrGT34B xylosyltransferase assay reaction products by MALDI-TOF MS
    Notes:
    1. Most mass spectrometric methods are not quantitative unless an internal standard is used that is in known amounts and is structurally identical to the reaction product. Consequently, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) described here can only be seen as a semi-quantitative method to determine GT activity. However, this procedure not only shows GT activity by detecting the expected reaction products but also allows the structural characterization of all reaction products generated by the enzyme PrGT34B.
    2. MALDI-TOF MS has been proven highly efficient for desorption and ionization of various biomolecules including oligosaccharides (Garozzo et al., 1995; Garozzo et al., 1997).
    1. Prepare the reaction mixture with extract containing the recombinant protein (or without recombinant protein for control reactions) exactly as described above (step 6F) but without adding radioactively labeled UDP-xylose.
    2. Incubate (25 °C for e.g. 17 h) the reaction mixture (50 µl), containing the extracted protein in HEPES-NaOH (pH 7.0) (50 mM), MnCl2 (5 mM), MgCl2 (5 mM), UDP-xylose (1 or 4 mM), and cello-oligosaccharides (1 mM).
      Note: The incubation time was chosen sufficiently long to allow for maximum incorporation of xylose.
    3. Stop the reaction by heating (5 min, 95 °C), remove precipitated protein by centrifugation (10,000 x g, 2 min, 4 °C) and transfer the supernatant with the reaction products to a fresh tube and place on ice.
    4. Mix an aliquot of the supernatant (1 µl) with the MALDI matrix 2,5-dihydroxybenzoic acid (10 mg/ml in ddH2O) (9 µl).
    5. Spot an aliquot of the sample-matrix mixture (1 µl) on a MALDI plate and air dry to allow crystallization of analyte and matrix molecules on the plate.
      Note: It is important to minimize the glycerol concentration in the mixture to make sure the molecules crystallize uniformly on the plate.
    6. Load the MALDI plate to a MALDI-TOF mass spectrometer such as the Voyager DETM PRO MALDI-TOF workstation.
      Notes:
      1. The spectrometer was calibrated using the three external standards des-ArgI-bradykinin (m/z = 904), angiotensin I (m/z = 1296) and [GluI]-fibrinopeptide B (m/z = 1570) diluted in 2,5-dihydroxybenzoic acid.
      2. Operate the spectrometer in the reflectron mode at an accelerating voltage of 20 kV with a delay time of 200 ns.
    7. Compile mass spectra for each sample by plotting the mass (m) to charge (z) ratio (x-axis) of all detected ion species against their measured intensities (y-axis) using any suitable software such as the Data Explorer (version 4.0.0.0).
      Note: Compilation of each mass spectrum should consist of the accumulated data from sufficient numbers of laser shots (e.g. 100 laser shots).
    8. Identify substrates and GT reaction products in the mass spectrum based on the molecular weights of their singly charged (z = 1) sodium adduct ions [M+Na]+.
      Notes:
      1. Because of the high NaCl concentration in the sample-matrix mixture (> 1 mM), the dominant ion species in positive ionization mode are the sodium adducts of the protonated molecular ions of the analyte.
      2. Use the mass spectra generated for control reactions done without enzyme to identify peaks that correlate to reaction products of the recombinant protein PrGT34B.
      3. Choose a suitable base peak intensity threshold to identify peaks that are above the noise in the mass spectrum.

Recipes

  1. PAGE loading dye
    SDS (1%)
    β-mercaptoethanol (1 mM)
    Glycerol (10%)
    Bromophenol blue
  2. Hypotonic lysis buffer
    50 mM HEPES-NaOH (pH 7.0)
    2 mM MgCl2
    10% glycerol (v/v)
    2% Triton X-100 (v/v)
    Benzonase® nuclease (10 U/ml)
    Protease inhibitors
    Prepare in double distilled water (ddH2O)
  3. Equilibration/wash buffer for IMAC
    50 mM HEPES-NaOH (pH 7.0)
    100 mM NaCl
    5 mM imidazole
    10 % glycerol (v/v)
    Prepare in ddH2O
  4. Sample preparation buffer for IMAC
    50 mM HEPES-NaOH (pH 7.0)
    500 mM NaCl
    25 mM imidazole
    10% glycerol (v/v)
    Prepare in ddH2O
  5. IMAC elution buffer
    50 mM HEPES-NaOH (pH 7.0)
    100 mM NaCl
    50 mM imidazole
    10% glycerol (v/v)
    Prepare in ddH2O
  6. Reaction buffer for enzymatic assays (with radioactively labeled UDP-xylose)
    50 mM HEPES-NaOH (pH 7.0)
    6.25 mM MnCl2
    6.25 mM MgCl2
    1.25 or 5 mM UDP-xylose
    1.25 mM oligosaccharide acceptor substrate (cellobiose, cellotetraose, cellopentaose or cellohexaose)
    UDP-[14C]xylose (9 kBq, 54,300 dpm, 150 pmol)
    Prepare in ddH2O
  7. Reaction buffer for enzymatic assays (without radioactively labeled UDP-xylose)
    50 mM HEPES-NaOH (pH 7.0)
    6.25 mM MnCl2
    6.25 mM MgCl2
    1.25 or 5 mM UDP-xylose
    1.25 mM oligosaccharide acceptor substrate (cellobiose, cellotetraose, cellopentaose or cellohexaose)
    Prepare in ddH2O

Acknowledgments

This work was supported by funding from the New Zealand Foundation for Research, Science and Technology, and the University of Auckland. We thank CarboSource Services (Complex Carbohydrate Research Center, Athens, GA) for the UDP-xylose and Scion Research for a doctoral stipend for Carsten P. Ade. Parts of the procedure described here were modified from Cavalier and Keegstra (2006).

References

  1. Ade, C. P., Bemm, F., Dickson, J. M., Walter, C. and Harris, P. J. (2014). Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine). Plant J 78(2): 305-318.
  2. Berger, I., Fitzgerald, D. J. and Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22(12): 1583-1587.
  3. Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V. and Henrissat, B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37(Database issue): D233-238.
  4. Cavalier, D. M. and Keegstra, K. (2006). Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexaose. J Biol Chem 281(45): 34197-34207.
  5. Condreay, J. P. and Kost, T. A. (2007). Baculovirus expression vectors for insect and mammalian cells. Curr Drug Targets 8(10): 1126-1131.
  6. Doblin, M. S., Pettolino, F. and Bacic, A. (2010). Evans Review: Plant cell walls: the skeleton of the plant world. Functional Plant Biology 37(5): 357-381.
  7. Driouich, A., Follet-Gueye, M. L., Bernard, S., Kousar, S., Chevalier, L., Vicre-Gibouin, M. and Lerouxel, O. (2012). Golgi-mediated synthesis and secretion of matrix polysaccharides of the primary cell wall of higher plants. Front Plant Sci 3: 79.
  8. Edwards, M. E., Dickson, C. A., Chengappa, S., Sidebottom, C., Gidley, M. J. and Reid, J. S. (1999). Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis. Plant J 19(6): 691-697.
  9. Egelund, J., Petersen, B. L., Motawia, M. S., Damager, I., Faik, A., Olsen, C. E., Ishii, T., Clausen, H., Ulvskov, P. and Geshi, N. (2006). Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II. Plant Cell 18(10): 2593-2607.
  10. Faik, A., Price, N. J., Raikhel, N. V. and Keegstra, K. (2002). An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis. Proc Natl Acad Sci U S A 99(11): 7797-7802.
  11. Garrozzo, D., Impallomeni, G., Spina, E., Sturiale, L. and Zanetti, F. (1995). Matrix‐assisted laser desorption/ionization mass spectrometry of polysaccharides. Rapid Commun Mass Spectrom 9(10): 937-941.
  12. Garozzo, D., Nasello, V., Spina, E. and Sturiale, L. (1997). Discrimination of isomeric oligosaccharides and sequencing of unknowns by post source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 11(14): 1561-1566.
  13. Keegstra, K. and Cavalier, D. (2011). Glycosyltransferases of the GT34 and GT37 families. In Annual Plant Reviews, Volume 41. In: Ulvskov, P., (ed). Plant Polysaccharides, Biosynthesis and Bioengineering Oxford. Blackwell Publishing, pp. 235-249.
  14. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. and Henrissat, B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42(Database issue): D490-495.
  15. Mast, S. W., Donaldson, L., Torr, K., Phillips, L., Flint, H., West, M., Strabala, T. J. and Wagner, A. (2009). Exploring the ultrastructural localization and biosynthesis of beta(1,4)-galactan in Pinus radiata compression wood. Plant Physiol 150(2): 573-583.
  16. Scheller, H. V. and Ulvskov, P. (2010). Hemicelluloses. Annu Rev Plant Biol 61: 263-289.

简介

糖基转移酶是含有催化模块的碳水化合物活性酶,其涉及催化寡糖和多糖和糖缀合物中糖苷键的生物合成。 Carbohydrate Active enZYmes最全面的集合之一是CAZy数据库( http://www.cazy.org ) ),其包含分配给96个家族的120,000个糖基转移酶,主要基于其保守和催化活性结构域的序列同源性(Cantarel等人,2009)。有趣的是,仅约1.6%的这些蛋白质的糖基转移酶活性已经通过实验表征(Lombard等人,2014)。近年来,已显示许多家族的膜结合糖基转移酶在植物细胞壁多糖的生物合成中起关键作用(Doblin等人,2010; Scheller和Ulvskov,2010 ; Driouich等人,2012)。它们催化糖基残基从供体核苷酸糖到受体的转移,形成相邻糖基残基之间的糖苷键。家族34含有已经显示参与木葡聚糖的生物合成并将木糖残基转移到(1→4)-β-葡聚糖链的糖基转移酶(Keegstra和Cavalier,2011)。我们以前的工作表明辐射蛋白质PrGT34B是木葡聚糖(1→6)-α-木糖基转移酶(Ade等人,2014)。在这里,我们描述了用于确定PrGT34B在体外的木糖基转移酶活性的程序。我们测量了在受控反应条件下木糖从供体底物UDP-木糖到不同的纤维寡糖受体底物的转移。测定包括放射性标记的反应产物的定量和它们通过质谱法的鉴定。我们还描述了异源表达的重组蛋白PrGT34B的纯化,鉴定和定量,为其在测定中的使用做准备。该方法可应用于许多不同植物物种中的宽范围的糖基转移酶。

材料和试剂

  1. 草地贪夜蛾(Spodoptera frugiperda)细胞(Sf9)(Life Technologies,Invitrogen TM ,目录号:11496-015)
  2. 载体pFastBacHTa (Life Technologies,Invitrogen TM ,目录号:10584-027)
  3. 大肠杆菌(大肠杆菌)菌株DH10Bac(Life Technologies,Invitrogen TM ,目录号:10359-016)
  4. 20%乙醇
  5. SDS
  6. β-巯基乙醇
  7. 甘油
  8. 溴酚蓝
  9. HEPES-NaOH(pH 7.0)
  10. MgCl 2
  11. MnCl 2
  12. Triton X-100
  13. Benzonase核酸酶(Novagen,目录号:70746)
  14. 蛋白酶抑制剂(完全不含EDTA,蛋白酶抑制剂混合物片)(Roche Diagnostics,目录号:04693159001)
  15. 咪唑
  16. Cellotetraose,cellopentaose和cellohexaose(Associates of Cape Cod,Northstar BioProducts ,目录号:400402-1,400404-1,400406-1)
  17. 纤维二糖(Sigma-Aldrich,目录号:C7252)
  18. UDP-D-木糖(CarboSource Services,Complex Carbohydrate Research Center,product code:UDP-Xylose)
  19. UDP- [sup 14 C-U]木糖(151.8mCi/mmol)(PerkinElmer,目录号:NEC543005UC)
  20. TALON金属亲和树脂(Takara Bio Company,Clontech,目录号:635501)
  21. BenchMark His标签蛋白标准品(Life Technologies,Invitrogen TM ,目录号:LC5606)
  22. Precision Plus蛋白质未标记标准品(Bio-Rad Laboratories,目录号:161-0363)
  23. 小鼠抗His 6单克隆抗体(GE Healthcare,产品代码:27-4710-01)
  24. 辣根过氧化物酶(HRP)缀合的次级绵羊抗小鼠抗体(GE Healthcare,产品代码:NA931-100UL)
  25. SYPRO Ruby蛋白凝胶染色(Life Technologies,Molecular Probes ,目录号:S-21900)
  26. 离子交换树脂Dowex 1x8-200(氯化物形式)(Sigma-Aldrich,目录号:217425)
  27. 2,5-二羟基苯甲酸(Sigma-Aldrich,目录号:85707)
  28. Starscint高闪点LSC鸡尾酒(PerkinElmer)
  29. PAGE装染料(见配方)
  30. 低渗裂解缓冲液(参见配方)
  31. IMAC的平衡/清洗缓冲液(参见配方)
  32. IMAC的样品制备缓冲液(参见配方)
  33. IMAC洗脱缓冲液(参见配方)
  34. 用于酶测定(用放射性标记的UDP-木糖)的反应缓冲液(参见配方)
  35. 用于酶测定(没有放射性标记的UDP-木糖)的反应缓冲液(参见配方)

设备

  1. 空重力流动色谱柱(20ml体积)(Bio-Rad Laboratories,目录号:732-1010)
  2. Micro Bio-Spin色谱柱(Bio-Rad Laboratories,目录号:732-6204)
  3. Amersham ECL检测系统(GE Healthcare,产品代码:RPN2105)
  4. PVDF转移膜(Amersham Hybond-P)(GE Healthcare,产品代码:10600023)
  5. NanoDrop ND-1000分光光度计(Thermo Fisher Scientific)
  6. LAS-3000 Lite成像器用软件LAS-3000图像读取器(Fujifilm Corporation)
  7. FLA 2000磷光计,带有软件FLA 2000G(Fujifilm Corporation)
  8. Rackbeta LKB Wallac(PerkinElmer,型号:1209)
  9. Voyager DE TM PRO MALDI-TOF Workstation(Life Technologies,Applied Biosystems )
    注意:我们认为实验的重复性不取决于上述精确设备,但可以使用任何其他等效仪器实现。

软件

  1. Software Multi Gauge v2.2(Fujifilm Corporation)
  2. 数据资源管理器(版本4.0.0.0)(Life Technologies,Applied Biosystems )
  3. 软件FLA 2000G(Fujifilm Corporation)
  4. 软件LAS-3000图像读取器(Fujifilm Corporation)

程序

  1. 糖基转移酶的异源表达
    注意:有大量的协议描述蛋白质的异源表达的程序。 在细菌,酵母和各种昆虫细胞系统中已经实现了植物糖基转移酶蛋白在酶分析中的成功表达。 我们不给出蛋白质PrGT34B的异源表达的详细描述,而仅仅概述我们使用的程序,并给出可能有用的一些信息。
    1. 在我们的研究中(Ade等人,2014),我们表达木糖基转移酶 PrgT34B在草地贪夜蛾(Spodoptera frugiperda)细胞(Sf9)中的表达 基于核多克隆病毒的杆状病毒表达 矢量系统。
      1. 细胞系Sf9先前已经用过 成功表达植物糖基转移酶(Egelund等人,2006)。 此外,Sf9细胞对于苜蓿银纹夜蛾(Autographa californica)核多糖病病毒的感染高度敏感,并且相应的 杆状病毒表达载体显示导致高表达 所需基因产物在昆虫细胞中的水平(Condreay和Kost, 2007)。
      2. 将感兴趣的序列克隆到载体中 pFastBacHTa ,在E中产生重组杆粒DNA。大肠杆菌菌株 DH10Bac或DH10MultiBac(Berger等人,2004),杆状病毒是 按Sf9细胞中的说明在Sf9细胞中产生和扩增 手动"Bac-to-Bac杆状病毒表达系统"。
      3. Sf9 用杆状病毒(1:1,000v/v的扩增培养基)接种起始浓度为2.0×10 6个细胞ml -1 -1的悬浮培养物(200ml) 病毒原种至Sf9细胞悬浮液),并在摇动(130rpm,   72小时,28℃)。 通过离心(500×g)收集感染的细胞,   10分钟,4℃),和用于蛋白质提取的细胞沉淀 以下详细)。
    2. 为了容易纯化重组体 蛋白在其用于酶测定之前,我们强烈建议 组氨酸标记的(6x组氨酸)融合蛋白。
    3. 在 此外,许多植物GT,包括木糖基转移酶PrGT34B II型膜结合蛋白与单个跨膜结构域(TMD) 接近肽的N-末端,其充当信号 锚定蛋白定位到高尔基体通过ER。   对于这样的蛋白质,我们建议表达N末端截短 版本的蛋白质,缺乏N末端TMD但含有 完整的功能域。 这可以导致更好的溶解性 异源表达的蛋白质,因此更容易从中提取   宿主细胞(详见下文)。
    4.  如果他的 6 标签是 融合到蛋白质的N末端,N末端的位置 必须仔细选择蛋白质的截短。 我们截断了 GT上游最突出的带电氨基酸之间   TMD和保守结构域以允许最佳表达 的组氨酸标签[参见Ade et al。(2014) 蛋白质截短的准确位置和His 6标记]。

  2. 从Sf9昆虫细胞中提取功能性异源蛋白
    1. 从8×10 8个新鲜沉淀物中提取天然重组蛋白PrGT34B   Sf9细胞,使用冰冷的低渗裂解缓冲液(20ml)。 裂解细胞   在冰上(40分钟),然后在定轨振荡器(50rpm,   h,4℃)。
    2. 通过离心澄清裂解物(14,000×g,5 min,4℃),并转移含有Triton X-100的上清液 溶解的蛋白质到新管中
    3. 进行净化 的来自总提取的His 6标记的重组蛋白PrGT34B 蛋白质。
      注意:我们保留了蛋白质提取物 冰不超过2小时,然后进行蛋白质纯化 避免蛋白质降解,蛋白质损失功能性蛋白质 沉淀或蛋白质变性。
    4. 另外,提取总  蛋白质从8×10 8个新鲜沉淀的Sf9细胞感染 非重组杆状病毒或用杆状病毒重组体空载 载体pFastBacHTa TM 用于计算所需的阴性对照反应  背景GT活动(见下文)。

  3. 使用固定化金属亲和层析(IMAC)纯化His6标记的重组GT 注意:TALON树脂由具有在电负性袋中保持Co 2 + 离子的四齿金属螯合剂的Sepharose珠组成。在中性pH下,靶蛋白的多组氨酸标签的组氨酸侧基未质子化并与螯合的钴离子结合。在这些条件下,在层析期间增加洗脱缓冲液中的咪唑浓度竞争性洗脱结合的多组氨酸标签蛋白 从树脂。 需要纯化GT以精确测定在酶测定中使用的特定蛋白质的量。 只有这样,才能计算GT的特定酶活性。
    1. 加载空重力流动色谱柱用2ml的50% (w/v)TALON金属亲和树脂浆液在20%乙醇中 温度
    2. 树脂沉降后,再涂一块玻璃料 并在室温下用双蒸水(ddH 2 O)(20ml)洗涤柱 温度
    3. 在室温下用平衡/洗涤缓冲液(10ml)平衡TALON树脂
    4. 混合总蛋白裂解液(20ml)(见上文)与冰冷的样品制备缓冲液(5ml)。
    5. 加载冰冷的25 ml可溶性蛋白裂解液,调节至50 mM   HEPES-NaOH(pH 7.0),100mM NaCl,5mM咪唑,10%甘油,1.6% Triton X-100,1.6mM MgCl 2,加到柱上 注意:增加 调节至100mM NaCl的样品的离子强度最小化 非特异性结合到树脂上并稳定可溶性蛋白质 在分馏期间的柱。 初始咪唑浓度为5   mM增加亲和结合的严格性并降低 背景蛋白结合。
    6. 用TALON树脂洗涤 平衡/洗涤缓冲液(20ml),然后洗脱His 6+标记的蛋白质 在室温下用IMAC洗脱缓冲液(15ml)从柱中洗脱
    7. 捕获洗脱蛋白的连续级分(1ml) 直接存储在冰上。 通过标准SDS-PAGE分析等分试样(10μl) 确定具有最高浓度和纯度的级分 重组蛋白用于随后的酶测定
    8. 在确定其酶活性之前,将洗脱的可溶性蛋白保持在4℃很短的时间 注意:我们保持纯化的蛋白质在冰上不超过24小时 之前使用它在GT测定中以避免功能蛋白的损失 蛋白质降解,蛋白质沉淀或蛋白质变性。

  4. 通过免疫印迹分析和肽质量指纹图谱(PMF)鉴定异源表达的重组蛋白PrGT34B
    注意:通过His 6标记蛋白的免疫印迹分析和质谱肽分析的组合来实现重组蛋白的同一性的验证。
    1. 使总蛋白提取物的等分试样(10μl)变性 从5.0×10 5 Sf9细胞提取的蛋白质)和纯化的IMAC  通过在PAGE装载染料中加热(在95℃5分钟)洗脱蛋白质
    2. 在12%聚丙烯酰胺凝胶上分离变性蛋白   合适的标记如BenchMark His-标记的蛋白标准品
    3. 使用任何种类的合适的电泳蛋白转移方法将蛋白转移到PVDF转移膜
    4. 用小鼠抗His 6单克隆抗体检测His 6 6 -Tagged蛋白 抗体(3000倍稀释)与辣根组合 过氧化物酶(HRP)结合的次级羊抗小鼠抗体 (7,000倍稀释)使用任何种类的合适的杂交方案
    5. 用膜显示膜上的化学发光信号 Amersham ECL检测系统和任何类型的合适分析系统 例如LAS-3000 Lite与软件LAS-3000图像读取器。
    6. 可选:除了鉴定His 6标记的蛋白质  总蛋白提取物和IMAC通过免疫印迹洗脱的级分 分析,分离异源纯化的IMAC的第二等分试样 通过标准SDS-PAGE表达蛋白。染色聚丙烯酰胺凝胶 与考马斯蓝,切除相关蛋白条带和受试者 PMF,使用任何类型的合适的商业提供者或学术 蛋白质组学设施 注意:我们建议PMF对纯化的蛋白质进行一次分析,以明确验证其身份。

  5. 纯化的蛋白PrGT34B的凝胶定量 注意:IMAC纯化后His 6标记的重组蛋白的纯度不是100%。因此,通过诸如NanoDrop ND-1000的分光光度法不可能准确定量蛋白质混合物中的一种特定蛋白质,并且需要备选的定量方法。
    1. 将等体积(10μl)的不同稀释液(例如,未稀释的,   10和20倍稀释)纯化的异源表达的IMAC 蛋白质与等体积(20μl)的不同稀释度的a 定量蛋白质质量梯度如Precision Plus蛋白 在12%聚丙烯酰胺凝胶上的未染色标准物
    2. 用任何种类的标准SDS-PAGE方法分离蛋白质
    3. 按照制造商的说明用定量荧光SYPRO Ruby蛋白凝胶染色法染色凝胶
    4. 用任何种类的合适的可视化荧光信号 分析系统,例如具有FLA 2000G的FLA 2000磷光成像仪 软件 注意:SYPRO Ruby蛋白染料的发射最大值为610 nm。
    5. 量化以前确定的特异性的蛋白质条带 蛋白质物质在凝胶上基于从中获得的校准曲线 在各种参考条带中已知量的蛋白质 稀释的定量蛋白梯使用软件Multi 规格v2.2。
      注意:未稀释的100,50和20 kDa条带 Precision Plus蛋白标准品(20μl)对应于300,1,500和300   ng /蛋白质。


      (C)

      说明
      LAU
      面积(mm 2
      BG
      LAU-BG
      凝胶上的蛋白质量(已装载)
      21
      背景
      27.19
      6.83
      27.19
      0
      0 ng
      1
      标准
      18.16
      2.72
      12.73
      5.44
      75.0 ng
      2
      标准
      19.80
      2.72
      10.81
      8.98
      112.5 ng
      3
      标准
      32.02
      4.23
      16.83
      15.19
      150.0 ng
      4
      标准
      43.80
      4.24
      16.88
      26.92
      225.0 ng
      5
      标准
      57.65
      4.54
      18.05
      39.60
      300.0 ng
      6
      标准
      90.78
      5.11
      20.34
      70.44
      375.0 ng
      7
      标准
      145.31
      6.87
      27.32
      117.99
      562.5 ng
      8
      标准
      188.50
      8.61
      34.27
      154.23
      750.0 ng
      9
      标准
      284.96
      11.20
      44.55
      240.40
      1125.0 ng
      10
      标准
      385.81
      12.92
      51.40
      334.42
      1500.0 ng
      11
      标准
      21.49
      3.97
      15.81
      5.68
      75.0 ng
      12
      标准
      26.14
      3.74
      14.88
      11.25
      112.5 ng
      13
      标准
      33.63
      4.98
      19.82
      13.81
      150.0 ng
      14
      标准
      53.90
      6.13
      24.40
      29.50
      225.0 ng
      15
      标准
      67.53
      6.66
      26.50
      41.04
      300.0 ng


      说明
      LAU
      面积(mm 2
      BG
      LAU-BG
      凝胶上蛋白质的量(计算)
      蛋白质浓度 的未稀释样品
      17
      10μl样品(20倍稀释)
      221.24
      733
      29.18
      192.06
      918.07 ng
      1.8361mg/ml
      18
      10μl样品(10倍稀释)
      474.50
      10.92
      43.44
      431.06 / 2060.52 ng
      2.0605mg/ml
      19
      10μl样品(5倍稀释)
      1031.81
      18.71
      74.45
      957.36
      4576.29 ng
      2.2881mg/ml
      20
      10μl样品(2倍稀释)
      2241.51
      24.06
      95.75
      2145.75
      10256.93 ng
      2.0514mg/ml






      浓度平均值:2.0590mg/ml
      图1.纯化的异源表达的IMAC的定量 蛋白通过SYPRO Ruby染色的SDS-凝胶的荧光分析。 等分试样(10μl)的纯化的重组蛋白的四个稀释液 和五种不同体积的蛋白质质量标准品Precision Plus 蛋白质标准品在12%丙烯酰胺凝胶上分离。凝胶是 用SYPRO Ruby蛋白凝胶染色(Molecular Probes)染色 荧光信号可视化(A)。 参考波段和波段   手动选择和定量的信号强度 选择的区域被检测为线性任意单位(LAU)。 已知 每个选择的参考条带和它们的蛋白质量 背景校正的信号强度值(LAU-BG) 生成校准曲线(B)。 所选样品中的蛋白质含量   使用校准曲线的方程计算频带。 的 测定样品中重组蛋白的最终浓度 通过考虑加载体积和稀释因子(C)。

  6. 辐射松木糖基转移酶PrGT34B的定量糖基转移酶测定
    一般信息:
    1. 此协议描述如何确定 体外 GT活动 木葡聚糖木糖基转移酶PrGT34B 反应条件,单糖部分从a 核苷酸糖例如UDP-木糖,用作供体底物 受体底物如纤维寡糖。
    2. 测定 25℃的温度和6.5-7.0的pH是最佳的 研究位于高尔基体的其他GTs的活性 辐射虫(Mast 等人 ,2009)。此外,最近的文献表明 高尔基体中的GT活性取决于微酸性 环境(Reyes和Orellana,2008)。然而,成功的酶  使用与蛋白PrGT34B密切相关的酶的体外测定 在以前的研究中在中性或微碱性条件下进行 (Edwards 等人,1999; Faik等人,2002; Cavalier和Keegstra,2006)。 因此,我们使用中性pH用于所述的所有测定 现行协议。
    3. 对于本文所述的所有GT活性测定 方案,蛋白PrGT34B是总Sf9蛋白提取物, 调整至低蛋白浓度为10mg/ml的总蛋白浓度 裂解缓冲液(参见上文),或通过IMAC纯化的蛋白质提取物中 用IMAC洗脱调节至总蛋白浓度为2.5mg/ml  缓冲区(见上文)。此外,通过纯化的提取物的稀释度 IMAC用含有IMAC洗脱缓冲液(见上文)制备 特异性PrGT34B浓度为2,000,100和40μg/ml。
    4. 总计  蛋白质提取物含有HEPES-NaOH(pH 7.0)(50mM),MgCl 2(2mM), 甘油(10%)和Triton X-100(2%)和IMAC纯化的蛋白质 提取物含有HEPES-NaOH(pH 7.0)(50mM),NaCl(100mM),咪唑 (50mM)和甘油(10%)。
      注意:木葡聚糖木糖基转移酶 测定异源表达的蛋白PrGT34B的活性 与放射性标记的UDP- [ 14]木糖和纤维二糖, 纤维四糖,纤维五糖或纤维六糖。
    1. 混合蛋白溶液(总蛋白提取物或IMAC纯化的提取物) (10μl)与含有UDP-木糖的反应缓冲液(40μl) UDP- [14 C]木糖和纤维寡糖作为受体底物。
    2. 孵育(25℃,例如1,17或44小时)所得反应混合物 (50mM),含有提取的蛋白质的HEPES-NaOH(pH7.0)(50mM)   MnCl 2(5mM),MgCl 2(5mM),UDP-木糖(1或4mM),UDP- [14 C] ]木糖 kBq,54,300dpm,150pmol)和纤维寡糖(1mM) 注意:
      1. 这些反应条件对应于50nmol受体底物 和50或200nmol未标记的UDP-糖供体底物 混合物,得到受体和供体的摩尔比为1:1或1:4。 加入到反应混合物中的150pmol UDP- [反式] 14 [木糖]木糖的量 对应于反应混合物中的浓度仅为3μM, 改变仅添加的UDP-木糖的总浓度 不显着。
      2. 该测定可以以任何其它摩尔比进行   在受体和供体底物之间。 其他GT活动可以 使用合适的核苷酸糖和供体底物测定 测定混合物如UDP-半乳糖和UDP- [ 14 C]半乳糖作为供体 底物和甘露寡糖作为受体底物进行测定 半乳甘露聚糖的半乳糖基转移酶活性 半乳糖基转移酶。
    3. 通过加热停止反应(5分钟,95℃) 通过离心(10,000×g,2分钟,4℃)除去沉淀的蛋白质 ℃),并将含有反应产物的上清液转移至新鲜 管和冰上放置
    4. 去除未并入的核苷酸糖 (UDP-木糖和UDP- [14 C]木糖)通过阴离子从反应混合物中分离 交换色谱(AEC),使用离子交换树脂Dowex 1x8-200   (氯化物)。
      注意:树脂的带正电荷的基团 充当在宽pH范围内保持电荷的强阴离子交换剂 并结合带负电荷的分子例如核苷酸糖 交换抗衡离子氯化物。
    5. 混合Dowex 1×8-200树脂 (1g)与水(10ml)混合,使树脂溶胀过夜,洗涤 树脂用淡水(10ml)洗涤两次,使树脂沉降(10分钟) min),弃去上清液并加入淡水得到50%(w/v) 树脂 - 水浆料
    6. 直接混合从澄清的上清液   测定反应(50μl)与50%(w/v)树脂 - 水浆液(400μl) 并在轨道摇床上(50rpm,室温下5分钟)孵育
    7. 将混合物转移至空的Micro Bio-Spin色谱 柱,其装有玻璃料以保留树脂并离心(700×g,   min,在室温下) 注意:流出物可以收集在a   新鲜微量离心管。 微生物旋转柱与空 微量离心管可以放入带有盖的15ml Falcon管中,这样   可以进行离心而没有污染的风险 离心机。
    8. 混合完全流出物(250-300μl),含 具有引入的放射性的不带电反应产物, 与闪烁计数液(Starscint高闪点 LSC混合物)(1ml)
    9. 用液体闪烁计数器测量结合的放射性,持续5分钟 注意:将测量的"每分钟计数"(cpm)转换为 "分解每分钟"(dpm)由自动完成 仪器使用通过绘图生成的校准曲线 检测已知活性的外标的cpm值 他们已知的光谱淬灭参数[SQP(E)]。
    10. 准备控制  用于与上述相同的酶测定的孵育,但是  无蛋白质和来自用Sf9细胞感染的蛋白质提取物 非重组病毒以确定背景放射性 由于在阴离子期间未掺入的UDP- [14 C]糖的不完全保留 交换色谱,酶独立降解UDP-糖和  UDP-糖水解背景活性。处理这些控制 孵育与样品相同,并测量剩余 非特异性背景放射性在AEC流出物中
    11. 计算木糖基转移酶活性如下。
      从反应产物中计算木糖到反应产物中的量 根据公式保留在样品中的总放射性 "每个测定中掺入的木糖到受体底物中的量(in pmol)=(A/B)* C"; 其中A表示保留在中的放射性 样品按阴离子交换色谱法(以dpm计),B代表 添加到测定中的总放射性(以dpm为单位),C表示 加入到测定中的UDP-糖(标记的+未标记的)的总量 (以pmol计)。
      1. GT活性可以显示为木糖结合 测定(以pmol /测定)或作为木糖掺入每时间(以pmol/h)   将所述测定孵育离散时间(例如1小时,17小时和44小时)。
      2. 对于含有已知量的异源蛋白质的测定 PrGT34B,特异性GT活性可以从数量(in pmol)木糖每时间和每次掺入受体底物 特异性酶的量(以pmol/h /μg计)。
      3. 背景 可以使用在对照测定中检测到的放射性(以dpm计) 计算样品中的非特异性活性或可以直接 从相应的实验样品中减去以提供a 背景校正的值用于计算酶活性。

  7. 通过MALDI-TOF MS鉴定和表征PrGT34B木糖基转移酶测定反应产物 注意:
    1. 大多数质谱方法不是定量的,除非内部 使用的是已知量并且在结构上相同的标准 到反应产物。 因此,使用基质辅助激光 解吸电离飞行时间质谱(MALDI-TOF MS) 这里描述的只能被看作是半定量的方法 确定GT活性。 然而,这个过程不仅显示GT 活性通过检测预期的反应产物,但也允许   结构表征所产生的所有反应产物 酶PrGT34B。
    2. MALDI-TOF MS已被证明是高效的   解吸和电离各种生物分子包括 寡糖(Garozzo等人,1995; Garozzo等人,1997)
    1. 用含有重组体的提取物制备反应混合物 蛋白(或没有重组蛋白用于对照反应) 如上所述(步骤6F),但不添加放射性标记 UDP-木糖。
    2. 孵育(25℃,例如17小时)反应混合物 (50μl),在HEPES-NaOH(pH7.0)中含有提取的蛋白质(50μl) (1mM或mM),MnCl 2(5mM),MgCl 2(5mM),UDP-木糖(1或4mM)和 纤维寡糖(1mM) 注意:孵育时间选择足够长以允许木糖的最大掺入。
    3. 通过加热(5分钟,95℃)停止反应,除去沉淀 蛋白通过离心(10,000×g,2分钟,4℃)并转移 将反应产物的上清液置于新管中并置于冰上
    4. 将上清液(1μl)的等分试样与MALDI基质2,5-二羟基苯甲酸(10mg/ml,在ddH 2 O中)(9μl)混合。
    5. 在MALDI板上点样样品 - 基质混合物(1μl)的等分试样   并空气干燥以允许分析物和基质分子的结晶   板。
      注意:使甘油最小化是重要的 以确保分子结晶 均匀地在盘子上。
    6. 将MALDI板装载到MALDI-TOF质谱仪,例如Voyager DE TM PRO MALDI-TOF工作站。
      注意:
      1. 使用三个外部标准物校准光谱仪 des-ArgI-缓激肽(m/z = 904),血管紧张素I(m/z = 1296)和 稀释在2,5-二羟基苯甲酸中的[GluI] - 纤维蛋白肽B(m/z = 1570) 酸。
      2. 在反射器模式下以20kV的加速电压和200ns的延迟时间操作光谱仪。
    7. 通过绘制质量(m)来计算每个样品的质谱图 电荷(z)比(x轴) 使用任何合适的软件例如测量的强度(y轴) 数据资源管理器(版本4.0.0.0)。
      注意:每个质量的编译 频谱应包括来自足够数量的累积数据 的激光照射(例如100次激光照射)。
    8. 识别底物和GT 反应产物的质谱基于分子量 的单电荷(z = 1)钠加合物离子[M + Na] + 。 注意:
      1. 因为样品 - 基质混合物中NaCl浓度高 (> 1mM),在正电离模式中的主要离子种类是 分析物质子化分子离子的钠加合物。
      2. 使用为没有进行的对照反应产生的质谱 以鉴定与所述反应产物相关的峰 重组蛋白PrGT34B。
      3. 选择合适的基本峰强度阈值,以识别质谱图中高于噪声的峰。

食谱

  1. PAGE装染料
    SDS(1%)
    β-巯基乙醇(1mM) 甘油(10%)
    溴酚蓝
  2. 低渗裂解缓冲液
    50mM HEPES-NaOH(pH 7.0)
    2mM MgCl 2/
    10%甘油(v/v) 2%Triton X-100(v/v) Benzonase 核酸酶(10U/ml)
    蛋白酶抑制剂
    在双蒸水(ddH 2 O)中制备
  3. IMAC的平衡/清洗缓冲区
    50mM HEPES-NaOH(pH 7.0)
    100 mM NaCl
    5mM咪唑 10%甘油(v/v) 准备在ddH 2 O
  4. IMAC的样本准备缓冲区
    50mM HEPES-NaOH(pH 7.0)
    500 mM NaCl
    25mM咪唑 10%甘油(v/v) 准备在ddH 2 O
  5. IMAC洗脱缓冲液
    50mM HEPES-NaOH(pH 7.0)
    100 mM NaCl
    50mM咪唑 10%甘油(v/v) 准备在ddH 2 O
  6. 用于酶测定的反应缓冲液(用放射性标记的UDP-木糖)
    50mM HEPES-NaOH(pH 7.0)
    6.25mM MnCl 2/
    6.25mM MgCl 2
    1.25或5mM UDP-木糖 1.25mM寡糖受体底物(纤维二糖,纤维四糖,cellopentaose或cellohexaose)
    UDP- [14 C]木糖(9kBq,54,300dpm,150pmol)
    准备在ddH 2 O
  7. 用于酶测定的反应缓冲液(无放射性标记的UDP-木糖)
    50mM HEPES-NaOH(pH 7.0)
    6.25mM MnCl 2/
    6.25mM MgCl 2
    1.25或5mM UDP-木糖 1.25mM寡糖受体底物(纤维二糖,纤维四糖,cellopentaose或cellohexaose)
    准备在ddH 2 O

致谢

这项工作得到了新西兰研究,科学和技术基金会以及奥克兰大学的资助。 我们感谢CarboSource Services(Complex Carbohydrate Research Center,Athens,GA)的UDP-木糖和Scion Research的Carsten P. Ade的博士津贴。 这里描述的部分程序从Cavalier和Keegstra(2006)修改。

参考文献

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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ade, C. P., Bemm, F., Dickson, J. M., Walter, C. and Harris, P. J. (2014). In vitro Assay of the Glycosyltransferase Activity of a Heterologously Expressed Plant Protein. Bio-protocol 4(21): e1285. DOI: 10.21769/BioProtoc.1285.
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