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Generation of IgG-Fc Glycovariants Using Recombinant Glycosidases and Glycosyltransferases
采用重组糖苷酶和糖基转移酶改变IgG-Fc糖基化修饰状态   

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Abstract

The immunoglobulin G (IgG) fragment crystallizable (Fc) domain contains a single, highly conserved asparagine 297 (N297) glycosylation site in the CH2 domain, which is buried within the hydrophobic core of each of the two heavy chains. The biantennary core glycan structure, composed of 2 N-acetylglucosamine (GlcNAc) and 3 mannose residues, can be further decorated with fucose, bisecting GlcNAc and terminal GlcNAc, galactose, and sialic acid. Presence or absence of distinct residues can alter IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Here, we provide a protocol for the generation of IgG-Fc de-galactosylated, galactosylated, de-sialylated and sialylated IgG antibodies using recombinant glycosidases and glycosyltransferases.

Keywords: Antibody glycosylation(抗体的糖基化), Galactosylation(半乳糖化), Sialylation(唾液酸), Fc glycan(FC聚糖), ST6Gal(ST6Gal)

Background

The use of glycosyltransferases for antibody glycan modification allows the attachment of sugar substrates to pre-existing glycan residues. Immunoglobulin G carries a single, highly conserved N-glycosylation site in each of its CH2 domains (Arnold et al., 2007) (Figure 1) allowing site-specific glycan modification with glycosyltransferases. Antibodies may carry additional N-glycans if their Fab domains contain Asn-X-Ser/Thr (X ≠ Pro) sequences (Mellquist et al., 1998). Careful selection of a monoclonal antibody lacking Fab glycosylation is therefore important for Fc-specific glycan modification. The protocol described herein was developed based on the following publications (Kingston, 2003; Kaneko et al., 2006; Anthony et al., 2008; Barb et al., 2009; Quast et al., 2015).



Figure 1. The IgG-Fc N-glycan. Schematic depiction of IgG with two fully processed IgG-Fc N-glycans (left) and composition of the glycan (right).

Materials and Reagents

  1. Amicon Ultra-4 Centrifugal Filter Units with 50 kDa MWCO (Merck Millipore Corporation, catalog number: UFC805096 )
  2. HisTrap HP column (GE Healthcare, catalog number: 29-0510-21 )
  3. StrepTrapTM HP column (GE Healthcare, catalog number: 29-0486-53 )
  4. Empty SPE tubes, 12 ml (Sigma-Aldrich, catalog number: 57176 )
  5. VisidryTM Female Luer Plug (Sigma-Aldrich, catalog number: 57098 )
  6. Caps for 12 ml SPE Tubes (Sigma-Aldrich, catalog number: 52174-U )
  7. 15 cm cell culture dish
  8. Spectra/Por 3 dialysis membrane (MWCO 3.5 kD), 45 mm width (VWR International, catalog number: 734-0686 )
  9. 10 clamps for dialysis tubing: Spectra/Por Closures, 55 mm (Spectrum, catalog number: 132737 )
  10. 0.2 µm syringe filters (Filtropur S plus 0.2) (Sarstedt AG, catalog number: 83.1826.102 )
  11. 0.2 µm bottle-top vacuum filter systems (Vakuumfiltration 1000 “rapid” Filtermax) (TTP, catalog number: 99950 )
  12. HEK293, HEK293T or HKB11 cells
  13. MES sodium salt (AppliChem GmbH, catalog number: A3101 )
  14. MES monohydrate (AppliChem GmbH, catalog number: A4730 )
  15. HEPES (Sigma-Aldrich, catalog number: H3375 )
  16. MOPS (AppliChem GmbH, catalog number: A1076 )
  17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9451 )
  18. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 306568 )
  19. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71380 )
  20. Sodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600-K )
  21. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  22. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
  23. MnCl2 tetrahydrate (AppliChem GmbH, catalog number: A2087 )
  24. MgCl2 hexahydrate (AppliChem GmbH, catalog number: A4425 )
  25. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  26. Sodium dihydrogen phosphate monohydrate (NaH2PO4) (AppliChem GmbH, catalog number: A1047 )
  27. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  28. Trizma® hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: T5941-500 G )
  29. Glycine (Sigma-Aldrich, catalog number: G8898 )
  30. Imidazole (AppliChem GmbH, catalog number: A1073 )
  31. Tween 20 (Sigma-Aldrich, catalog number: P7949 )
  32. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  33. Sodium azide (AppliChem GmbH, catalog number: A1430 )
  34. D(+)-Lactose monohydrate (AppliChem GmbH, catalog number: A0880 )
  35. 1 M Tris, pH 8.8 (AppliChem GmbH, catalog number: A4265 )
  36. d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411-500 MG )
  37. Penicillin and streptomycin, liquid (Thermo Fischer Scientific, GibcoTM, catalog number: 15070-063 )
  38. UDP-galactose (Merck Millipore Corporation, Calbiochem®, catalog number: 670111-50 MG )
  39. CMP-sialic acid (Merck Millipore Corporation, Calbiochem®, catalog number: 233264-5 MG )
  40. StarGate® pCSG-IBA-144 Acceptor Vectors (IBA, catalog number: 5-5144-001 )
  41. StarGate® transfer reagent kit (IBA, catalog number: 5-1603-001 )
  42. Western Blocking Reagent, Solution (Sigma-Aldrich, catalog number: 11921673001 )
  43. Neuraminidase from Clostridium perfringes, recombinantly expressed in E. coli (New England Biolabs, catalog number: P0720S )
  44. β1-4-galactosidase from Streptococcus pneumoniae, recombinantly expressed in E. coli (Merck Millipore Corporation, Calbiochem®, catalog number: 345806-50 MIU )
  45. DMEM high glucose (4.5 g/L) (Thermo Fischer Scientific, GibcoTM, catalog number: 11965092 )
  46. Protein G sepharose for fast flow (GE healthcare, catalog number: 17-0618-01 )
  47. 20 µM polyethylene (PE) frits (Sigma-Aldrich, catalog number: 57181 )
  48. Biotinylated Lens Culinaris agglutinin (LCA) (Reactolab, catalog number: B-1045 )
  49. Biotinylated Erythrina Cristagalli lectin (ECL) (Reactolab, catalog number: B-1145 )
  50. Biotinylated Elderberry bark lectin (Sambucus nigra agglutinin, SNA) (Reactolab, catalog number: B-1305 )
  51. Agarose bound Sambucus Nigra Lectin (SNA), Vector Laboratories (Reactolab, catalog number: AL1303 )
  52. Streptavidin-HRP (Mabtech, catalog number: 3310-9 )
  53. MEM NEAA (non-essential amino acids) 100x (Thermo Fischer Scientific, GibcoTM, catalog number: 1140050 )
  54. Sodium pyruvate solution (100 mM) (Sigma-Aldrich, catalog number: S8636 )
  55. PierceTM ECL Western Blotting Substrate (Thermo Fischer Scientific, catalog number: 32106 )
  56. SuperSignalTM West Pico Chemiluminescent Substrate (Thermo Fischer Scientific catalog number: 34080 )
  57. General
    1. Tris-buffered saline (TBS) (see Recipes)
    2. Phosphate-buffered saline (PBS) (see Recipes)
  58. For purification of recombinant glycosyltransferases
    1. 293, 293T and HKB11 cell culture medium (see Recipes)
    2. 293, 293T and HKB11 cell protein expression medium (see Recipes)
    3. 2x HEPES-buffered saline (HeBS) (see Recipes)
    4. 2.5 M CaCl2 (see Recipes)
    5. Sterp-trap column elution buffer (see Recipes)
    6. His-trap elution buffer (see Recipes)
    7. His-trap wash buffer (see Recipes)
    8. His-trap supernatant dilution buffer (see Recipes)
  59. For enzymatic galactosylation
    1. Galactosyltransferase buffer (see Recipes)
  60. For enzymatic sialylation
    1. Sialyltransferase buffer (see Recipes)
  61. For enrichment of highly-sialylated antibodies using SNA-agarose
    1. SNA-agarose binding/wash buffer (see Recipes)
    2. SNA-agarose elution Buffer (see Recipes)
    3. SNA-agarose clearing Buffer (see Recipes)
    4. 0.08% NaN3 (see Recipes)
  62. For de-sialylation using recombinant neuraminidase
    1. Neuraminidase buffer (see Recipes)
  63. For de-galactosylation using recombinant β1-4-galactosidase
    1. β1-4-galactosidase buffer (see Recipes)
  64. For purification of glyco-modified antibodies using gravity flow protein-G sepharose columns
    1. Protein G column elution buffer (see Recipes)
    2. Protein G eluate neutralization buffer (see Recipes)
  65. For analysis of antibody glycosylation by lectin-blotting
    1. Lectin blot incubation buffer (see Recipes)
    2. Lectin blot wash buffer (see Recipes)

Equipment

  1. Spectrophotometer (Thermo Scientific, NanoDrop, model: 1000 )
  2. Tabletop centrifuge capable of > 20,000 x g centrifugation and cooling to 4 °C (Eppendorf)
  3. GE ÄKTAprime plus (GE Healthcare, catalog number: 11-0013-13 )
  4. Mini-PROTEAN® Tetra vertical electrophoresis cell, 4-gel, for 1.0 mm thick handcast gels, with PowerPacTM Basic power supply (Bio-Rad, catalog number: 1658025FC )
  5. Fusion FX7 gel imaging system (Vilber, model: Fusion FX7 )

Procedure

  1. Purification of recombinant glycosyltransferases
    To obtain pure, functional sialyl- and galactosyltransferases, the respective coding sequences are cloned into eukaryotic expression vectors. These vectors contain a BM40 signal sequence resulting in secretion of the recombinant protein as well as N- and C-terminal affinity tags which are used for purification of the glycosyltransferases from cell culture supernatants of transiently transfected eukaryotic cells.
    1. Clone the extracellular domain of human ST6Gal1 (NCBI Reference Sequence: NP_003023.1; from amino acid sequence KEKKKGS until GFRTIHC) and bovine β1,4GalT (GenBank: AAI20416.1 from amino acid sequence GSNLTSA until VDIGTPS) into pCSG-IBA-144 expression vectors according to the manufacturer’s recommendations. This results in a fusion protein containing an N-terminal Twin-Strep®-tag and a C-terminal 6xHistidine-tag.
    2. Transiently transfect HEK293, HEK293T or HKB11 cells with calcium-phosphate precipitated plasmids [adapted from (Kingston, 2003)]:
      1. Plate 6 x 106 cells per 15 cm cell culture dish in 20 ml DMEM high glucose containing P/S and 10% FCS.
      2. On the next day, freshly precipitate the plasmid DNA (values are given for one 15 cm cell culture dish and can be scaled up):
        1. Mix 20 µg plasmid DNA with 50 µl 2.5 M CaCl2 by pipetting up and down and fill up to 500 µl with ddH2O. Add 500 µl 2x HeBS in a 15 ml tube and slowly add the DNA-CaCl2 mix while creating bubbles using an electronic pipettor (Figure 2).
        2. Incubate for 30 min at room temperature.


        Figure 2. Mixing DNA-CaCl2 into 2x HeBS

      3. Distribute equally over the plate seeded on the day before and mix by carefully tilting the plate.
      4. After 12-16 h, suck-off the medium and carefully replace it with 30 ml DMEM (4.5 g/L glucose) containing P/S, 10% FCS, NEAA and sodium pyruvate.
      5. Incubate for 4-5 days.
      6. Harvest the supernatant and sterile-filter through 0.2 µm.
      7. Immediately proceed to HisTrap and SterpTrap column mediated purification.
    3. Purify the recombinant glycosyltransferases using HisTrap (nickel affinity) and StrepTrapTM (SII-tag affinity) columns
      1. Dilute the cell culture supernatant 1:2 with PBS containing 40 mM imidazole (pH 7.4).
      2. Apply to a 1 or 5 ml HIS-trap column (GE) using a GE ÄKTAprime plus.
      3. Wash with 10 column volumes PBS containing 20 mM imidazole.
      4. Elute with 10 column volumes 300 mM imidazole.
      5. Collect the entire eluate (the first 5-10 column volumes) and slowly (< 0.5 ml/min flow rate) apply to a StrepTrapTM column using GE ÄKTAprime plus.
      6. Wash with 10 column volumes PBS.
      7. Elute with 10 column volumes 2.5 mM d-desthiobiotin in PBS.
      8. Dialyze the protein-containing fractions by filling them into a Spectra/Por 3 Dialysis Membrane (MWCO 3.5 kD) tube and overnight incubation in at least 1 liter 0.2 M MES pH 6.5 (β1-4GalT) or 25 mM MOPS 100 mM KCl pH 7.2 (St6Gal).
      9. Determine the protein concentration using nano-drop. The absorption at 280 nm for a 1% (10 g/L) solution is 13.1 for β 1-4GalT and 17.1 for ST6Gal1. If necessary, concentrate using Amicon Ultra-4 Centrifugal Filter Units to reach a final concentration between 0.5 and 1.5 mg/ml.
      10. Sterilize by 0.2 µm filtration, add 20% sterile glycerol, aliquot and store at -20 °C.

  2. Enzymatic galactosylation
    Recombinant galactosyltransferase and UDP-galactose is used to add galactose to the IgG-Fc N-glycans of antibodies. 
    1. Buffer-exchange the antibody by dialysis to 0.2 M MES pH 6.5.
    2. Remove the antibody from dialysis and store the dialysis buffer at 4 °C for later use. Add 5 µg β 1-4GalT per mg antibody, add UDP-galactose to a final concentration of 10 mM, MnCl2 to a final concentration of 20 mM and supplement with 0.02% NaN3.
    3. Incubate for 48 h at 37 °C.
    4. Centrifuge for 2 h at 4 °C and > 20,000 x g to remove aggregates.
    5. Purify the galactosylated antibody using gravity-flow protein-G sepharose columns or continue to sialylation. A schematic depiction of galactosylated IgG is shown in Figure 3.


      Figure 3. Schematic depiction of enzymatically glycomodified IgG. Starting from a typical glycan profile of a recombinantly expressed IgG (upper panel, center) the glycan composition after de-galactosylation (upper panel, left) and galactosylation (upper panel, right) is shown. After the addition of galactose, sialylation results in the addition of terminal sialic acid to most galactose residues (lower panel, center). Finally de-sialylation can be used to remove sialic acid (lower panel, right).

  3. Enzymatic sialylation
    Recombinant sialyltransferase and CMP-sialic acid is used to add sialic acid to galactose-containing IgG-Fc N-glycans. 
    1. Dialyze the entire galactosylation reaction overnight at 4 °C to 0.2 M MES, pH 6.5 using the same buffer as used for the initial dialysis in (B).
    2. Dialyze overnight at 4 °C to 25 mM MOPS, 100 mM KCl, pH 7.2.
    3. Remove the reaction from the dialysis and determine the protein concentration using Nano-Drop.
    4. Add 50 µg ST6Gal1 per mg antibody and CMP-sialic acid to a final concentration of 1.5 mM.
    5. Incubate for 24 h at 37 °C.
    6. Dialyze overnight at 4 °C to TBS.
    7. Purify the antibody using gravity-flow protein-G sepharose columns or enrich for highly sialylated antibodies using SNA-agarose. A schematic depiction of sialylated IgG is shown in Figure 3.

  4. Enrichment of highly-sialylated antibodies using SNA-agarose
    The efficiency of enzymatic galactosylation and in particular sialylation can vary depending on the enzyme batch. The use of agarose bound Sambucus Nigra Lectin (SNA) which specifically binds to antibodies containing at least 2 sialic acid residues in the Fc-linked glycan region (Stadelmann et al., 2009) allows to enrich for antibodies with high sialic acid content.
    1. Pour 1-2 ml SNA-agarose in an SPE tube containing a polyethylene (PE) frit with 20 µm porosity and let the slurry settle (this takes around 5 min). Place an additional PE frit on top of the slurry (Figure 4).


      Figure 4. Set up of SNA-agarose gravity flow column

    2. Wash 3 times with 5 column volumes TBS + 0.1 M CaCl2.
    3. Add CaCl2 to a final concentration of 0.1 M to the sialylated antibodies previously dialyzed to TBS.
    4. Apply to the SNA-agarose column and collect the flow-through.
    5. Re-apply the flow through to the column.
    6. Wash 3 times with 5 column volumes TBS + 0.1 M CaCl2.
    7. Elute with 5 column volumes 0.5 M lactose in TBS (SNA agarose elution buffer) and collect the eluate in fractions corresponding to 1 column volume.
    8. Elute with 5 column volumes 0.5 M lactose/0.2 M acetic acid (SNA agarose clearing buffer) and collect the eluate in fractions corresponding to 1 column volume.
      Regenerate the column by washing with 10 column volumes 0.5 M lactose/0.2 M acetic acid followed by 10 column volumes TBS and 5 column volumes SNA agarose storage buffer. Close the bottom of the column with a female luer plug, add 2 ml SNA agarose storage buffer and close the top with an SPE tube Cap. Store the column at 4 °C.
    9. Dialyze the eluates to TBS.
    10. Purify using a protein-G sepharose column.

  5. De-sialylation using recombinant neuraminidase
    1. Dialyze the antibody to 50 mM sodium citrate, pH 6.0 (neuraminidase buffer).
    2. Add 70 units neuraminidase per mg antibody (or at least 300 U per ml).
    3. Incubate 48 h at 37 °C.
    4. Purify the antibody using protein-G sepharose. A schematic depiction of de-sialylated IgG is shown in Figure 3.

  6. De-galactosylation using recombinant β1-4-galactosidase
    β-1-4-galactosidase can only remove terminal galactose (King et al., 2006). Therefore, neuraminidase treatment has to be done first if the antibody’s IgG-Fc glycan is sialylated.
    1. Dialyze the antibody to 50 mM sodium phosphate buffer pH 6.0 (galactosidase buffer).
    2. Add 6 mU β1-4-galactosidase per 100 µg antibody.
    3. Incubate for 6 h at room temperature followed by 1 h at 37 °C.
    4. Purify the antibody using Protein-G sepharose. A schematic depiction of de-galactosylated IgG is shown in Figure 3.

  7. Purification of glyco-modified antibodies using gravity flow protein-G sepharose columns
    This step is used to remove the glycosidases and/or glycosyltransferases added during glycol-modification procedures. Pour protein-G sepharose slurry (1-2 ml, depending on the amount of antibody to be purified) in an SPE tube containing a PE frit with 20 µm porosity. After the slurry has settled (this takes around 5 min), insert an additional SPE frit on top. The column is set up as shown for SNA agarose in Figure 4.
    1. Apply the glycosylated antibody by gravity flow.
    2. Wash extensively with TBS.
    3. Elute with 0.1 M glycine, pH 2.5 and collect the eluates in 1 ml tubes containing the appropriate amount of 1 M Tris, pH 8.8 to result in a pH of 7.4.
    4. Dialyze to TBS or PBS.

  8. Analysis of antibody glycosylation by lectin-blotting
    Immunoblotting with the use of recombinant biotinylated lectins (referred to as lectin-blotting) can be used to detect the presence of specific glycan residues. This is a fast and reliable method to confirm the glycol-modification of antibodies.
    1. Load 1 to 2 µg of each glycovariant on a 10% poly-acrylamide gel and transfer the protein to a 0.45 µm nitrocellulose membrane.
      Note: Throughout the lectin-blotting procedure, avoid contact of the protein-containing side of the membrane with other surfaces, as this would lead to increased background signals in particular for the detection of sialic acid using biotinylated SNA. Therefore, use individual trays for each membrane throughout the procedure.
    2. Block the membrane with Western Blocking Reagent (10% purified casein protein in maleic-acid buffer) diluted 1:10 in TBS at room temperature for 1 h or at 4 °C overnight.
    3. Remove the blocking solution and add the respective lectin for the glycan of interest (ECL for galactose, LCA for mannose and SNA for sialic acid) diluted 1:1,000 (resulting in 5 µg/ml for ECL, 5 µg/ml for LCA and 2 µg/ml for SNA) in TBS containing 1 mM CaCl2, MgCl2, MnCl2 and 1% Western Blocking Reagent (lectin incubation buffer).
    4. Incubate at room temperature for 1 h.
    5. Wash at least 6 x 5 min with TBS containing 0.1% Tween 20.
    6. Incubate with TBS containing 1% Western Blocking Reagent and Streptavidin-HRP (1:1,000) at room temperature for 1 h.
    7. Wash at least 6 x 5 min with TBS containing 0.1% Tween 20.
    8. Apply a chemiluminescent peroxide substrate and detect the signal using a gel imaging system or x-ray film. LCA and SNA work well using PierceTM ECL as substrate, ECL using SuperSignalTM West Pico (Figure 5).


      Figure 5. Exemplary lectin-blot analysis of antibody glycosylation. Shown is a 293T cell expressed Fc-humanized IgG1 antibody before glycosylation (unmodified), after galactosylation and purification using protein G sepharose (galactosylated) and after galactosylation, sialylation and purification using protein G sepharose (sialylated). Coomassie staining and immunoblotting for the core glycan component mannose is used to confirm purity and equal loading, respectively. ECL detects only terminal galactose and the signal is therefore lost upon sialylation.

Recipes

  1. General
    1. Tris-buffered saline (TBS)
      0.05 M Tris-HCl
      0.15 M NaCl
      Adjust pH to 7.4
    2. Phosphate-buffered saline (PBS)
      2.7 mM KCl
      8 mM Na2HPO4
      1.8 mM KH2PO4
      137 mM NaCl
      Adjust pH to 7.4

  2. For purification of recombinant glycosyltransferases
    1. 293, 293T and HKB11 cell culture medium
      DMEM high glucose
      Penicillin/streptomycin (P/S, 50 U/ml)
      1 mM sodium pyruvate
      10% fetal calf serum
    2. 293, 293T and HKB11 cell protein expression medium
      DMEM high glucose
      Penicillin/streptomycin (P/S, 50 U/ml)
      1x NEAA
      1 mM sodium pyruvate
      10% fetal calf serum
    3. 2x HEPES-buffered saline (HeBS)
      0.28 M CaCl2
      0.05 M HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid)
      1.5 mM Na2PO4
      Adjust pH to 7.05 with 5 M NaOH and sterilize by 0.2 µm filtration
    4. 2.5 M CaCl2
      2.5 M CaCl2 in ddH2O
    5. StrepTrapTM column elution buffer
      2.5 mM D-desthiobiotin in PBS
    6. His-trap elution buffer
      300 mM imidazole in PBS, adjust pH to 7.4
    7. His-trap wash buffer
      20 mM imidazole in PBS, adjust pH to 7.4
    8. His-trap supernatant dilution buffer
      40 mM imidazole in PBS, adjust pH to 7.4

  3. For enzymatic galactosylation
    1. Galactosyltransferase buffer
      25 mM MOPS
      100 mM KCl
      Adjust pH to pH 7.2 with KOH

  4. For enzymatic sialylation
    1. Sialyltransferase buffer
      0.2 M MES
      Prepare a 0.2 M MES-Sodium salt solution and a 0.2 M MES monohydrate solution and mix the two components to reach pH 6.5 (this requires around two fold excess of MES-sodium salt solution)

  5. For enrichment of highly-sialylated antibodies using SNA-agarose
    1. SNA-agarose binding/wash buffer
      TBS + 0.1 M CaCl2
    2. SNA-agarose elution Buffer
      0.5 M lactose in TBS
      Adjust pH to 7.5
    3. SNA-agarose clearing Buffer
      0.5 M lactose in 0.2 M acetic acid
    4. SNA-agarose storage Buffer
      10 mM HEPES
      0.15 M NaCl
      0.1 mM CaCl2
      0.08 % NaN3
      20 mM lactose
      Adjust pH to 7.5 with NaOH

  6. For de-sialylation using recombinant neuraminidase
    1. Neuraminidase buffer
      50 mM Sodium citrate, pH 6.0
      Adjust pH to 6 with 50 mM citric acid

  7. For de-galactosylation using recombinant β1-4-galactosidase
    1. β1-4-galactosidase buffer
      50 mM sodium phosphate, pH 6.0
      Prepare a 50 mM Na2HPO4 solution and a 50 mM NaH2PO4 solution and mix the two components to reach pH 6.5 (this requires around 7 fold excess of 50 mM NaH2PO4)

  8. For purification of glyco-modified antibodies using gravity flow protein-G sepharose columns
    1. Protein G column elution buffer
      0.2 M Glycine
      Adjust pH to 2.5 with HCl
    2. Protein G eluate neutralization buffer
      1 M Tris, pH 8.8

  9. For analysis of antibody glycosylation by lectin-blotting
    1. Lectin blot incubation buffer
      TBS containing 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2 and 1% Western Blocking Reagent
    2. Lectin blot wash buffer
      TBS + 0.1% Tween 20

Acknowledgments

We thank Dr. Falk Nimmerjahn (Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany) for his help to establishing this protocol and Patrick Weber (Institute of Experimental Immunology, Laboratory of Neuroinflammation, University of Zürich) for expert technical assistance. I. Quast was supported by a DOC scholarship provided by the Austrian Academy of Sciences (ÖAW). This protocol was adapted from Kingston, 2003; Kaneko et al., 2006; Anthony et al., 2008; Barb et al., 2009; Quast et al., 2015.

References

  1. Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M. and Dwek, R. A. (2007). The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 25: 21-50.
  2. Anthony, R. M., Nimmerjahn, F., Ashline, D. J., Reinhold, V. N., Paulson, J. C. and Ravetch, J. V. (2008). Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG fc. Science 320(5874): 373-376.
  3. Barb, A. W., Brady, E. K. and Prestegard, J. H. (2009). Branch-specific sialylation of IgG-Fc glycans by ST6Gal-I. Biochemistry 48(41): 9705-9707.
  4. Kaneko, Y., Nimmerjahn, F. and Ravetch, E. V. (2006). Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313(5787): 670-673.
  5. King, S. J., Hippe, K. R. and Weiser, J. N. (2006). Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae. Molecular Microbiology 59(3): 961-974.
  6. Kingston, R. E., (2003). Introduction of DNA into mammalian cells. Current Protocols in Molecular Biology, Chapter 9 (DOI: 10.1002/0471142727.mb0900s64).
  7. Mellquist, J. L., Kasturi, L., Spitalnik, S. L. and Shakin-Eshleman, S. H. (1998). The amino acid following an Asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. Biochemistry 37(19): 6833-6837.
  8. Quast, I., Keller, C. W., Maurer, M. A., Giddens, J. P., Tackenberg, B., Wang, L. X., Munz, C., Nimmerjahn, F., Dalakas, M. C. and Lunemann, J. D. (2015). Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity. Journal of Clinical Investigation 125(11): 4160-4170.
  9. Stadlmann, J., Weber, A., Pabst, M., Anderle, H., Kunert, R., Ehrlich, H. J., Peter Schwarz, H. and Altmann, F. (2009). A close look at human IgG sialylation and subclass distribution after lectin fractionation. Proteomics 9(17): 4143-4153.

简介

免疫球蛋白G(IgG)片段可结晶(Fc)结构域在CH2结构域中包含单个,高度保守的天冬酰胺297(N297)糖基化位点,其掩埋在两条重链的每一条的疏水核内。由2个N-乙酰葡萄糖胺(GlcNAc)和3个甘露糖残基组成的双触角核心聚糖结构可以进一步用岩藻糖,二等分GlcNAc和末端GlcNAc,半乳糖和唾液酸装饰。不同残基的存在或不存在可以改变IgG效应子功能,例如抗体依赖性细胞介导的细胞毒性(ADCC)或补体依赖性细胞毒性(CDC)。在这里,我们提供使用重组糖苷酶和糖基转移酶产生IgG-Fc去半乳糖基化,半乳糖基化,去唾液酸化和唾液酸化IgG抗体的方案。

[背景] 糖基转移酶用于抗体聚糖修饰的用途允许将糖底物连接到预先存在的聚糖残基上。免疫球蛋白G在其每个CH2结构域中携带单个高度保守的N-糖基化位点(Arnold等人,2007)(图1),允许用糖基转移酶进行位点特异性聚糖修饰。如果抗体的Fab结构域含有Asn-X-Ser/Thr(X≠Pro)序列(Mellquist等人,1998),则抗体可携带额外的N-聚糖。因此,仔细选择缺少Fab糖基化的单克隆抗体对于Fc特异性聚糖修饰是重要的。本文所述的方案是基于以下出版物开发的(Kingston,2003; Kaneko等人,2006; Anthony等人,2008; Barb等人。,2009; Quast 等。,2015)。




图1. IgG-Fc N-聚糖。两个完全加工的IgG的示意图IgG-Fc N-聚糖(左)和聚糖的组成(右)。


关键字:抗体的糖基化, 半乳糖化, 唾液酸, FC聚糖, ST6Gal

材料和试剂

  1. 具有50kDa MWCO的Amicon Ultra-4离心过滤器单元(Merck Millipore Corporation,目录号:UFC805096)
  2. HisTrap HP柱(GE Healthcare,目录号:29-0510-21)
  3. StrepTrap HP柱(GE Healthcare,目录号:29-0486-53)
  4. 空SPE管,12ml(Sigma-Aldrich,目录号:57176)
  5. Visidry Female Luer Plug(Sigma-Aldrich,目录号:57098)
  6. 用于12ml SPE管(Sigma-Aldrich,目录号:52174-U)的帽
  7. 15厘米细胞培养皿
  8. Spectra/Por 3透析膜(MWCO 3.5kD),45mm宽(VWR International,目录号:734-0686)
  9. 10用于透析管的夹具:Spectra/Por Closures,55mm(Spectrum,目录号:132737)
  10. 0.2μm注射器过滤器(Filtropur S加0.2)(Sarstedt AG,目录号:83.1826.102)
  11. 0.2μm瓶顶真空过滤系统(Vakuumfiltration 1000"rapid"Filtermax)(TTP,目录号:99950)
  12. HEK293,HEK293T或HKB11细胞
  13. MES钠盐(AppliChem GmbH,目录号:A3101)
  14. MES一水合物(AppliChem GmbH,目录号:A4730)
  15. HEPES(Sigma-Aldrich,目录号:H3375)
  16. MOPS(AppliChem GmbH,目录号:A1076)
  17. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9451)
  18. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:306568)
  19. 氯化钠(NaCl)(Sigma-Aldrich,目录号:71380)
  20. 柠檬酸钠二水合物(Sigma-Aldrich,目录号:W302600-K)
  21. 柠檬酸(Sigma-Aldrich,目录号:251275)
  22. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C7902)
  23. MnCl 2·4水合物(AppliChem GmbH,目录号:A2087)
  24. MgCl 2·6H 2 O(AppliChem GmbH,目录号:A4425)
  25. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  26. 磷酸二氢钠一水合物(NaH 2 PO 4)(AppliChem GmbH,目录号:A1047)
  27. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662)
  28. 盐酸盐(Tris-HCl)(Sigma-Aldrich,目录号:T5941-500G)
  29. 甘氨酸(Sigma-Aldrich,目录号:G8898)
  30. 咪唑(AppliChem GmbH,目录号:A1073)
  31. 吐温20(Sigma-Aldrich,目录号:P7949)
  32. 甘油(Sigma-Aldrich,目录号:G5516)
  33. 叠氮化钠(AppliChem GmbH,目录号:A1430)
  34. D(+) - 乳糖一水合物(AppliChem GmbH,目录号:A0880)
  35. 1M Tris,pH8.8(AppliChem GmbH,目录号:A4265)
  36. d-Desthiobiotin(Sigma-Aldrich,目录号:D1411-500MG)
  37. 青霉素和链霉素,液体(Thermo Fischer Scientific,Gibco TM ,目录号:15070-063)
  38. UDP-半乳糖(Merck Millipore Corporation,Calbiochem ,目录号:670111-50MG)
  39. CMP-唾液酸(Merck Millipore Corporation,Calbiochem ,目录号:233264-5MG)
  40. StarGate ? pCSG-IBA-144受体载体(IBA,目录号:5-5144-001)
  41. StarGate ?转移试剂盒(IBA,目录号:5-1603-001)
  42. Western阻断试剂溶液(Sigma-Aldrich,目录号:11921673001)
  43. 来自产气荚膜梭菌的神经氨酸酶,在E中重组表达。大肠杆菌(New England Biolabs,目录号:P0720S)
  44. 来自肺炎链球菌的β1-4-半乳糖苷酶,在E中重组表达。大肠杆菌(Merck Millipore Corporation,Calbiochem ,目录号:345806-50MIU)
  45. DMEM高葡萄糖(4.5g/L)(Thermo Fischer Scientific,Gibco TM ,目录号:11965092)
  46. 用于快速流动的蛋白G琼脂糖凝胶(GE healthcare,目录号:17-0618-01)
  47. 20μM聚乙烯(PE)玻璃料(Sigma-Aldrich,目录号:57181)
  48. 生物素化的透明凝集素(LCA)(Reactolab,目录号:B-1045)
  49. 生物素化的Erythrina Cristagalli凝集素(ECL)(Reactolab,目录号:B-1145)
  50. 生物素化接骨木树皮凝集素(接骨木花凝集素,SNA)(Reactolab,目录号:B-1305)
  51. 琼脂糖结合的接骨木果胶(SNA),Vector Laboratories(Reactolab,目录号:AL1303)
  52. 链霉亲和素-HRP(Mabtech,目录号:3310-9)
  53. MEM NEAA(非必需氨基酸)100x(Thermo Fischer Scientific,Gibco TM ,目录号:1140050)
  54. 丙酮酸钠溶液(100mM)(Sigma-Aldrich,目录号:S8636)
  55. Pierce TM ECL Western Blotting Substrate(Thermo Fischer Scientific,目录号:32106)
  56. SuperSignal TM West Pico Chemiluminescent Substrate(Thermo Fischer Scientific目录号:34080)
  57. 一般
    1. Tris缓冲盐水(TBS)(见Recipes)
    2. 磷酸盐缓冲盐水(PBS)(见配方)
  58. 用于重组糖基转移酶的纯化
    1. 293,293T和HKB11细胞培养基(见配方)
    2. 293,293T和HKB11细胞蛋白表达培养基(参见Recipes)
    3. 2x HEPES缓冲盐水(HeBS)(参见Recipes)
    4. 2.5 M CaCl 2(参见配方)
    5. Sterp-trap柱洗脱缓冲液(参见配方)
    6. His-trap洗脱缓冲液(参见配方)
    7. His-trap洗涤缓冲液(参见配方)
    8. His-trap上清液稀释缓冲液(参见配方)
  59. 用于酶促半乳糖基化
    1. 半乳糖基转移酶缓冲液(参见配方)
  60. 用于酶促唾液酸化
    1. 唾液酸转移酶缓冲液(参见配方)
  61. 用于使用SNA-琼脂糖富集高度唾液酸化的抗体
    1. SNA-琼脂糖结合/洗涤缓冲液(参见配方)
    2. SNA琼脂糖洗脱缓冲液(参见配方)
    3. SNA琼脂糖清除缓冲液(参见配方)
    4. 0.08%NaN 3 (参见配方)
  62. 用于使用重组神经氨酸酶的脱唾液酸化
    1. 神经氨酸酶缓冲液(见配方)
  63. 用于使用重组β1-4-半乳糖苷酶的去半乳糖基化
    1. β1-4-半乳糖苷酶缓冲液(参见配方)
  64. 用于使用重力流动蛋白-G琼脂糖柱来纯化糖修饰的抗体
    1. 蛋白G柱洗脱缓冲液(参见配方)
    2. 蛋白G洗脱中和缓冲液(参见配方)
  65. 用于通过凝集素印迹分析抗体糖基化
    1. 凝集素印迹孵育缓冲液(参见配方)
    2. 凝集素印迹洗涤缓冲液(见配方)

设备

  1. 分光光度计(Thermo Scientific,NanoDrop,型号:1000)
  2. 台式离心机能够> 20,000×g离心并冷却至4℃(Eppendorf)
  3. ?KTAprimeplus(GE Healthcare,目录号:11-0013-13)
  4. 使用PowerPac TM基本电源(Bio-Rad,目录号:1658025FC)的用于1.0mm厚的手摇凝胶的Mini-PROTEAN Tetra TM垂直电泳槽4-凝胶,
  5. Fusion FX7凝胶成像系统(Vilber,型号:Fusion FX7)

程序

  1. 重组糖基转移酶的纯化
    为了获得纯的功能性唾液酸和半乳糖基转移酶,将各自的编码序列克隆到真核表达载体中。这些载体含有导致重组蛋白分泌的BM40信号序列以及用于从瞬时转染的真核细胞的细胞培养物上清液中纯化糖基转移酶的N-和C-末端亲和标签。
    1. 将人ST6Gal1(NCBI参考序列:NP_003023.1;从氨基酸序列KEKKKGS直到GFRTIHC)和牛β1,4GalT(GenBank:AAI20416.1从氨基酸序列GSNLTSA直到VDIGTPS)的胞外结构域克隆到pCSG-IBA-144表达载体根据制造商的建议。这导致含有N末端双链Stag-标记和C-末端6x组氨酸标签的融合蛋白。
    2. 用磷酸钙沉淀的质粒瞬时转染HEK293,HEK293T或HKB11细胞[改编自(Kingston,2003)]:
      1. 板在含有P/S和10%FCS的20ml DMEM高葡萄糖中每15cm细胞培养皿中6×10 6个细胞。
      2. 第二天,新鲜沉淀质粒DNA(对于一个15cm细胞培养皿给出值,并且可以放大):
        1. 用移液管向上和向下混合20μg质粒DNA和50μl2.5M CaCl 2,并用ddH 2 O填充至500μl。在15ml管中加入500μl2x HeBS,并使用电子移液器(图2)缓慢加入DNA-CaCl 2 2混合物,同时产生气泡。
        2. 在室温下孵育30分钟。


        图2.将DNA-CaCl <2>混合到2x HeBS 中

      3. 平均分配在前一天种植的板上,仔细倾斜板混合
      4. 12-16小时后,吸出培养基并小心地用含有P/S,10%FCS,NEAA和丙酮酸钠的30ml DMEM(4.5g/L葡萄糖)替换。
      5. 孵育4-5天。
      6. 收获上清液和无菌过滤通过0.2微米
      7. 立即进行HisTrap和SterpTrap柱介导纯化。
    3. 使用HisTrap(镍亲和力)和StrepTrap TM(SII标签亲和力)柱纯化重组糖基转移酶
      1. 用含有40mM咪唑(pH 7.4)的PBS稀释细胞培养上清液1:2
      2. 使用GE?KTAprimeplus。应用于1或5 ml HIS-捕获柱(GE)
      3. 用10个柱体积的含有20mM咪唑的PBS洗涤
      4. 用10个柱体积的300mM咪唑洗脱
      5. 使用GE?KTAprimeplus,将整个洗脱液(前5-10柱体积)和缓慢地(<0.5ml/min流速)应用于StrepTrap TM柱。
      6. 用10倍柱体积的PBS冲洗
      7. 用10倍柱体积的2.5mM d-脱硫生物素的PBS溶液洗脱
      8. 通过将含蛋白质的级分填充到Spectra/Por 3透析膜(MWCO 3.5kD)管中并在至少1升0.2M MES pH 6.5(β1-4GalT)或25mM MOPS 100mM KCl pH7.2 (St6Gal)。
      9. 使用纳米滴定确定蛋白质浓度。 1%(10g/L)溶液在280nm的吸收对于β1-4GalT为13.1,对于ST6Gal1为17.1。如有必要,使用Amicon Ultra-4离心过滤器单元浓缩至0.5至1.5mg/ml的最终浓度。
      10. 通过0.2μm过滤灭菌,加入20%无菌甘油,等分并储存在-20℃。

  2. 酶半乳糖基化
    重组半乳糖基转移酶和UDP-半乳糖用于向抗体的IgG-Fc N-聚糖添加半乳糖。 
    1. 通过透析缓冲液交换抗体至0.2M MES pH 6.5。
    2. 从透析中取出抗体,并将透析缓冲液储存在4℃以备后用。加入5μgβ1-4GalT/mg抗体,加入UDP-半乳糖至终浓度为10mM,MnCl 2至终浓度为20mM,并补充0.02%NaN 3,/sub>。
    3. 在37℃孵育48小时。
    4. 在4℃下离心2小时, 20,000 x g 删除聚合。
    5. 使用重力流动蛋白G琼脂糖柱纯化半乳糖基化抗体或继续唾液酸化。半乳糖基化IgG的示意图显示于图3中

      图3.酶促糖基化IgG的示意图。从重组表达的IgG(上图,中心)的典型聚糖谱开始,在去半乳糖基化(上图,左)和半乳糖基化上图,右图)。在加入半乳糖后,唾液酸化导致末端唾液酸加到大多数半乳糖残基(下图,中心)。最后脱唾液酸化可用于除去唾液酸(下图,右)
  3. 酶唾液酸化
    重组唾液酸转移酶和CMP-唾液酸用于向含半乳糖的IgG-Fc N-聚糖添加唾液酸。
    1. 使用与用于(B)中的初始透析相同的缓冲液,在4℃下将整个半乳糖基化反应透析过夜至0.2M MES,pH 6.5。
    2. 在4℃至25mM MOPS,100mM KCl,pH7.2透析过夜
    3. 从透析中去除反应,并使用Nano-Drop确定蛋白质浓度。
    4. 加入50μgST6Gal1/mg抗体和CMP-唾液酸至终浓度1.5mM
    5. 在37℃孵育24小时。
    6. 在4℃下透析至TBS过夜。
    7. 使用重力流蛋白-G琼脂糖柱纯化抗体或使用SNA-琼脂糖富集高度唾液酸化的抗体。唾液酸化IgG的示意图显示于图3中
  4. 使用SNA-琼脂糖富集高度唾液酸化的抗体 酶半乳糖基化的效率和特别是唾液酸化可以根据酶批次而变化。使用与Fc连接的聚糖区域中含有至少2个唾液酸残基的抗体特异性结合的琼脂糖结合的接骨木花青素凝集素(SNA)(Stadelmann等人, 2009)允许富集具有高唾液酸含量的抗体
    1. 在含有20μm孔隙度的聚乙烯(PE)玻璃料的SPE管中倒入1-2ml SNA-琼脂糖,并使浆液沉降(这需要约5分钟)。在浆料顶部放置一块额外的PE玻璃料(图4)。


      图4.设置SNA琼脂糖重力流动柱

    2. 用5个柱体积的TBS + 0.1M CaCl 2洗涤3次
    3. 将CaCl 2加至预先用TBS透析的唾液酸化抗体的终浓度为0.1M。
    4. 应用于SNA琼脂糖柱并收集流出物。
    5. 重新将流量应用到列。
    6. 用5个柱体积的TBS + 0.1M CaCl 2洗涤3次
    7. 用5个柱体积的0.5M乳糖在TBS(SNA琼脂糖洗脱缓冲液)中洗脱,并收集相应于1个柱体积的洗脱液。
    8. 用5个柱体积的0.5M乳糖/0.2M乙酸(SNA琼脂糖清除缓冲液)洗脱,并收集相应于1个柱体积的洗脱液。
      通过用10个柱体积的0.5M乳糖/0.2M乙酸,然后10个柱体积的TBS和5个柱体积的SNA琼脂糖储存缓冲液洗涤来再生柱。关闭柱的底部与母鲁尔插头,加入2毫升SNA琼脂糖储存缓冲液,并关闭顶部与SPE管帽。将色谱柱保存在4°C。
    9. 将洗脱物透析到TBS。
    10. 使用蛋白-G琼脂糖柱进行纯化
  5. 使用重组神经氨酸酶的脱唾液酸化
    1. 将抗体透析至50mM柠檬酸钠,pH 6.0(神经氨酸酶缓冲液)
    2. 每mg抗体加入70单位神经氨酸酶(或至少300U/ml)
    3. 在37℃孵育48小时。
    4. 使用蛋白-G琼脂糖纯化抗体。去唾液酸化的IgG的示意图显示在图3中
  6. 使用重组β1-4-半乳糖苷酶进行去半乳糖基化 β-1-4-半乳糖苷酶只能去除末端半乳糖(King等人,2006)。因此,如果抗体的IgG-Fc聚糖被唾液酸化,则必须首先进行神经氨酸酶处理
    1. 将抗体透析至50mM磷酸钠缓冲液pH 6.0(半乳糖苷酶缓冲液)
    2. 每100μg抗体加入6 mUβ1-4-半乳糖苷酶
    3. 在室温下孵育6小时,然后在37℃下孵育1小时
    4. 使用蛋白-G琼脂糖纯化抗体。去半乳糖基化IgG的示意图如图3所示
  7. 使用重力流动蛋白-G琼脂糖柱纯化糖修饰的抗体
    该步骤用于除去在乙二醇修饰过程中加入的糖苷酶和/或糖基转移酶。在含有20μm孔隙度的PE玻璃料的SPE管中倒入蛋白-G琼脂糖浆(1-2ml,取决于待纯化的抗体的量)。在浆液沉降后(这需要约5分钟),在顶部插入另外的SPE玻璃料。该柱设置如图4中所示的SNA琼脂糖。
    1. 通过重力流应用糖基化抗体
    2. 用TBS广泛洗涤。
    3. 用0.1M甘氨酸(pH2.5)洗脱,并将洗脱液收集在含有适量的1M Tris(pH8.8)的1ml管中,得到pH为7.4。
    4. 透析到TBS或PBS。

  8. 通过凝集素印迹分析抗体糖基化 使用重组生物素化凝集素的免疫印迹(称为凝集素印迹)可用于检测特定聚糖残基的存在。这是一种快速可靠的方法来确认抗体的乙二醇修饰。
    1. 在10%聚丙烯酰胺凝胶上加载1到2μg的每种糖变体,并将蛋白转移到0.45μm的硝酸纤维素膜上。
      注意:在整个凝集素印迹过程中,避免膜的含蛋白质侧与其他表面接触,因为这将导致增加的背景信号,特别是用于使用生物素化SNA检测唾液酸。因此,在整个程序中为每个膜使用单独的托盘。
    2. 用Western Blocking Reagent(在马来酸缓冲液中10%纯化的酪蛋白,在TBS中1:10稀释)在室温下封闭膜1小时或在4℃过夜。
    3. 除去封闭溶液,加入相应的凝集素(ECL用于半乳糖,LCA用于甘露糖,SNA用于唾液酸),稀释度为1:1000(导致ECL为5μg/ml,LCA为5μg/ml, μg/ml用于SNA)在含有1mM CaCl 2,MgCl 2,MnCl 2和1%Western阻断试剂的TBS(凝集素孵育缓冲液)。
    4. 在室温下孵育1小时
    5. 用含有0.1%吐温20的TBS洗涤至少6×5分钟
    6. 在室温下用含有1%西方阻断试剂和链霉亲和素-HRP(1:1,000)的TBS孵育1小时。
    7. 用含有0.1%吐温20的TBS洗涤至少6×5分钟
    8. 应用化学发光过氧化物底物,并使用凝胶成像系统或X射线胶片检测信号。 LCA和SNA使用Pierce TM ECL作为底物,ECL使用SuperSignal TM West Pico工作良好(图5)。

      图5.抗体糖基化的示例性凝集素印迹分析。显示在糖基化(未修饰)之后,在使用蛋白G琼脂糖(半乳糖基化)进行半乳糖基化和纯化后和在半乳糖基化后,293T细胞表达的Fc-人源化IgG1抗体,唾液酸化和使用蛋白G琼脂糖(唾液酸化)纯化。核心聚糖组分甘露糖的考马斯染色和免疫印迹分别用于确认纯度和相等的加载。 ECL仅检测末端半乳糖,因此唾液酸化后信号丢失

食谱

  1. 一般
    1. Tris缓冲盐水(TBS)
      0.05M Tris-HCl
      0.15 M NaCl
      将pH调节到7.4
    2. 磷酸盐缓冲盐水(PBS)
      2.7 mM KCl
      8mM Na 2 HPO 4
      1.8mM KH 2 PO 4 sub/
      137 mM NaCl 将pH调节至7.4

  2. 用于重组糖基转移酶的纯化
    1. 293,293T和HKB11细胞培养基 DMEM高血糖
      青霉素/链霉素(P/S,50U/ml) 1mM丙酮酸钠 10%胎牛血清
    2. 293,293T和HKB11细胞蛋白表达培养基 DMEM高血糖
      青霉素/链霉素(P/S,50U/ml) 1x NEAA
      1mM丙酮酸钠 10%胎牛血清
    3. 2x HEPES缓冲盐水(HeBS)
      0.28M CaCl 2
      0.05 M HEPES(N-2-羟乙基哌嗪-N'-2-乙磺酸) 1.5mM Na 2 PO 4/dn 用5M NaOH调节pH至7.05,并通过0.2μm过滤灭菌
    4. 2.5 M CaCl 2/
      2.5M CaCl 2在ddH 2 O 2中的浓度
    5. StrepTrap TM 柱洗脱缓冲液
      2.5mM D-脱硫生物素的PBS溶液中
    6. His-trap洗脱缓冲液
      300mM咪唑的PBS溶液,调节pH至7.4
    7. 他的陷阱洗涤缓冲液
      20mM咪唑的PBS溶液,调节pH至7.4
    8. His-trap上清液稀释缓冲液
      40mM咪唑的PBS溶液,调节pH至7.4
  3. 对于酶促半乳糖基化
    1. 半乳糖转移酶缓冲液
      25 mM MOPS
      100 mM KCl
      用KOH调节pH至7.2
  4. 对于酶促唾液酸化
    1. 唾液酸转移酶缓冲液
      0.2 M MES
      制备0.2M MES-钠盐溶液和0.2M MES一水合物溶液,并混合两种组分以达到pH6.5(这需要约两倍过量的MES-钠盐溶液)。
  5. 使用SNA-琼脂糖富集高度唾液酸化的抗体
    1. SNA-琼脂糖结合/洗涤缓冲液
      TBS + 0.1M CaCl 2
    2. SNA琼脂糖洗脱缓冲液
      0.5 M乳糖在TBS中 将pH调节至7.5
    3. SNA琼脂糖清除缓冲液
      0.5M乳糖在0.2M乙酸中的溶液
    4. SNA琼脂糖贮存缓冲液
      10 mM HEPES
      0.15 M NaCl
      0.1mM CaCl 2/v/v 0.08%NaN 3
      20mM乳糖 用NaOH调节pH至7.5
  6. 对于使用重组神经氨酸酶的脱唾液酸化
    1. 神经氨酸酶缓冲液
      50 mM柠檬酸钠,pH 6.0
      用50mM柠檬酸调节pH至6
  7. 使用重组β1-4-半乳糖苷酶进行去半乳糖基化
    1. β1-4-半乳糖苷酶缓冲液 50mM磷酸钠,pH 6.0
      制备50mM Na 2 HPO 4溶液和50mM NaH 2 PO 4 PO 4溶液,并混合两种组分达到pH 6.5(这需要约7倍过量的50mM NaH 2 PO 4)。

  8. 使用重力流动蛋白-G琼脂糖柱纯化糖修饰的抗体
    1. 蛋白G柱洗脱缓冲液
      0.2 M甘氨酸 用HCl
      调节pH至2.5
    2. 蛋白G洗脱液中和缓冲液
      1M Tris,pH 8.8

  9. 用于通过凝集素印迹分析抗体糖基化
    1. 凝集素印迹孵育缓冲液
      含有1mM CaCl 2,1mM MnCl 2,1mM MgCl 2和1%Western阻断试剂的TBS。
    2. 凝集素印迹洗涤缓冲液
      TBS + 0.1%吐温20

致谢

我们感谢Falk Nimmerjahn博士(Erlangen-Nuremberg大学生物系,德国Erlangen)帮助建立这个协议,Patrick Weber(苏黎世大学Neuroinflammation实验免疫学研究所)为专家技术援助。 I. Quast由奥地利科学院(?AW)提供的DOC奖学金支持。该协议改编自Kingston,2003; Kaneko等人,2006; Anthony等人。,2008; Barb 。,2009; Quast 。,2015。

参考文献

  1. Arnold,JN,Wormald,MR,Sim,RB,Rudd,PM and Dwek,RA(2007)。  糖基化对人免疫球蛋白的生物学功能和结构的影响。 Annu Rev Immunol 25:21-50。
  2. Anthony,RM,Nimmerjahn,F.,Ashli??ne,DJ,Reinhold,VN,Paulson,JC和Ravetch,JV(2008)。  用重组IgG fc重现IVIG抗炎活性。 Science 320(5874):373-376。
  3. Barb,AW,Brady,EK和Prestegard,JH(2009)。  通过ST6Gal-I的IgG-Fc聚糖的分支特异性唾液酸化。生物化学48(41):9705-9707。
  4. Kaneko,Y.,Nimmerjahn,F。和Ravetch,EV(2006)。  由Fc唾液酸化产生的免疫球蛋白G的抗炎活性。科学 313(5787):670-673。
  5. King,SJ,Hippe,KR和Weiser,JN(2006)。  通过由肺炎链球菌表达的外切糖苷酶的连续活性对人糖缀合物的糖基化。 Molecular Microbiology 59(3):961-974。
  6. Kingston,RE,(2003)。  DNA into mammalian cells。 ,,第9章(DOI:10.1002/0471142727.mb0900s64)。
  7. Mellquist,JL,Kasturi,L.,Spitalnik,SL和Shakin-Eshleman,SH(1998)。  IgG Fc结构域的唾液酸化削弱补体依赖性细胞毒性 Journal of Clinical Investigation 125(11):4160-4170。
  8. Stadlmann,J.,Weber,A.,Pabst,M.,Anderle,H.,Kunert,R。,Ehrlich,HJ,Peter Schwarz,H.and Altmann,F。(2009)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19688751"target ="_ blank">仔细观察凝集素分离后的人IgG唾液酸化和亚类分布。 em> Proteomics 9(17):4143-4153。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Quast, I., Maurer, M. A. and Lünemann, J. D. (2016). Generation of IgG-Fc Glycovariants Using Recombinant Glycosidases and Glycosyltransferases. Bio-protocol 6(15): e1886. DOI: 10.21769/BioProtoc.1886.
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