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
发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1886 浏览次数: 12379
评审: Jia LiOmar AkilElizabeth V. Clarke
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参见作者原研究论文
The authors used this protocol in:
Nov 2015
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
- Amicon Ultra-4 Centrifugal Filter Units with 50 kDa MWCO (Merck Millipore Corporation, catalog number: UFC805096 )
- HisTrap HP column (GE Healthcare, catalog number: 29-0510-21 )
- StrepTrapTM HP column (GE Healthcare, catalog number: 29-0486-53 )
- Empty SPE tubes, 12 ml (Sigma-Aldrich, catalog number: 57176 )
- VisidryTM Female Luer Plug (Sigma-Aldrich, catalog number: 57098 )
- Caps for 12 ml SPE Tubes (Sigma-Aldrich, catalog number: 52174-U )
- 15 cm cell culture dish
- Spectra/Por 3 dialysis membrane (MWCO 3.5 kD), 45 mm width (VWR International, catalog number: 734-0686 )
- 10 clamps for dialysis tubing: Spectra/Por Closures, 55 mm (Spectrum, catalog number: 132737 )
- 0.2 µm syringe filters (Filtropur S plus 0.2) (Sarstedt AG, catalog number: 83.1826.102 )
- 0.2 µm bottle-top vacuum filter systems (Vakuumfiltration 1000 “rapid” Filtermax) (TTP, catalog number: 99950 )
- HEK293, HEK293T or HKB11 cells
- MES sodium salt (AppliChem GmbH, catalog number: A3101 )
- MES monohydrate (AppliChem GmbH, catalog number: A4730 )
- HEPES (Sigma-Aldrich, catalog number: H3375 )
- MOPS (AppliChem GmbH, catalog number: A1076 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9451 )
- Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 306568 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71380 )
- Sodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600-K )
- Citric acid (Sigma-Aldrich, catalog number: 251275 )
- Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
- MnCl2 tetrahydrate (AppliChem GmbH, catalog number: A2087 )
- MgCl2 hexahydrate (AppliChem GmbH, catalog number: A4425 )
- Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
- Sodium dihydrogen phosphate monohydrate (NaH2PO4) (AppliChem GmbH, catalog number: A1047 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
- Trizma® hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: T5941-500 G )
- Glycine (Sigma-Aldrich, catalog number: G8898 )
- Imidazole (AppliChem GmbH, catalog number: A1073 )
- Tween 20 (Sigma-Aldrich, catalog number: P7949 )
- Glycerol (Sigma-Aldrich, catalog number: G5516 )
- Sodium azide (AppliChem GmbH, catalog number: A1430 )
- D(+)-Lactose monohydrate (AppliChem GmbH, catalog number: A0880 )
- 1 M Tris, pH 8.8 (AppliChem GmbH, catalog number: A4265 )
- d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411-500 MG )
- Penicillin and streptomycin, liquid (Thermo Fischer Scientific, GibcoTM, catalog number: 15070-063 )
- UDP-galactose (Merck Millipore Corporation, Calbiochem®, catalog number: 670111-50 MG )
- CMP-sialic acid (Merck Millipore Corporation, Calbiochem®, catalog number: 233264-5 MG )
- StarGate® pCSG-IBA-144 Acceptor Vectors (IBA, catalog number: 5-5144-001 )
- StarGate® transfer reagent kit (IBA, catalog number: 5-1603-001 )
- Western Blocking Reagent, Solution (Sigma-Aldrich, catalog number: 11921673001 )
- Neuraminidase from Clostridium perfringes, recombinantly expressed in E. coli (New England Biolabs, catalog number: P0720S )
- β1-4-galactosidase from Streptococcus pneumoniae, recombinantly expressed in E. coli (Merck Millipore Corporation, Calbiochem®, catalog number: 345806-50 MIU )
- DMEM high glucose (4.5 g/L) (Thermo Fischer Scientific, GibcoTM, catalog number: 11965092 )
- Protein G sepharose for fast flow (GE healthcare, catalog number: 17-0618-01 )
- 20 µM polyethylene (PE) frits (Sigma-Aldrich, catalog number: 57181 )
- Biotinylated Lens Culinaris agglutinin (LCA) (Reactolab, catalog number: B-1045 )
- Biotinylated Erythrina Cristagalli lectin (ECL) (Reactolab, catalog number: B-1145 )
- Biotinylated Elderberry bark lectin (Sambucus nigra agglutinin, SNA) (Reactolab, catalog number: B-1305 )
- Agarose bound Sambucus Nigra Lectin (SNA), Vector Laboratories (Reactolab, catalog number: AL1303 )
- Streptavidin-HRP (Mabtech, catalog number: 3310-9 )
- MEM NEAA (non-essential amino acids) 100x (Thermo Fischer Scientific, GibcoTM, catalog number: 1140050 )
- Sodium pyruvate solution (100 mM) (Sigma-Aldrich, catalog number: S8636 )
- PierceTM ECL Western Blotting Substrate (Thermo Fischer Scientific, catalog number: 32106 )
- SuperSignalTM West Pico Chemiluminescent Substrate (Thermo Fischer Scientific catalog number: 34080 )
- General
- Tris-buffered saline (TBS) (see Recipes)
- Phosphate-buffered saline (PBS) (see Recipes)
- Tris-buffered saline (TBS) (see Recipes)
- For purification of recombinant glycosyltransferases
- 293, 293T and HKB11 cell culture medium (see Recipes)
- 293, 293T and HKB11 cell protein expression medium (see Recipes)
- 2x HEPES-buffered saline (HeBS) (see Recipes)
- 2.5 M CaCl2 (see Recipes)
- Strep-trap column elution buffer (see Recipes)
- His-trap elution buffer (see Recipes)
- His-trap wash buffer (see Recipes)
- His-trap supernatant dilution buffer (see Recipes)
- 293, 293T and HKB11 cell culture medium (see Recipes)
- For enzymatic galactosylation
- Galactosyltransferase buffer (see Recipes)
- Galactosyltransferase buffer (see Recipes)
- For enzymatic sialylation
- Sialyltransferase buffer (see Recipes)
- Sialyltransferase buffer (see Recipes)
- For enrichment of highly-sialylated antibodies using SNA-agarose
- SNA-agarose binding/wash buffer (see Recipes)
- SNA-agarose elution Buffer (see Recipes)
- SNA-agarose clearing Buffer (see Recipes)
- 0.08% NaN3 (see Recipes)
- SNA-agarose binding/wash buffer (see Recipes)
- For de-sialylation using recombinant neuraminidase
- Neuraminidase buffer (see Recipes)
- Neuraminidase buffer (see Recipes)
- For de-galactosylation using recombinant β1-4-galactosidase
- β1-4-galactosidase buffer (see Recipes)
- β1-4-galactosidase buffer (see Recipes)
- For purification of glyco-modified antibodies using gravity flow protein-G sepharose columns
- Protein G column elution buffer (see Recipes)
- Protein G eluate neutralization buffer (see Recipes)
- Protein G column elution buffer (see Recipes)
- For analysis of antibody glycosylation by lectin-blotting
- Lectin blot incubation buffer (see Recipes)
- Lectin blot wash buffer (see Recipes)
- Lectin blot incubation buffer (see Recipes)
Equipment
- Spectrophotometer (Thermo Scientific, NanoDrop, model: 1000 )
- Tabletop centrifuge capable of > 20,000 x g centrifugation and cooling to 4 °C (Eppendorf)
- GE ÄKTAprime plus (GE Healthcare, catalog number: 11-0013-13 )
- 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 )
- Fusion FX7 gel imaging system (Vilber, model: Fusion FX7 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
分子生物学 > 蛋白质 > 蛋白质-蛋白质相互作用
免疫学 > 抗体分析 > 抗体修饰
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