Home › Immunology › Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
Immunology JoVE (Open Access) Citable · DOI

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

DOI: 10.3791/57750-v
What you'll learn
  • ✓ Purify soluble glycoproteins from mammalian cell culture supernatant
  • ✓ Crystallize glycoproteins and perform ligand-soaking experiments
  • ✓ Apply biolayer interferometry and isothermal titration calorimetry for binding characterization
  • ✓ Interpret X-ray crystallography data for glycoprotein-ligand interactions
Protocol

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Difficulty
advanced
Total time
~2–4 weeks (purification ~3–5 days, crystallization screening ~1–3 weeks, data collection ~1–2 days)
Model organism
HEK293
Biosafety
BSL-1

Steps

1
Purify soluble glycoprotein from HEK293 supernatant

Harvest conditioned medium from HEK293 cells expressing the glycoprotein of interest and apply purification methods (affinity chromatography, size-exclusion) to isolate soluble protein to homogeneity.

▶ 00:38
2
Crystallize glycoproteins using vapor diffusion

Screen crystallization conditions for purified glycoprotein and optimize crystal growth parameters to obtain diffraction-quality crystals suitable for X-ray data collection.

▶ 02:08
3
Soak glycoprotein crystals with ligand

Incubate grown protein crystals in solutions containing the ligand of interest to allow diffusion and complex formation within the crystal lattice.

▶ 03:44
4
Characterize Fab and small molecule binding kinetics

Use biolayer interferometry and isothermal titration calorimetry to measure binding constants, kinetic rates, and thermodynamic parameters for interactions between the glycoprotein and Fab fragments or small molecules.

▶ 04:33
5
Interpret structural and biophysical characterization results

Analyze X-ray crystallography electron density maps, refine atomic models, and correlate structural data with kinetic and thermodynamic measurements to elucidate glycoprotein-ligand interactions.

▶ 07:34
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