Purification and characterization of the N-terminal domains of Als1 and Als3 proteins from Candida albicans

Candida albicans Als1 and Als3 proteins are members of ALS (agglutinin-like sequence) gene family and they have been shown to adhere to epithelial and endothelial human cells (Sheppart et al. 2004). Als proteins have three-domain gene structure with a N-terminal part that is substrate-specific (Hoyer, 2001).
The N-terminal parts of the ALS1, ALS3 were inserted into pYEX-S1 vector using the In-FusionTM method. Proteins were secreted to the medium and purified using affinity chromatography (Ni-NTA column) and gel filtration (Superdex HR 75). The purity of proteins and the level of expression have been visualized using SDS-PAGE electrophoresis. To test the binding capacity of Als proteins, a bead adhesion assay has been performed. Tosyl-activated M-280 Dynabeads (Invitrogen) have been coated with fibronectin. Subsequently, Als1 and Als3 proteins have been incubated with fibronectin-coated beads to observe binding of both proteins. SDS-PAGE gel and Western blot results confirmed that both proteins bound to fibronectin, which confirms their activity.
To obtain information about protein folding and secondary structure of the N-terminal part of Als1p and Als3p, circular dichroic spectra were recorded. The CD spectrum of the N-terminal part of Als1p showed a dichroic minimum at 218 nm and a strong positive dichroic maximum near 200 nm, which is in accordance with results obtained by Sheppard et al. (2004). The CD spectrum for the N-terminal part of Als3p showed a dichroic minimum at 217 nm and a maximum at 200 nm. These results suggest that both proteins consist mostly of ?-sheet (50.1%) component, contain disordered structures (26.9%), turn structures (19.3%) as well as ?-helix (3.7%).
The equilibrium dissociation constant (KD) for the interaction of Als1p with immobilized fibronectin was determined by surface plasmon resonance (SPR) using a BIAcore 3000 system (GE Healthcare). Fibronectin was covalently immobilized on a CM5 sensor chip via amine coupling using an amine-coupling kit (BIAcore). Increasing concentrations of the analyte (Als1 protein) were used (0.1 nM-50 ?M). The equilibrium dissociation constant is KD - 1.3 ?M.
To determine the structure of the N-terminal part of Als1p and Als3p, four crystal screens (Hampton Research 1&2, JB Cryo, JB Classic 1-4 and 5-8) have been used to screen for optimal conditions for crystal growth. Sitting and hanging drop methods have been used. Screens were placed and stored either at room temperature (20°C) or at 4°C. Protein concentrations were varied between 2 mg/ml and 11.6 mg/ml for both Als1p and Als3p. Currently, screens are under observation.