Hitrap deae

SbcCD proteins used in this study were purified and analyzed as described elsewhere⁴⁹ . Briefly, SbcCD protein complex was overexpressed from SbcCD-overexpressing plasmid pDL761 in E. coli DL776. Cell extracts were prepared using lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl, 10 mM β-mercaptoethanol, 10% (w/v) sucrose, 1 mM phenylmethylsulfonyl fluoride) by sonication followed by polyethyleneimine precipitation. SbcCD was further purified by HiTrap DEAE (GE Healthcare), ammonium precipitation, Superose 6 HR 10/30 (Amersham) and Mono Q HR 5/5 (Amersham). The peak fraction was dialyzed in SbcCD strage buffer (50 mM Tris,-HCl pH 7.5, 1 mM EDTA, 50 mM NaCl, 10 mM 2–mercaptoethanol, 10% glycerol) and stored at −80 °C.

To concentrate secreted May1, YNB conditioned media was prepared as described above from 32-hour cultures of wild-type C. neoformans. The media was then diluted 2.7-fold into buffer A (50 mM Tris base pH 8), chosen to increase the pH in order to limit May1 autoproteolysis, dilute salts in the media (final conductivity ~6%), and confer a negative charge to the peptidase domain. The media was then loaded onto a 1 ml HiTrap DEAE fast flow column (GE Healthcare) using a fast protein liquid chromatography system with a flow rate of 1.5 ml/min. May1 was eluted using a 30 minute linear gradient of 0–100% buffer B (50 mM Tris base, 1 M NaCl, pH 8). Active fractions were determined by measuring activity using the substrate IQ-2. They were then combined and approximate May1 concentration determined by active-site titration.

The culture medium was centrifuged at 5,000g for 10 min to obtain the culture supernatant, which was then filtered through a 0.2-μm membrane. The culture supernatants were separated with a molecular weight cutoff spin column (GE Healthcare). The >0.5 kDa fraction was obtained by dialysis with a Micro Float-A-Lyzer Dialysis Device (Spectrum Laboratories). The culture supernatant was separated using an AKTA Design HPLC system (GE Healthcare) using a Superdex peptide column (GE Healthcare) and eluted with distilled water at a flow rate of 1 ml min⁻¹. The fraction was applied to an L-column (Chemicals Evaluation and Research Institute, Japan) and eluted with 0.1% formic acid and 0.1% formic acid/acetonitrile in a linear gradient at a flow rate of 0.2 ml min⁻¹. Ion exchange chromatography was performed using a HiTrap-DEAE (GE Healthcare), CM (GE Healthcare) and an SP column (GE Healthcare). The elution buffer for DEAE was 0 M and 1.0 M NaCl in 20 mM Tris-HCl (pH 8.0). The elution buffer for SP and CM was 0 M and 1.0 M NaCl in 50 mM acetic acid in a linear gradient at a flow rate of 1 ml min⁻¹. Finally, the sample was separated using a ZIC-HILIC column (Merck Millipore, Germany) and eluted with 70% acetonitrile and 10% acetonitrile in a linear gradient at a flow rate of 0.5 ml min⁻¹. The eluent was monitored by ultraviolet spectrophotometry at 210 nm.