Phenyl sepharose

The genes HiGulD (Uniprot ID Q57517) and HiUxuA (Uniprot ID P44488) were amplified from H. influenzae strain Rd KW20 (ATCC 51907) genomic DNA. PCR was performed using KOD Extreme DNA Polymerase (Novagen) according to the manufacturer’s guidelines. The conditions were: 2 min at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 66°C, and 20 s at 72°C. Primers are listed in Supplementary file 7. The amplified fragment was cloned into the C-terminal TEV cleavable 10x-Histag containing vector pNYCOMPS-LIC-TH10-ccdB (C-term) such that the tag is out of frame (pNYCOMPSC-tagless), by ligation-independent cloning (Aslanidis and de Jong, 1990 (link); Savitsky et al., 2010 (link)).
The pNYCOMPSC-tagless HiGulD and HiUxuA constructs were transformed into E. coli BL21 (DE3) for expression. Both HiGulD and HiUxuA were purified from 1 L of culture using DEAE Sepharose, Q‐Sepharose, and phenyl-Sepharose columns (all Amersham Biosciences) as previously described (Wichelecki et al., 2014 (link)). Proteins were concentrated to 15 g/L and 6 g/L, respectively, flash frozen in liquid nitrogen, and stored at −80°C.

The proteasome active fractions, recovered after each purification step, were detected by measuring the CT-like activity using the specific fluorogenic substrate LLVY. The T. bernacchii hemolysates were loaded on a DEAE Sepharose Fast Flow column, previously equilibrated in 25 mM Tris/HCl, pH 7.5 (Buffer A), and connected to an AKTA_FPLC system (Amersham Biosciences). Bound proteins were eluted using an ionic strength gradient from 0 to 1 M NaCl in Buffer A at a flow rate of 1 ml·min⁻¹. The active fractions were pooled, dialysed extensively against 25 mM Tris/HCl, pH 7.5 and 1 M ammonium sulfate (Buffer A) and then loaded on to a Phenyl Sepharose (Amersham) column, connected to an AKTA_FPLC system (Amersham Biosciences), equilibrated in the same buffer. Bound proteins were eluted with a linear gradient (0–100%) of 25 mM Tris/HCl (pH 7.5) (buffer B) at a flow rate of 1 ml·min⁻¹. The active fractions were pooled, dialysed against 25 mM Tris/HCl (pH 7.5) and then applied to a Superdex 200 PC 3.2/30 column connected to a SMART System (Pharmacia), equilibrated in 25 mM Tris/HCl, pH 7.5 and 50 mM NaCl. Finally, active fractions were pooled and the purified proteasome was stored in 25 mM Tris/HCl pH 7.5, containing 5% glycerol.

Tuberculin PPD (RT 50) was obtained from Statens Serum Institute, and PHA from In vivogen. LAM and PIMs were prepared as previously described in detail (5 (link)). Briefly, heat-killed bacteria were frozen and thawed several times, sonicated and extracted in 40% hot phenol for 1 h at 70°C. ManLAM and PIM were obtained from the water and phenol PHAses respectively. The dialyzed water PHAse was submitted to affinity chromatography on Concanavalin A-SePHArose. After elution, bound material was subjected to hydrophobic interaction chromatography on Phenyl-SePHArose (Amersham, Sweden). Bound ManLAM was eluted and further separated from other glycolipids by gel filtration on SePHAcryl S-100 (Amersham, Sweden). The phenol PHAse obtained above was washed 3 times with PBS and extracted with an equal volume of 2% SDS in PBS overnight at room temperature. The resulting water PHAse was precipitated with of ice-cold ethanol. PIMs contained in the precipitate were purified to homogeneity by gel filtration on SePHAcryl S-100. PIM contains both PIM₂ and PIM₆ isoforms, differing in number of fatty acyl constituents (5 (link)).